Ferrer M

References (44)

Title : Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor - Fernandez-Lopez_2023_Chem.Commun.(Camb)__
Author(s) : Fernandez-Lopez L , Cea-Rama I , Alvarez-Malmagro J , Ressmann AK , Gonzalez-Alfonso JL , Coscolin C , Shahgaldian P , Plou FJ , Modregger J , Pita M , Sanz-Aparicio J , Ferrer M
Ref : Chem Commun (Camb) , : , 2023
Abstract : Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.
ESTHER : Fernandez-Lopez_2023_Chem.Commun.(Camb)__
PubMedSearch : Fernandez-Lopez_2023_Chem.Commun.(Camb)__
PubMedID: 37376994
Gene_locus related to this paper: 9zzzz-a0a2k8jn75

Title : The Mobility of the Cap Domain Is Essential for the Substrate Promiscuity of a Family IV Esterase from Sorghum Rhizosphere Microbiome - Distaso_2023_Appl.Environ.Microbiol__e0180722
Author(s) : Distaso M , Cea-Rama I , Coscolin C , Chernikova TN , Tran H , Ferrer M , Sanz-Aparicio J , Golyshin PN
Ref : Applied Environmental Microbiology , :e0180722 , 2023
Abstract : Metagenomics offers the possibility to screen for versatile biocatalysts. In this study, the microbial community of the Sorghum bicolor rhizosphere was spiked with technical cashew nut shell liquid, and after incubation, the environmental DNA (eDNA) was extracted and subsequently used to build a metagenomic library. We report the biochemical features and crystal structure of a novel esterase from the family IV, EH(0), retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH(0) (optimum temperature [T(opt)], 50 degreesC; melting temperature [T(m)], 55.7 degreesC; optimum pH [pH(opt)], 9.5) was stable in the presence of 10 to 20% (vol/vol) organic solvents and exhibited hydrolytic activity against p-nitrophenyl esters from acetate to palmitate, preferably butyrate (496 U mg(-1)), and a large battery of 69 structurally different esters (up to 30.2 U mg(-1)), including bis(2-hydroxyethyl)-terephthalate (0.16 +/- 0.06 U mg(-1)). This broad substrate specificity contrasts with the fact that EH(0) showed a long and narrow catalytic tunnel, whose access appears to be hindered by a tight folding of its cap domain. We propose that this cap domain is a highly flexible structure whose opening is mediated by unique structural elements, one of which is the presence of two contiguous proline residues likely acting as possible hinges, which together allow for the entrance of the substrates. Therefore, this work provides a new role for the cap domain, which until now was thought to be an immobile element that contained hydrophobic patches involved in substrate prerecognition and in turn substrate specificity within family IV esterases. IMPORTANCE A better understanding of structure-function relationships of enzymes allows revelation of key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH(0), a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH(0) revealed the potential of the sorghum rhizosphere microbiome as a source of enzymes with interesting properties, such as pH and solvent tolerance and remarkably broad substrate promiscuity. Its structure resembled those of homologous proteins from mesophilic Parvibaculum and Erythrobacter spp. and hyperthermophilic Pyrobaculum and Sulfolobus spp. and had a very narrow, single-entry access tunnel to the active site, with access controlled by a capping domain that includes a number of nonconserved proline residues. These structural markers, distinct from those of other substrate-promiscuous esterases, can help in tuning substrate profiles beyond tunnel and active site engineering.
ESTHER : Distaso_2023_Appl.Environ.Microbiol__e0180722
PubMedSearch : Distaso_2023_Appl.Environ.Microbiol__e0180722
PubMedID: 36602332
Gene_locus related to this paper: 9bact-EH0

Title : Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins - Distaso_2023_Appl.Environ.Microbiol__e0170422
Author(s) : Distaso MA , Chernikova TN , Bargiela R , Coscolin C , Stogios P , Gonzalez-Alfonso JL , Lemak S , Khusnutdinova AN , Plou FJ , Evdokimova E , Savchenko A , Lunev EA , Yakimov MM , Golyshina OV , Ferrer M , Yakunin AF , Golyshin PN
Ref : Applied Environmental Microbiology , :e0170422 , 2023
Abstract : Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70 degreesC of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90 degreesC, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80 degreesC). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical alpha/beta-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 A revealed the presence of a N-terminal beta-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest effective volumes among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90 degreesC) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.
ESTHER : Distaso_2023_Appl.Environ.Microbiol__e0170422
PubMedSearch : Distaso_2023_Appl.Environ.Microbiol__e0170422
PubMedID: 36719236
Gene_locus related to this paper: 9bact-estC55.8n1 , 9bact-IS10

Title : Enhancing the Hydrolytic Activity of a Lipase towards Larger Triglycerides through Lid Domain Engineering - Fernandez-Lopez_2023_Int.J.Mol.Sci_24_13768
Author(s) : Fernandez-Lopez L , Roda S , Robles-Martin A , Munoz-Tafalla R , Almendral D , Ferrer M , Guallar V
Ref : Int J Mol Sci , 24 : , 2023
Abstract : Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or Lip(MRD)) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (Lip(MRD) numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation.
ESTHER : Fernandez-Lopez_2023_Int.J.Mol.Sci_24_13768
PubMedSearch : Fernandez-Lopez_2023_Int.J.Mol.Sci_24_13768
PubMedID: 37762071
Gene_locus related to this paper: 9pseu-a0a1l7F3D7

Title : Assigning Functions of Unknown Enzymes by High-Throughput Enzyme Characterization - Molina-Espeja_2023_Methods.Mol.Biol_2555_181
Author(s) : Molina-Espeja P , Fernandez-Lopez L , Golyshin PN , Ferrer M
Ref : Methods Mol Biol , 2555 :181 , 2023
Abstract : The discovery of new enzymes is strongly enabled by the implementation of high-throughput screening methods to detect enzymatic activity in single organisms or clone expression libraries, or to benchmark their performances against known prototypes. In this chapter, a number of methods, applicable at high-throughput scale, are described that allow the screening and characterization of enzymes relevant to biotechnology, particularly, ester-hydrolases (esterases, lipases, phospholipases, and polyester hydrolases).
ESTHER : Molina-Espeja_2023_Methods.Mol.Biol_2555_181
PubMedSearch : Molina-Espeja_2023_Methods.Mol.Biol_2555_181
PubMedID: 36306087

Title : Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation - Vidal_2022_Front.Microbiol_13_868839
Author(s) : Vidal P , Martinez-Martinez M , Fernandez-Lopez L , Roda S , Mendez-Garcia C , Golyshina OV , Guallar V , Pelaez AI , Ferrer M
Ref : Front Microbiol , 13 :868839 , 2022
Abstract : Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naive and in silico metagenomic approaches, we retrieved 16 esterases from the alpha/beta-hydrolase fold superfamily with the closest match from uncultured acidophilic Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium, and Ferrimicrobium), Acidiphilium, and other Proteobacteria inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of -2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in Escherichia coli. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33-68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30-65 degreesC, retaining 20-61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 +/- 0.6 degreesC). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 +/- 75 U/g at pH 8.0 and 30 degreesC) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.
ESTHER : Vidal_2022_Front.Microbiol_13_868839
PubMedSearch : Vidal_2022_Front.Microbiol_13_868839
PubMedID: 35663881
Gene_locus related to this paper: 9zzzz-t1a3k4 , 9zzzz-t1ci96 , 9zzzz-t1b379 , 9zzzz-t1be47 , 9zzzz-t1d4I7 , 9actn-KY010298 , 9bact-KY010297

Title : EP-Pred: A Machine Learning Tool for Bioprospecting Promiscuous Ester Hydrolases - Xiang_2022_Biomolecules_12_
Author(s) : Xiang R , Fernandez-Lopez L , Robles-Martin A , Ferrer M , Guallar V
Ref : Biomolecules , 12 : , 2022
Abstract : When bioprospecting for novel industrial enzymes, substrate promiscuity is a desirable property that increases the reusability of the enzyme. Among industrial enzymes, ester hydrolases have great relevance for which the demand has not ceased to increase. However, the search for new substrate promiscuous ester hydrolases is not trivial since the mechanism behind this property is greatly influenced by the active site's structural and physicochemical characteristics. These characteristics must be computed from the 3D structure, which is rarely available and expensive to measure, hence the need for a method that can predict promiscuity from sequence alone. Here we report such a method called EP-pred, an ensemble binary classifier, that combines three machine learning algorithms: SVM, KNN, and a Linear model. EP-pred has been evaluated against the Lipase Engineering Database together with a hidden Markov approach leading to a final set of ten sequences predicted to encode promiscuous esterases. Experimental results confirmed the validity of our method since all ten proteins were found to exhibit a broad substrate ambiguity.
ESTHER : Xiang_2022_Biomolecules_12_
PubMedSearch : Xiang_2022_Biomolecules_12_
PubMedID: 36291739

Title : Crystal structure of a family VIII beta-lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope - Cea-Rama_2022_FEBS.J__
Author(s) : Cea-Rama I , Coscolin C , Gonzalez-Alfonso JL , Raj J , Vasiljevic M , Plou FJ , Ferrer M , Sanz-Aparicio J
Ref : Febs J , : , 2022
Abstract : Family VIII esterases present similarities to class C beta-lactamases, which show nucleophilic serines located at the S-X-X-K motif instead of the G-X-S-X-G or G-D-S-(L) motif shown by other carboxylesterase families. Here, we report the crystal structure of a novel family VIII (subfamily VIII. I) esterase (EH(7) ; denaturing temperature, 52.6+/-0.3 degreesC; pH optimum 7.0-9.0) to deepen its broad substrate range. Indeed, the analysis of the substrate specificity revealed its capacity to hydrolyse nitrocefin as a model chromogenic cephalosporin substrate (40.4 +/- 11.4 units/g), as well as a large battery of 66 structurally different esters (up to 1730 min(-1) ), including bis(2-hydroxyethyl)-terephthalate (241.7 +/- 8.5 units/g) and the mycotoxin T-2 (1220 +/- 52 units/g). It also showed acyltransferase activity through the synthesis of benzyl 3-oxobutanoate (40.4 +/- 11.4 units/g) from benzyl alcohol and vinyl acetoacetate. Such a broad substrate scope is rare among family VIII esterases and lipolytic enzymes. Structural analyses of free and substrate-bound forms of this homo-octamer esterase suggest that EH(7) presents a more opened and exposed S1 site having no steric hindrance for the entrance of substrates to the active site, more flexible R1, R2 and R3 regions allowing the binding of a wide spectrum of substrates into the active site, as well as small residues in the conserved motif Y-X-X containing the catalytic Tyr enabling the entrance of large substrates. These unique structural elements in combination with docking experiments allowed us to gain valuable insights into the substrate specificity of this esterase and possible others belonging to family VIII.
ESTHER : Cea-Rama_2022_FEBS.J__
PubMedSearch : Cea-Rama_2022_FEBS.J__
PubMedID: 35694902

Title : Crystal structures of a novel family IV esterase in free and substrate-bound form - Hoppner_2021_FEBS.J_288_3570
Author(s) : Hoppner A , Bollinger A , Kobus S , Thies S , Coscolin C , Ferrer M , Jaeger KE , Smits SHJ
Ref : Febs J , 288 :3570 , 2021
Abstract : Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an alpha-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV.
ESTHER : Hoppner_2021_FEBS.J_288_3570
PubMedSearch : Hoppner_2021_FEBS.J_288_3570
PubMedID: 33342083
Gene_locus related to this paper: pseth-a0a1m6y2k1

Title : Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase - Cea-Rama_2021_Comput.Struct.Biotechnol.J_19_2307
Author(s) : Cea-Rama I , Coscolin C , Katsonis P , Bargiela R , Golyshin PN , Lichtarge O , Ferrer M , Sanz-Aparicio J
Ref : Comput Struct Biotechnol J , 19 :2307 , 2021
Abstract : Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH(3)) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.
ESTHER : Cea-Rama_2021_Comput.Struct.Biotechnol.J_19_2307
PubMedSearch : Cea-Rama_2021_Comput.Struct.Biotechnol.J_19_2307
PubMedID: 33995922
Gene_locus related to this paper: 9zzzz-a0a2k8jn75

Title : Computationally Driven Rational Design of Substrate Promiscuity on Serine Ester Hydrolases - Roda_2021_ACS.Catal_11_3590
Author(s) : Roda S , Fernandez-Lopez L , Canadas R , Santiago G , Ferrer M , Guallar V
Ref : ACS Catal , 11 :3590 , 2021
Abstract : Enzymes with a broad substrate specificity are of great interest both at the basic and applied level. Understanding the main parameters that make an enzyme substrate ambiguous could be thus important not only for their selection from the ever-increasing amount of sequencing data but also for engineering a more substrate promiscuous variant. This issue, which remains unresolved, was herein investigated by targeting a serine ester hydrolase (EH102), which exhibits a narrow substrate spectrum, being only capable of hydrolyzing 16 out of 96 esters tested. By using a modeling approach, we demonstrated that one can rationalize active site parameters defining substrate promiscuity, and that based on them the substrate specificity can be significantly altered. This was accomplished by designing two variants, EH102DM2 and EH102TM2, that hydrolyze 51 and 63 esters, respectively, while maintaining similar or higher turnover rates compared to the original enzyme. We hypothesized that the parameters identified here (the volume, size, exposure, enclosure, hydrophobicity, and hydrophilicity of the active site cavity and its tightness) can serve in the future to expand the substrate spectra of esterases and thus expand their use in biotechnology and synthetic chemistry.
ESTHER : Roda_2021_ACS.Catal_11_3590
PubMedSearch : Roda_2021_ACS.Catal_11_3590
PubMedID:

Title : Promiscuous Esterases Counterintuitively Are Less Flexible than Specific Ones - Nutschel_2021_J.Chem.Inf.Model__
Author(s) : Nutschel C , Coscolin C , David B , Mulnaes D , Ferrer M , Jaeger KE , Gohlke H
Ref : J Chem Inf Model , : , 2021
Abstract : Understanding mechanisms of promiscuity is increasingly important from a fundamental and application point of view. As to enzyme structural dynamics, more promiscuous enzymes generally have been recognized to also be more flexible. However, examples for the opposite received much less attention. Here, we exploit comprehensive experimental information on the substrate promiscuity of 147 esterases tested against 96 esters together with computationally efficient rigidity analyses to understand the molecular origin of the observed promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly less flexible than specific ones, are significantly more thermostable, and have a significantly increased specific activity. These results may be reconciled with a model according to which structural flexibility in the case of specific esterases serves for conformational proofreading. Our results signify that an esterase sequence space can be screened by rigidity analyses for promiscuous esterases as starting points for further exploration in biotechnology and synthetic chemistry.
ESTHER : Nutschel_2021_J.Chem.Inf.Model__
PubMedSearch : Nutschel_2021_J.Chem.Inf.Model__
PubMedID: 33949194

Title : Tuning the Properties of Natural Promiscuous Enzymes by Engineering Their Nano-environment - Giunta_2020_ACS.Nano__
Author(s) : Giunta CI , Cea-Rama I , Alonso S , Briand ML , Bargiela R , Coscolin C , Corvini PF , Ferrer M , Sanz-Aparicio J , Shahgaldian P
Ref : ACS Nano , : , 2020
Abstract : Owing to their outstanding catalytic properties, enzymes represent powerful tools for carrying out a wide range of (bio)chemical transformations with high proficiency. In this context, enzymes with high biocatalytic promiscuity are somewhat neglected. Here, we demonstrate that a meticulous modification of a synthetic shell that surrounds an immobilized enzyme possessing broad substrate specificity allows the resulting nanobiocatalyst to be endowed with enantioselective properties while maintaining a high level of substrate promiscuity. Our results show that control of the enzyme nano-environment enables tuning of both substrate specificity and enantioselectivity. Further, we demonstrate that our strategy of enzyme supramolecular engineering allows the enzyme to be endowed with markedly enhanced stability in an organic solvent (i.e., acetonitrile). The versatility of the method was assessed with two additional substrate-promiscuous and structurally different enzymes, for which improvements in enantioselectivity and stability were confirmed. We expect this method to promote the use of supramolecularly engineered promiscuous enzymes in industrially relevant biocatalytic processes.
ESTHER : Giunta_2020_ACS.Nano__
PubMedSearch : Giunta_2020_ACS.Nano__
PubMedID: 33306346
Gene_locus related to this paper: 9zzzz-a0a2k8jn75

Title : A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri - Structural and Functional Insights - Bollinger_2020_Front.Microbiol_11_114
Author(s) : Bollinger A , Thies S , Knieps-Grunhagen E , Gertzen C , Kobus S , Hoppner A , Ferrer M , Gohlke H , Smits SHJ , Jaeger KE
Ref : Front Microbiol , 11 :114 , 2020
Abstract : Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14T. The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 A resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical alpha/beta-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 A resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.
ESTHER : Bollinger_2020_Front.Microbiol_11_114
PubMedSearch : Bollinger_2020_Front.Microbiol_11_114
PubMedID: 32117139
Gene_locus related to this paper: 9psed-peh

Title : Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis - Bollinger_2020_Appl.Environ.Microbiol_86_e00106
Author(s) : Bollinger A , Molitor R , Thies S , Koch R , Coscolin C , Ferrer M , Jaeger KE
Ref : Applied Environmental Microbiology , 86 :e00106 , 2020
Abstract : Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
ESTHER : Bollinger_2020_Appl.Environ.Microbiol_86_e00106
PubMedSearch : Bollinger_2020_Appl.Environ.Microbiol_86_e00106
PubMedID: 32111588
Gene_locus related to this paper: 9psed-peh , alcbs-q0vt77 , alcbs-q0vtl7 , aneth-d3xb96 , alcbs-q0vl36 , alcbs-q0vq49 , 9psed-CE24 , 9psed-CE23 , 9psed-CE22 , 9psed-CE20 , 9psed-CE18 , 9psed-CE15 , 9psed-CE13 , alcbs-q0vmp2 , alcbs-q0vlp6 , marav-a1u5n0 , alcbs-q0vlk5 , 9psed-a0a1h5udv9

Title : Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis - Alonso_2020_Nat.Catal_3_319
Author(s) : Alonso S , Santiago G , Cea-Rama I , Fernandez-Lopez L , Coscolin C , Modregger J , Ressmann A , Martinez-Martinez M , Marrero H , Bargiela R , Pita M , Gonzalez-Alfonso JL , Briand M , Rojo D , Barbas C , Plou FJ , Golyshin PN , Shahgaldian P , Sanz-Aparicio J , Guallar V , Ferrer M
Ref : Nature Catalysis , 3 :319 , 2019
Abstract : Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on pro-teins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel-Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (>=83%; e.e. >99.9%) were achieved in one pot with this artificial multifunc-tional metalloenzyme.
ESTHER : Alonso_2020_Nat.Catal_3_319
PubMedSearch : Alonso_2020_Nat.Catal_3_319
PubMedID:
Gene_locus related to this paper: 9bact-LAE6

Title : The Thaumarchaeon N. gargensis carries functional bioABD genes and has a promiscuous E. coli DeltabioH-complementing esterase EstN1 - Chow_2018_Sci.Rep_8_13823
Author(s) : Chow J , Danso D , Ferrer M , Streit WR
Ref : Sci Rep , 8 :13823 , 2018
Abstract : Biotin is an essential cofactor required for carboxylation and decarboxylation reactions in all domains of life. While biotin biosynthesis in most Bacteria and Eukarya is well studied, the complete pathway for this vitamer in Archaea is still not known. Detailed genome searches indicated the presence of possible bio gene clusters only in Methanococcales and Thaumarchaeota. Therefore, we analysed the functionality of the predicted genes bioA, bioB, bioD and bioF in the Thaumarchaeon Nitrososphaera gargensis Ga2.9 which are essential for the later steps of biotin synthesis. In complementation tests, the gene cluster-encoded N. gargensis bioABD genes except bioF restored growth of corresponding E. coli Rosetta-gami 2 (DE3) deletion mutants. To find out how biotin biosynthesis is initiated, we searched the genome for a possible bioH analogue encoding a pimeloyl-ACP-methylester carboxylesterase. The respective amino acid sequence of the ORF estN1 showed weak conserved domain similarity to this class of enzymes (e-value 3.70e(-42)). Remarkably, EstN1 is a promiscuous carboxylesterase that complements E. coli DeltabioH and Mesorhizobium loti DeltabioZ mutants for growth on biotin-free minimal medium. Additional 3D-structural models support the hypothesis that EstN1 is a BioH analogue. Thus, this is the first report providing experimental evidence that Archaea carry functional bio genes.
ESTHER : Chow_2018_Sci.Rep_8_13823
PubMedSearch : Chow_2018_Sci.Rep_8_13823
PubMedID: 30218044

Title : Determinants and prediction of esterase substrate promiscuity patterns - Martinez-Martinez_2018_ACS.Chem.Biol_13_225
Author(s) : Martinez-Martinez M , Coscolin C , Santiago G , Chow J , Stogios PJ , Bargiela R , Gertler C , Navarro-Fernandez J , Bollinger A , Thies S , Mendez-Garcia C , Popovic A , Brown G , Chernikova TN , Garcia-Moyano A , Bjergah GE , Perez-Garcia P , Hai T , Del Pozo MV , Stokke R , Steen IH , Cui H , Xu X , Nocek BP , Alcaide M , Distaso M , Mesa V , Pelaez AI , Sanchez J , Buchholz PCF , Pleiss J , Fernandez-Guerra A , Glockner FO , Golyshina OV , Yakimov MM , Savchenko A , Jaeger KE , Yakunin AF , Streit WR , Golyshin PN , Guallar V , Ferrer M
Ref : ACS Chemical Biology , 13 :225 , 2018
Abstract : Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps ranking (classifying) promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence datasets.
ESTHER : Martinez-Martinez_2018_ACS.Chem.Biol_13_225
PubMedSearch : Martinez-Martinez_2018_ACS.Chem.Biol_13_225
PubMedID: 29182315
Gene_locus related to this paper: 9zzzz-a0a2k8jn75 , 9zzzz-a0a2k8jt94 , 9zzzz-a0a0g3fj44 , 9zzzz-a0a0g3fh10 , 9zzzz-a0a0g3fh03 , 9bact-a0a1s5qkj8 , 9zzzz-a0a0g3feh5 , 9zzzz-a0a0g3fkz4 , 9zzzz-a0a0g3fh07 , 9zzzz-a0a0g3fh34 , 9zzzz-a0a0g3fh31 , 9bact-KY458167 , alcbs-q0vqa3 , 9bact-a0a1s5qki8 , 9zzzz-a0a0g3feq8 , 9zzzz-a0a0g3feh8 , 9zzzz-a0a0g3fh19 , 9bact-KY203037 , 9bact-a0a1s5ql22 , 9bact-a0a1s5qm34 , 9bact-KY203034 , 9bact-r9qzg0 , 9bact-a0a1s5qly8 , 9zzzz-a0a0g3fkz8 , 9zzzz-a0a0g3feg9 , 9zzzz-KY203033 , 9zzzz-a0a0g3fes4 , 9zzzz-a0a0g3fh42 , 9bact-a0a1s5qlx2 , 9zzzz-KY483651 , 9bact-a0a1s5qmh4 , 9zzzz-KY203032 , 9zzzz-EH87 , 9zzzz-a0a0g3fei1 , 9zzzz-a0a0g3fet2 , 9zzzz-KY483647 , 9zzzz-EH82 , 9zzzz-a0a0g3fe15 , 9bact-KY203031 , 9bact-t1w006 , 9zzzz-a0a0g3fet6 , 9bact-KY458164 , geoth-g8myf3 , 9bact-a0a1s5ql04 , 9gamm-a0a1y0ihk7 , 9bact-a0a1s5qly6 , 9bact-a0a1s5qkg4 , 9bact-a0a1s5qkm4 , 9gamm-s5tv80 , 9gamm-a0a0c4zhg2 , 9zzzz-t1b379 , 9gamm-KY483646 , 9bact-KY458160 , 9zzzz-a0a0g3fj57 , 9gamm-s5t8349 , 9arch-KY203036 , 9bact-KY458168 , 9zzzz-a0a0g3fes0 , 9zzzz-t1be47 , 9bact-KY458159 , 9zzzz-a0a0g3fh39 , 9bact-t1vzd5 , 9prot-EH41 , 9bact-Lip114 , alcbs-q0vt77 , 9bact-a0a1s5qke6 , 9bact-a0a1s5qkf3 , 9prot-SRP030024 , 9gamm-s5t532 , 9bact-a0a1s5qkl2 , 9bact-a0a1s5qkk8 , 9zzzz-KY203030 , 9zzzz-t1d4I7 , 9prot-KY019260 , 9bact-a0a1s5qm38 , 9arch-KY458161 , 9prot-KY010302 , 9zzzz-a0a0g3fl25 , 9actn-KY010298 , 9gamm-s5u059 , 9bact-a0a1s5qmi7 , 9bact-KY010297 , 9bact-KY483642 , 9bact-a0a1s5qkj1 , 9bact-KY010299 , 9bact-KY483648 , alcbs-q0vtl7 , 9bact-a0a1s5qf1 , 9bact-a0a1s5qkg0 , 9bact-a0a0h4tgu6 , 9bact-MilE3 , 9bact-LAE6 , 9alte-MGS-MT1 , 9bact-r9qzf7 , 9gamm-k0c6t6 , alcbs-q0vl36 , alcbs-q0vlq1 , alcbs-q0vq49 , bacsu-pnbae , canar-LipB , canan-lipasA , geost-lipas , marav-a1u5n0 , pseps-i7k8x5 , staep-GEHD , symth-q67mr3 , altma-s5cfn7 , cycsp-k0c2b8 , alcbs-q0vlk5 , 9bact-k7qe48 , 9bact-MGS-M1 , 9bact-MGS-M2 , 9bact-a0a0b5kns5 , 9zzzz-a0a0g3fej4 , 9zzzz-a0a0g3fj60 , 9zzzz-a0a0g3fej0 , 9zzzz-a0a0g3fj64 , 9bact-a0a0b5kc16 , 9zzzz-a0a0g3feg6 , 9zzzz-a0a0g3feu6

Title : Functional-Based Screening Methods for Detecting Esterase and Lipase Activity Against Multiple Substrates - Reyes-Duarte_2018_Methods.Mol.Biol_1835_109
Author(s) : Reyes-Duarte D , Coscolin C , Martinez-Martinez M , Ferrer M , Garcia-Arellano H
Ref : Methods Mol Biol , 1835 :109 , 2018
Abstract : Functional screens have been extensively used for searching native enzymes or mutant variants in clone libraries. Esterases and lipases are the most retrieved enzymes, because they are within the more demanded industrial enzymes and because a number of simple and generic screening methods can be applied for their screen. Here, we describe the use of a generic pH indicator assay protocol which unambiguously allows detecting in high-throughput manner esterase and lipase activity and quantifying specific activities using an ester concentration above 0.5 mM. The described method is simple and generic to allow the selection of esterases and lipases targeting desired esters.
ESTHER : Reyes-Duarte_2018_Methods.Mol.Biol_1835_109
PubMedSearch : Reyes-Duarte_2018_Methods.Mol.Biol_1835_109
PubMedID: 30109647

Title : Rational Engineering of Multiple Active Sites in an Ester Hydrolase - Santiago_2018_Biochemistry_57_2245
Author(s) : Santiago G , Martinez-Martinez M , Alonso S , Bargiela R , Coscolin C , Golyshin PN , Guallar V , Ferrer M
Ref : Biochemistry , 57 :2245 , 2018
Abstract : Effects of altering the properties of an active site in an enzymatic homogeneous catalyst have been extensively reported. However, the possibility of increasing the number of such sites, as commonly done in heterogeneous catalytic materials, remains unexplored, particularly because those have to accommodate appropriate residues in specific configurations. This possibility was investigated by using a serine ester hydrolase as the target enzyme. By using the Protein Energy Landscape Exploration software, which maps ligand diffusion and binding, we found a potential binding pocket capable of holding an extra catalytic triad and oxyanion hole contacts. By introducing two mutations, this binding pocket became a catalytic site. Its substrate specificity, substrate preference, and catalytic activity were different from those of the native site of the wild type ester hydrolase and other hydrolases, due to the differences in the active site architecture. Converting the binding pocket into an extra catalytic active site was proven to be a successful approach to create a serine ester hydrolase with two functional reactive groups. Our results illustrate the accuracy and predictive nature of modern modeling techniques, opening novel catalytic opportunities coming from the presence of different catalytic environments in single enzymes.
ESTHER : Santiago_2018_Biochemistry_57_2245
PubMedSearch : Santiago_2018_Biochemistry_57_2245
PubMedID: 29600855
Gene_locus related to this paper: 9bact-LAE6

Title : Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families - Popovic_2017_Sci.Rep_7_44103
Author(s) : Popovic A , Hai T , Tchigvintsev A , Hajighasemi M , Nocek B , Khusnutdinova AN , Brown G , Glinos J , Flick R , Skarina T , Chernikova TN , Yim V , Bruls T , Paslier DL , Yakimov MM , Joachimiak A , Ferrer M , Golyshina OV , Savchenko A , Golyshin PN , Yakunin AF
Ref : Sci Rep , 7 :44103 , 2017
Abstract : Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
ESTHER : Popovic_2017_Sci.Rep_7_44103
PubMedSearch : Popovic_2017_Sci.Rep_7_44103
PubMedID: 28272521
Gene_locus related to this paper: 9zzzz-a0a0g3fj39 , 9zzzz-a0a0g3fj48 , 9zzzz-A0A0G3FEJ8

Title : High Throughput Screening of Esterases, Lipases and Phospholipases in Mutant and Metagenomic Libraries: A Review - Pena-Garcia_2016_Comb.Chem.High.Throughput.Screen_19_605
Author(s) : Pena-Garcia C , Martinez-Martinez M , Reyes-Duarte D , Ferrer M
Ref : Comb Chem High Throughput Screen , 19 :605 , 2016
Abstract : Nowadays, enzymes can be efficiently identified and screened from metagenomic resources or mutant libraries. A set of a few hundred new enzymes can be found using a simple substrate within few months. Hence, the establishment of collections of enzymes is no longer a big hurdle. However, a key problem is the relatively low rate of positive hits and that a timeline of several years from the identification of a gene to the development of a process is the reality rather than the exception. Major problems are related to the time-consuming and cost-intensive screening process that only very few enzymes finally pass. Accessing to the highest possible enzyme and mutant diversity by different, but complementary approaches is increasingly important. The aim of this review is to deliver state-of-art status of traditional and novel screening protocols for targeting lipases, esterases and phospholipases of industrial relevance, and that can be applied at high throughput scale (HTS) for at least 200 distinct substrates, at a speed of more than 105 - 108 clones/day. We also review fine-tuning sequence analysis pipelines and in silico tools, which can further improve enzyme selection by an unprecedent speed (up to 1030 enzymes). If the hit rate in an enzyme collection could be increased by HTS approaches, it can be expected that also the very further expensive and time-consuming enzyme optimization phase could be significantly shortened, as the processes of enzyme-candidate selection by such methods can be adapted to conditions most likely similar to the ones needed at industrial scale.
ESTHER : Pena-Garcia_2016_Comb.Chem.High.Throughput.Screen_19_605
PubMedSearch : Pena-Garcia_2016_Comb.Chem.High.Throughput.Screen_19_605
PubMedID: 26552433

Title : Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats - Alcaide_2015_Environ.Microbiol_17_332
Author(s) : Alcaide M , Stogios PJ , Lafraya A , Tchigvintsev A , Flick R , Bargiela R , Chernikova TN , Reva ON , Hai T , Leggewie CC , Katzke N , La Cono V , Matesanz R , Jebbar M , Jaeger KE , Yakimov MM , Yakunin AF , Golyshin PN , Golyshina OV , Savchenko A , Ferrer M
Ref : Environ Microbiol , 17 :332 , 2015
Abstract : The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5 degrees C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65 degrees C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16 degrees C, was shown to contain halopiezophilic-like enzymes that are most active at 70 degrees C and with denaturing temperatures of 71.4 degrees C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.
ESTHER : Alcaide_2015_Environ.Microbiol_17_332
PubMedSearch : Alcaide_2015_Environ.Microbiol_17_332
PubMedID: 25330254
Gene_locus related to this paper: 9alte-MGS-MT1 , 9bact-MGS-M1 , 9bact-MGS-M2 , 9bact-a0a0b5kns5

Title : Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase - Placido_2015_Appl.Microbiol.Biotechnol_99_10031
Author(s) : Placido A , Hai T , Ferrer M , Chernikova TN , Distaso M , Armstrong D , Yakunin AF , Toshchakov SV , Yakimov MM , Kublanov IV , Golyshina OV , Pesole G , Ceci LR , Golyshin PN
Ref : Applied Microbiology & Biotechnology , 99 :10031 , 2015
Abstract : A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct alpha/beta-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new alpha-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-alpha-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the alpha/beta-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 degrees C. Furthermore, enzymes were active in organic solvents (e.g., >30 % methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.
ESTHER : Placido_2015_Appl.Microbiol.Biotechnol_99_10031
PubMedSearch : Placido_2015_Appl.Microbiol.Biotechnol_99_10031
PubMedID: 26266751
Gene_locus related to this paper: 9bact-a0a0h4tgu6 , 9bact-a0a0k1z4z5

Title : Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage - Mendez-Garcia_2014_ISME.J_8_1259
Author(s) : Mendez-Garcia C , Mesa V , Sprenger RR , Richter M , Diez MS , Solano J , Bargiela R , Golyshina OV , Manteca A , Ramos JL , Gallego JR , Llorente I , Martins dos Santos VA , Jensen ON , Pelaez AI , Sanchez J , Ferrer M
Ref : Isme J , 8 :1259 , 2014
Abstract : Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH approximately 2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including 'ARMAN' (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with approximately 44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O2 concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO2, CH4, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called 'ARMAN'-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H2, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H2 in acidic suboxic macroscopic growths.
ESTHER : Mendez-Garcia_2014_ISME.J_8_1259
PubMedSearch : Mendez-Garcia_2014_ISME.J_8_1259
PubMedID: 24430486
Gene_locus related to this paper: 9zzzz-t1a3k4 , 9zzzz-t1ci96 , 9zzzz-t0yxy9 , 9zzzz-t1bz84

Title : Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters - Martinez-Martinez_2014_Microb.Biotechnol_7_184
Author(s) : Martinez-Martinez M , Lores I , Pena-Garcia C , Bargiela R , Reyes-Duarte D , Guazzaroni ME , Pelaez AI , Sanchez J , Ferrer M
Ref : Microb Biotechnol , : , 2014
Abstract : Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the alpha/beta-hydrolase family. The protein shares low-to-medium identity (</= 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25-30 degrees C and pH 8.5; it retains approximately 55% of its activity at 4 degrees C and less than 8% at >/= 55 degrees C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other alpha/beta-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200-21 000 units g-1 protein at 40 degrees C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0-55 000 units g-1 protein), including (+/-)-menthyl-acetate, (+/-)-neomenthyl acetate, (+/-)-pantolactone, (+/-)-methyl-mandelate, (+/-)-methyl-lactate and (+/-)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available.
ESTHER : Martinez-Martinez_2014_Microb.Biotechnol_7_184
PubMedSearch : Martinez-Martinez_2014_Microb.Biotechnol_7_184
PubMedID: 24418210
Gene_locus related to this paper: 9bacl-h6nd87

Title : Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the alpha\/beta hydrolase family - Alcaide_2013_Biochem.J_454_157
Author(s) : Alcaide M , Tornes J , Stogios PJ , Xu X , Gertler C , Di Leo R , Bargiela R , Lafraya A , Guazzaroni ME , Lopez-Cortes N , Chernikova TN , Golyshina OV , Nechitaylo TY , Plumeier I , Pieper DH , Yakimov MM , Savchenko A , Golyshin PN , Ferrer M
Ref : Biochemical Journal , 454 :157 , 2013
Abstract : Several members of the C-C MCP (meta-cleavage product) hydrolase family demonstrate an unusual ability to hydrolyse esters as well as the MCPs (including those from mono- and bi-cyclic aromatics). Although the molecular mechanisms responsible for such substrate promiscuity are starting to emerge, the full understanding of these complex enzymes is far from complete. In the present paper, we describe six distinct alpha/beta hydrolases identified through genomic approaches, four of which demonstrate the unprecedented characteristic of activity towards a broad spectrum of substrates, including p-nitrophenyl, halogenated, fatty acyl, aryl, glycerol, cinnamoyl and carbohydrate esters, lactones, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate and 2-hydroxy-6-oxohepta-2,4-dienoate. Using structural analysis and site-directed mutagenesis we have identified the three residues (Ser32, Val130 and Trp144) that determine the unusual substrate specificity of one of these proteins, CCSP0084. The results may open up new research avenues into comparative catalytic models, structural and mechanistic studies, and biotechnological applications of MCP hydrolases.
ESTHER : Alcaide_2013_Biochem.J_454_157
PubMedSearch : Alcaide_2013_Biochem.J_454_157
PubMedID: 23750508
Gene_locus related to this paper: 9bact-r9qzf7 , 9gamm-k0c6t6

Title : Microbiota from the distal guts of lean and obese adolescents exhibit partial functional redundancy besides clear differences in community structure - Ferrer_2013_Environ.Microbiol_15_211
Author(s) : Ferrer M , Ruiz A , Lanza F , Haange SB , Oberbach A , Till H , Bargiela R , Campoy C , Segura MT , Richter M , von Bergen M , Seifert J , Suarez A
Ref : Environ Microbiol , 15 :211 , 2013
Abstract : Recent research has disclosed a tight connection between obesity, metabolic gut microbial activities and host health. Obtaining a complete understanding of this relationship remains a major goal. Here, we conducted a comparative metagenomic and metaproteomic investigation of gut microbial communities in faecal samples taken from an obese and a lean adolescent. By analysing the diversity of 16S rDNA amplicons (10% operational phylogenetic units being common), 22 Mbp of consensus metagenome sequences (~70% common) and the expression profiles of 613 distinct proteins (82% common), we found that in the obese gut, the total microbiota was more abundant on the phylum Firmicutes (94.6%) as compared with Bacteroidetes (3.2%), although the metabolically active microbiota clearly behaves in a more homogeneous manner with both contributing equally. The lean gut showed a remarkable shift towards Bacteroidetes (18.9% total 16S rDNA), which become the most active fraction (81% proteins). Although the two gut communities maintained largely similar gene repertoires and functional profiles, improved pili- and flagella-mediated host colonization and improved capacity for both complementary aerobic and anaerobic de novo B(12) synthesis, 1,2-propanediol catabolism (most likely participating in de novo B(12) synthesis) and butyrate production were observed in the obese gut, whereas bacteria from lean gut seem to be more engaged in vitamin B(6) synthesis. Furthermore, this study provides functional evidence that variable combinations of species from different phyla could 'presumptively' fulfil overlapping and/or complementary functional roles required by the host, a scenario where minor bacterial taxa seem to be significant active contributors.
ESTHER : Ferrer_2013_Environ.Microbiol_15_211
PubMedSearch : Ferrer_2013_Environ.Microbiol_15_211
PubMedID: 22891823
Gene_locus related to this paper: 9bact-d1ped1 , 9bact-d1pg85 , 9firm-a5z5y4 , 9firm-a5z5y5 , bacun-a7v041 , 9bact-d1p9d7

Title : Biochemical diversity of carboxyl esterases and lipases from lake arreo (Spain): a metagenomic approach - Martinez-Martinez_2013_Appl.Environ.Microbiol_79_3553
Author(s) : Martinez-Martinez M , Alcaide M , Tchigvintsev A , Reva O , Polaina J , Bargiela R , Guazzaroni ME , Chicote A , Canet A , Valero F , Rico Eguizabal E , Guerrero Mdel C , Yakunin AF , Ferrer M
Ref : Applied Environmental Microbiology , 79 :3553 , 2013
Abstract : The esterases and lipases from the alpha/beta hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42 degrees 46'N, 2 degrees 59'W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40 degrees C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated.
ESTHER : Martinez-Martinez_2013_Appl.Environ.Microbiol_79_3553
PubMedSearch : Martinez-Martinez_2013_Appl.Environ.Microbiol_79_3553
PubMedID: 23542620
Gene_locus related to this paper: 9bact-LAE6

Title : Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica - Kube_2013_Nat.Commun_4_2156
Author(s) : Kube M , Chernikova TN , Al-Ramahi Y , Beloqui A , Lopez-Cortez N , Guazzaroni ME , Heipieper HJ , Klages S , Kotsyurbenko OR , Langer I , Nechitaylo TY , Lunsdorf H , Fernandez M , Juarez S , Ciordia S , Singer A , Kagan O , Egorova O , Petit PA , Stogios P , Kim Y , Tchigvintsev A , Flick R , Denaro R , Genovese M , Albar JP , Reva ON , Martinez-Gomariz M , Tran H , Ferrer M , Savchenko A , Yakunin AF , Yakimov MM , Golyshina OV , Reinhardt R , Golyshin PN
Ref : Nat Commun , 4 :2156 , 2013
Abstract : Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
ESTHER : Kube_2013_Nat.Commun_4_2156
PubMedSearch : Kube_2013_Nat.Commun_4_2156
PubMedID: 23877221
Gene_locus related to this paper: olean-olei00960 , olean-r4ym14 , olean-r4yv64 , olean-r4ys13

Title : Genome Sequence of Thalassolituus oleivorans MIL-1 (DSM 14913T) - Golyshin_2013_Genome.Announc_1_e0014113
Author(s) : Golyshin PN , Werner J , Chernikova TN , Tran H , Ferrer M , Yakimov MM , Teeling H , Golyshina OV
Ref : Genome Announc , 1 :e0014113 , 2013
Abstract : Thalassolituus oleivorans is one of the most prevalent marine gammaproteobacteria in microbial communities, emerging after oil spills in coastal, estuarine, and surface seawaters. Here, we present the assembled genome of strain T. oleivorans MIL-1 (DSM 14913(T)), which is 3,920,328 bp with a G+C content of 46.6%.
ESTHER : Golyshin_2013_Genome.Announc_1_e0014113
PubMedSearch : Golyshin_2013_Genome.Announc_1_e0014113
PubMedID: 23599290
Gene_locus related to this paper: 9gamm-m5dq85 , 9gamm-m5dt68 , 9gamm-m5dm97

Title : Gene sets for utilization of primary and secondary nutrition supplies in the distal gut of endangered Iberian lynx - Alcaide_2012_PLoS.One_7_e51521
Author(s) : Alcaide M , Messina E , Richter M , Bargiela R , Peplies J , Huws SA , Newbold CJ , Golyshin PN , Simon MA , Lopez G , Yakimov MM , Ferrer M
Ref : PLoS ONE , 7 :e51521 , 2012
Abstract : Recent studies have indicated the existence of an extensive trans-genomic trans-mural co-metabolism between gut microbes and animal hosts that is diet-, host phylogeny- and provenance-influenced. Here, we analyzed the biodiversity at the level of small subunit rRNA gene sequence and the metabolic composition of 18 Mbp of consensus metagenome sequences and activity characteristics of bacterial intra-cellular extracts, in wild Iberian lynx (Lynx pardinus) fecal samples. Bacterial signatures (14.43% of all of the Firmicutes reads and 6.36% of total reads) related to the uncultured anaerobic commensals Anaeroplasma spp., which are typically found in ovine and bovine rumen, were first identified. The lynx gut was further characterized by an over-representation of 'presumptive' aquaporin aqpZ genes and genes encoding 'active' lysosomal-like digestive enzymes that are possibly needed to acquire glycerol, sugars and amino acids from glycoproteins, glyco(amino)lipids, glyco(amino)glycans and nucleoside diphosphate sugars. Lynx gut was highly enriched (28% of the total glycosidases) in genes encoding alpha-amylase and related enzymes, although it exhibited low rate of enzymatic activity indicative of starch degradation. The preponderance of beta-xylosidase activity in protein extracts further suggests lynx gut microbes being most active for the metabolism of beta-xylose containing plant N-glycans, although beta-xylosidases sequences constituted only 1.5% of total glycosidases. These collective and unique bacterial, genetic and enzymatic activity signatures suggest that the wild lynx gut microbiota not only harbors gene sets underpinning sugar uptake from primary animal tissues (with the monotypic dietary profile of the wild lynx consisting of 80-100% wild rabbits) but also for the hydrolysis of prey-derived plant biomass. Although, the present investigation corresponds to a single sample and some of the statements should be considered qualitative, the data most likely suggests a tighter, more coordinated and complex evolutionary and nutritional ecology scenario of carnivore gut microbial communities than has been previously assumed.
ESTHER : Alcaide_2012_PLoS.One_7_e51521
PubMedSearch : Alcaide_2012_PLoS.One_7_e51521
PubMedID: 23251564
Gene_locus related to this paper: 9zzzz-j9g462 , 9zzzz-j9bqr9 , 9zzzz-j9gjr1 , 9zzzz-j9gm47 , 9zzzz-j9ddn5 , 9zzzz-j9g9e5 , 9zzzz-j9fnv6

Title : Functional-based screening methods for lipases, esterases, and phospholipases in metagenomic libraries - Reyes-Duarte_2012_Methods.Mol.Biol_861_101
Author(s) : Reyes-Duarte D , Ferrer M , Garcia-Arellano H
Ref : Methods Mol Biol , 861 :101 , 2012
Abstract : The use of metagenomic techniques for enzyme discovery constitutes a powerful approach. Functional screens, in contrast to sequence homology search, enable us to select enzymes based on their activity. It is noteworthy that they additionally guarantee the identification of genes coding for enzymes that exhibited no sequence similarity to known counterparts from public databases and that even do not match any putative catalytic residues, involved in the selected catalytic function. Therefore, this strategy not only provides new enzymes for new biotechnological applications, but also allows functional assignment of many proteins, found in abundance in the databases, currently designated as "hypothetical" or "conserved hypothetical" proteins. In the past decade, there has been an exponential increase in the design of functional screening programmes, the majority of them established for hydrolases and oxidoreductases. Here, functional screening methods that guarantee the greatest enzyme diversity, for mining esterases and lipases, are described.
ESTHER : Reyes-Duarte_2012_Methods.Mol.Biol_861_101
PubMedSearch : Reyes-Duarte_2012_Methods.Mol.Biol_861_101
PubMedID: 22426714

Title : Microbial beta-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail - Del Pozo_2012_Biotechnol.Biofuels_5_73
Author(s) : Del Pozo MV , Fernandez-Arrojo L , Gil-Martinez J , Montesinos A , Chernikova TN , Nechitaylo TY , Waliszek A , Tortajada M , Rojas A , Huws SA , Golyshina OV , Newbold CJ , Polaina J , Ferrer M , Golyshin PN
Ref : Biotechnol Biofuels , 5 :73 , 2012
Abstract : BACKGROUND: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product.
RESULTS: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45-55 degrees C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 +/- 1.7% vs. 34.5 +/- 1.5% in control conditions) after 96-120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50 degrees C and pH 5.2) and 2-38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays.
CONCLUSIONS: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases.
ESTHER : Del Pozo_2012_Biotechnol.Biofuels_5_73
PubMedSearch : Del Pozo_2012_Biotechnol.Biofuels_5_73
PubMedID: 22998985
Gene_locus related to this paper: 9bact-m4pza5

Title : Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, Central Spain) - Ferrer_2011_Microb.Ecol_62_824
Author(s) : Ferrer M , Guazzaroni ME , Richter M , Garcia-Salamanca A , Yarza P , Suarez-Suarez A , Solano J , Alcaide M , van Dillewijn P , Molina-Henares MA , Lopez-Cortes N , Al-Ramahi Y , Guerrero C , Acosta A , de Eugenio LI , Martinez V , Marques S , Rojo F , Santero E , Genilloud O , Perez-Perez J , Rossello-Mora R , Ramos JL
Ref : Microb Ecol , 62 :824 , 2011
Abstract : The phylogenetic and functional structure of the microbial community residing in a Ca(2+)-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO(4) x 2 H(2)O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca(2+) and SO (4) (2-) being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical-chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.
ESTHER : Ferrer_2011_Microb.Ecol_62_824
PubMedSearch : Ferrer_2011_Microb.Ecol_62_824
PubMedID: 21735153
Gene_locus related to this paper: 9zzzz-d9pjn2

Title : Inter-conversion of catalytic abilities in a bifunctional carboxyl\/feruloyl-esterase from earthworm gut metagenome - Vieites_2010_Microb.Biotechnol_3_48
Author(s) : Vieites JM , Ghazi A , Beloqui A , Polaina J , Andreu JM , Golyshina OV , Nechitaylo TY , Waliczek A , Yakimov MM , Golyshin PN , Ferrer M
Ref : Microb Biotechnol , 3 :48 , 2010
Abstract : Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k(cat)/K(m))](CE)/[(k(cat)/K(m))](FAE) factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys(281), Asp(282), Asn(316) and Lys(317)) situated close to the catalytic core (Ser(143)/Asp(273)/His(305)) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of approximately 0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.
ESTHER : Vieites_2010_Microb.Biotechnol_3_48
PubMedSearch : Vieites_2010_Microb.Biotechnol_3_48
PubMedID: 21255305

Title : Novel hybrid esterase-haloacid dehalogenase enzyme -
Author(s) : Beloqui A , Polaina J , Vieites JM , Reyes-Duarte D , Torres R , Golyshina OV , Chernikova TN , Waliczek A , Aharoni A , Yakimov MM , Timmis KN , Golyshin PN , Ferrer M
Ref : Chembiochem , 11 :1975 , 2010
PubMedID: 20715265

Title : Diversity of glycosyl hydrolases from cellulose-depleting communities enriched from casts of two earthworm species - Beloqui_2010_Appl.Environ.Microbiol_76_5934
Author(s) : Beloqui A , Nechitaylo TY , Lopez-Cortes N , Ghazi A , Guazzaroni ME , Polaina J , Strittmatter AW , Reva O , Waliczek A , Yakimov MM , Golyshina OV , Ferrer M , Golyshin PN
Ref : Applied Environmental Microbiology , 76 :5934 , 2010
Abstract : The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-beta-glucanases, beta-glucosidases, beta-cellobiohydrolases, beta-galactosidase, and beta-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of beta-galactosidases/alpha-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).
ESTHER : Beloqui_2010_Appl.Environ.Microbiol_76_5934
PubMedSearch : Beloqui_2010_Appl.Environ.Microbiol_76_5934
PubMedID: 20622123
Gene_locus related to this paper: 9zzzz-d8vn29

Title : The 'pH optimum anomaly' of intracellular enzymes of Ferroplasma acidiphilum - Golyshina_2006_Environ.Microbiol_8_416
Author(s) : Golyshina OV , Golyshin PN , Timmis KN , Ferrer M
Ref : Environ Microbiol , 8 :416 , 2006
Abstract : A wide range of microorganisms, the so-called acidophiles, inhabit acidic environments and grow optimally at pH values between 0 and 3. The intracellular pH of these organisms is, however, close to neutrality or slightly acidic. It is to be expected that enzymatic activities dedicated to extracellular functions would be adapted to the prevailing low pH of the environment (0-3), whereas intracellular enzymes would be optimally active at the near-neutral pH of the cytoplasm (4.6-7.0). The genes of several intracellular or cell-bound enzymes, a carboxylesterase and three alpha-glucosidases, from Ferroplasma acidiphilum, a cell wall-lacking acidophilic archaeon with a growth optimum at pH 1.7, were cloned and expressed in Escherichia coli, and their products purified and characterized. The Ferroplasmaalpha-glucosidases exhibited no sequence similarity to known glycosyl hydrolases. All enzymes functioned and were stable in vitro in the pH range 1.7-4.0, and had pH optima much lower than the mean intracellular pH of 5.6. This 'pH optimum anomaly' suggests the existence of yet-undetected cellular compartmentalization providing cytoplasmic pH patchiness and low pH environments for the enzymes we have analysed.
ESTHER : Golyshina_2006_Environ.Microbiol_8_416
PubMedSearch : Golyshina_2006_Environ.Microbiol_8_416
PubMedID: 16478448
Gene_locus related to this paper: 9eury-q2pce5

Title : Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis - Schneiker_2006_Nat.Biotechnol_24_997
Author(s) : Schneiker S , Martins dos Santos VA , Bartels D , Bekel T , Brecht M , Buhrmester J , Chernikova TN , Denaro R , Ferrer M , Gertler C , Goesmann A , Golyshina OV , Kaminski F , Khachane AN , Lang S , Linke B , McHardy AC , Meyer F , Nechitaylo T , Puhler A , Regenhardt D , Rupp O , Sabirova JS , Selbitschka W , Yakimov MM , Timmis KN , Vorholter FJ , Weidner S , Kaiser O , Golyshin PN
Ref : Nat Biotechnol , 24 :997 , 2006
Abstract : Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation-related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.
ESTHER : Schneiker_2006_Nat.Biotechnol_24_997
PubMedSearch : Schneiker_2006_Nat.Biotechnol_24_997
PubMedID: 16878126
Gene_locus related to this paper: alcbo-Q9F9H0 , alcbs-q0vl04 , alcbs-q0vl36 , alcbs-q0vl92 , alcbs-q0vlp5 , alcbs-q0vlq1 , alcbs-q0vlt9 , alcbs-q0vm92 , alcbs-q0vmd2 , alcbs-q0vmd6 , alcbs-q0vmn9 , alcbs-q0vnu3 , alcbs-q0vp43 , alcbs-q0vpa9 , alcbs-q0vpc7 , alcbs-q0vpg7 , alcbs-q0vpn2 , alcbs-q0vps0 , alcbs-q0vq49 , alcbs-q0vsg4 , alcbs-q0vth9 , marav-a1u5n0 , alcbs-q0vp59 , alcbs-q0vlk5

Title : Conversion of a carboxylesterase into a triacylglycerol lipase by a random mutation -
Author(s) : Reyes-Duarte D , Polaina J , Lopez-Cortes N , Alcalde M , Plou FJ , Elborough K , Ballesteros A , Timmis KN , Golyshin PN , Ferrer M
Ref : Angew Chem Int Ed Engl , 44 :7553 , 2005
PubMedID: 16254934

Title : Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora - Ferrer_2005_Environ.Microbiol_7_1996
Author(s) : Ferrer M , Golyshina OV , Chernikova TN , Khachane AN , Reyes-Duarte D , Santos VA , Strompl C , Elborough K , Jarvis G , Neef A , Yakimov MM , Timmis KN , Golyshin PN
Ref : Environ Microbiol , 7 :1996 , 2005
Abstract : A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.
ESTHER : Ferrer_2005_Environ.Microbiol_7_1996
PubMedSearch : Ferrer_2005_Environ.Microbiol_7_1996
PubMedID: 16309396
Gene_locus related to this paper: 9zzzz-q2yi75 , 9zzzz-q2yi81 , 9zzzz-q2yi93

Title : Microbial enzymes mined from the Urania deep-sea hypersaline anoxic basin - Ferrer_2005_Chem.Biol_12_895
Author(s) : Ferrer M , Golyshina OV , Chernikova TN , Khachane AN , Martins dos Santos VA , Yakimov MM , Timmis KN , Golyshin PN
Ref : Chemical Biology , 12 :895 , 2005
Abstract : We created a metagenome expression library from the brine:seawater interface of the Urania hypersaline basin, screened it for esterases, and characterized five of these. Two had no significant sequence homology to known esterases, hydrolyzed both carboxylesters and thioesters, and exhibited unusual, habitat-specific characteristics (preference for high hydrostatic pressure and salinity). One has an unusual structural signature incorporating three catalytic active centers mediating distinct hydrolytic activities and an adaptive tertiary-quaternary structure that alters between three molecular states, according to the prevailing physicochemical conditions. Some of the esterases have high activities, specificities, enantioselectivities, and exceptional stability in polar solvents, and they are therefore potentially useful for industrial biotransformations. One possesses the highest enantioselectivity toward an ester of the important chiral synthon solketal (E: 126[S]; 98%ee).
ESTHER : Ferrer_2005_Chem.Biol_12_895
PubMedSearch : Ferrer_2005_Chem.Biol_12_895
PubMedID: 16125101
Gene_locus related to this paper: 9zzzz-q335p2 , 9zzzz-q335p5

Title : Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain - Ferrer_2004_Appl.Environ.Microbiol_70_4499
Author(s) : Ferrer M , Chernikova TN , Timmis KN , Golyshin PN
Ref : Applied Environmental Microbiology , 70 :4499 , 2004
Abstract : A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E. coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)). The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5 degrees C).
ESTHER : Ferrer_2004_Appl.Environ.Microbiol_70_4499
PubMedSearch : Ferrer_2004_Appl.Environ.Microbiol_70_4499
PubMedID: 15294778
Gene_locus related to this paper: olean-q6a2s8