Yakunin AF

References (20)

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 : Harnessing extremophilic carboxylesterases for applications in polyester depolymerisation and plastic waste recycling - Williams_2023_Essays.Biochem__
Author(s) : Williams GB , Ma H , Khusnutdinova AN , Yakunin AF , Golyshin PN
Ref : Essays Biochem , : , 2023
Abstract : The steady growth in industrial production of synthetic plastics and their limited recycling have resulted in severe environmental pollution and contribute to global warming and oil depletion. Currently, there is an urgent need to develop efficient plastic recycling technologies to prevent further environmental pollution and recover chemical feedstocks for polymer re-synthesis and upcycling in a circular economy. Enzymatic depolymerization of synthetic polyesters by microbial carboxylesterases provides an attractive addition to existing mechanical and chemical recycling technologies due to enzyme specificity, low energy consumption, and mild reaction conditions. Carboxylesterases constitute a diverse group of serine-dependent hydrolases catalysing the cleavage and formation of ester bonds. However, the stability and hydrolytic activity of identified natural esterases towards synthetic polyesters are usually insufficient for applications in industrial polyester recycling. This necessitates further efforts on the discovery of robust enzymes, as well as protein engineering of natural enzymes for enhanced activity and stability. In this essay, we discuss the current knowledge of microbial carboxylesterases that degrade polyesters (polyesterases) with focus on polyethylene terephthalate (PET), which is one of the five major synthetic polymers. Then, we briefly review the recent progress in the discovery and protein engineering of microbial polyesterases, as well as developing enzyme cocktails and secreted protein expression for applications in the depolymerisation of polyester blends and mixed plastics. Future research aimed at the discovery of novel polyesterases from extreme environments and protein engineering for improved performance will aid developing efficient polyester recycling technologies for the circular plastics economy.
ESTHER : Williams_2023_Essays.Biochem__
PubMedSearch : Williams_2023_Essays.Biochem__
PubMedID: 37334661

Title : Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases - Khusnutdinova_2023_FEBS.J_290_4966
Author(s) : Khusnutdinova AN , Batyrova KA , Brown G , Fedorchuk T , Chai YS , Skarina T , Flick R , Petit AP , Savchenko A , Stogios P , Yakunin AF
Ref : Febs J , 290 :4966 , 2023
Abstract : Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography-mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 from Dechloromonas aromatica and NOS0089 from Nostoc sp. were determined in the apo-state along with the DAR3835 H274N glycolyl intermediate. Structure-based site-directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein-ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.
ESTHER : Khusnutdinova_2023_FEBS.J_290_4966
PubMedSearch : Khusnutdinova_2023_FEBS.J_290_4966
PubMedID: 37437000
Gene_locus related to this paper: anasp-ALR0039 , decar-q479b8

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 : Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters - Hajighasemi_2018_Environ.Sci.Technol_52_12388
Author(s) : Hajighasemi M , Tchigvintsev A , Nocek B , Flick R , Popovic A , Hai T , Khusnutdinova AN , Brown G , Xu X , Cui H , Anstett J , Chernikova TN , Bruls T , Le Paslier D , Yakimov MM , Joachimiak A , Golyshina OV , Savchenko A , Golyshin PN , Edwards EA , Yakunin AF
Ref : Environ Sci Technol , 52 :12388 , 2018
Abstract : The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 A resolution and revealed a modified alpha/beta hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.
ESTHER : Hajighasemi_2018_Environ.Sci.Technol_52_12388
PubMedSearch : Hajighasemi_2018_Environ.Sci.Technol_52_12388
PubMedID: 30284819
Gene_locus related to this paper: 9zzzz-a0a0g3fj39 , 9zzzz-a0a0g3fj48 , 9zzzz-A0A0G3FEJ8 , 9bact-a4uz10

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 : Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases - Hajighasemi_2016_Biomacromolecules_17_2027
Author(s) : Hajighasemi M , Nocek BP , Tchigvintsev A , Brown G , Flick R , Xu X , Cui H , Hai T , Joachimiak A , Golyshin PN , Savchenko A , Edwards EA , Yakunin AF
Ref : Biomacromolecules , 17 :2027 , 2016
Abstract : Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial alpha/beta-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble alpha-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 A resolution and revealed a classical alpha/beta-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.
ESTHER : Hajighasemi_2016_Biomacromolecules_17_2027
PubMedSearch : Hajighasemi_2016_Biomacromolecules_17_2027
PubMedID: 27087107
Gene_locus related to this paper: marav-a1u5n0 , rhopa-q6n9m9 , alcbs-q0vlq1

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
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 : The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes - Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
Author(s) : Tchigvintsev A , Tran H , Popovic A , Kovacic F , Brown G , Flick R , Hajighasemi M , Egorova O , Somody JC , Tchigvintsev D , Khusnutdinova A , Chernikova TN , Golyshina OV , Yakimov MM , Savchenko A , Golyshin PN , Jaeger KE , Yakunin AF
Ref : Applied Microbiology & Biotechnology , 99 :2165 , 2015
Abstract : Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against alpha-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 degrees C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.
ESTHER : Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
PubMedSearch : Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
PubMedID: 25194841

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 : 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 : Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica - Lemak_2012_Biochem.J_445_193
Author(s) : Lemak S , Tchigvintsev A , Petit P , Flick R , Singer AU , Brown G , Evdokimova E , Egorova O , Gonzalez CF , Chernikova TN , Yakimov MM , Kube M , Reinhardt R , Golyshin PN , Savchenko A , Yakunin AF
Ref : Biochemical Journal , 445 :193 , 2012
Abstract : The uncharacterized alpha/beta-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate alpha-naphthyl acetate at 5-30 degrees C with maximal activity at 15-20 degrees C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 A resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45 degrees C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
ESTHER : Lemak_2012_Biochem.J_445_193
PubMedSearch : Lemak_2012_Biochem.J_445_193
PubMedID: 22519667
Gene_locus related to this paper: olean-d0vwz4

Title : Mapping the reaction coordinates of enzymatic defluorination - Chan_2011_J.Am.Chem.Soc_133_7461
Author(s) : Chan PW , Yakunin AF , Edwards EA , Pai EF
Ref : Journal of the American Chemical Society , 133 :7461 , 2011
Abstract : The carbon-fluorine bond is the strongest covalent bond in organic chemistry, yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiological conditions. Elucidating the molecular basis of this rare biocatalytic activity will provide the fundamental chemical insights into how this formidable feat is achieved. Here, we present a series of high-resolution (1.15-1.80 A) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate, and enzyme-product complex. We demonstrate that enzymatic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion but also is finely tailored for the smaller fluorine halogen atom to establish selectivity toward fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymatic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated organic compounds, which in turn will assist the synthesis, detoxification, biodegradation, disposal, recycling, and regulatory strategies for the growing markets of organofluorines across major industrial sectors.
ESTHER : Chan_2011_J.Am.Chem.Soc_133_7461
PubMedSearch : Chan_2011_J.Am.Chem.Soc_133_7461
PubMedID: 21510690
Gene_locus related to this paper: rhopa-q6nam1

Title : Sequence- and activity-based screening of microbial genomes for novel dehalogenases - Chan_2010_Microb.Biotechnol_3_107
Author(s) : Chan WY , Wong M , Guthrie J , Savchenko AV , Yakunin AF , Pai EF , Edwards EA
Ref : Microb Biotechnol , 3 :107 , 2010
Abstract : Dehalogenases are environmentally important enzymes that detoxify organohalogens by cleaving their carbon-halogen bonds. Many microbial genomes harbour enzyme families containing dehalogenases, but a sequence-based identification of genuine dehalogenases with high confidence is challenging because of the low sequence conservation among these enzymes. Furthermore, these protein families harbour a rich diversity of other enzymes including esterases and phosphatases. Reliable sequence determinants are necessary to harness genome sequencing-efforts for accelerating the discovery of novel dehalogenases with improved or modified activities. In an attempt to extract dehalogenase sequence fingerprints, 103 uncharacterized potential dehalogenase candidates belonging to the alpha/beta hydrolase (ABH) and haloacid dehalogenase-like hydrolase (HAD) superfamilies were screened for dehalogenase, esterase and phosphatase activity. In this first biochemical screen, 1 haloalkane dehalogenase, 1 fluoroacetate dehalogenase and 5 l-2-haloacid dehalogenases were found (success rate 7%), as well as 19 esterases and 31 phosphatases. Using this functional data, we refined the sequence-based dehalogenase selection criteria and applied them to a second functional screen, which identified novel dehalogenase activity in 13 out of only 24 proteins (54%), increasing the success rate eightfold. Four new L-2-haloacid dehalogenases from the HAD superfamily were found to hydrolyse fluoroacetate, an activity never previously ascribed to enzymes in this superfamily.
ESTHER : Chan_2010_Microb.Biotechnol_3_107
PubMedSearch : Chan_2010_Microb.Biotechnol_3_107
PubMedID: 21255311
Gene_locus related to this paper: rhopa-q6nam1

Title : Mining bacterial genomes for novel arylesterase activity - Wang_2010_Microb.Biotechnol_3_677
Author(s) : Wang L , Mavisakalyan V , Tillier ER , Clark GW , Savchenko AV , Yakunin AF , Master ER
Ref : Microb Biotechnol , 3 :677 , 2010
Abstract : One hundred and seventy-one genes encoding potential esterases from 11 bacterial genomes were cloned and overexpressed in Escherichia coli; 74 of the clones produced soluble proteins. All 74 soluble proteins were purified and screened for esterase activity; 36 proteins showed carboxyl esterase activity on short-chain esters, 17 demonstrated arylesterase activity, while 38 proteins did not exhibit any activity towards the test substrates. Esterases from Rhodopseudomonas palustris (RpEST-1, RpEST-2 and RpEST-3), Pseudomonas putida (PpEST-1, PpEST-2 and PpEST-3), Pseudomonas aeruginosa (PaEST-1) and Streptomyces avermitilis (SavEST-1) were selected for detailed biochemical characterization. All of the enzymes showed optimal activity at neutral or alkaline pH, and the half-life of each enzyme at 50 degrees C ranged from < 5 min to over 5 h. PpEST-3, RpEST-1 and RpEST-2 demonstrated the highest specific activity with pNP-esters; these enzymes were also among the most stable at 50 degrees C and in the presence of detergents, polar and non-polar organic solvents, and imidazolium ionic liquids. Accordingly, these enzymes are particularly interesting targets for subsequent application trials. Finally, biochemical and bioinformatic analyses were compared to reveal sequence features that could be correlated to enzymes with arylesterase activity, facilitating subsequent searches for new esterases in microbial genome sequences.
ESTHER : Wang_2010_Microb.Biotechnol_3_677
PubMedSearch : Wang_2010_Microb.Biotechnol_3_677
PubMedID: 21255363

Title : Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis - Brown_2008_Biochemistry_47_5724
Author(s) : Brown G , Singer A , Proudfoot M , Skarina T , Kim Y , Chang C , Dementieva I , Kuznetsova E , Gonzalez CF , Joachimiak A , Savchenko A , Yakunin AF
Ref : Biochemistry , 47 :5724 , 2008
Abstract : Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.
ESTHER : Brown_2008_Biochemistry_47_5724
PubMedSearch : Brown_2008_Biochemistry_47_5724
PubMedID: 18459799

Title : Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG - Gonzalez_2006_J.Biol.Chem_281_14514
Author(s) : Gonzalez CF , Proudfoot M , Brown G , Korniyenko Y , Mori H , Savchenko AV , Yakunin AF
Ref : Journal of Biological Chemistry , 281 :14514 , 2006
Abstract : The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.
ESTHER : Gonzalez_2006_J.Biol.Chem_281_14514
PubMedSearch : Gonzalez_2006_J.Biol.Chem_281_14514
PubMedID: 16567800
Gene_locus related to this paper: ecoli-yaim , ecoli-yeiG

Title : Enzyme genomics: Application of general enzymatic screens to discover new enzymes - Kuznetsova_2005_FEMS.Microbiol.Rev_29_263
Author(s) : Kuznetsova E , Proudfoot M , Sanders SA , Reinking J , Savchenko A , Arrowsmith CH , Edwards AM , Yakunin AF
Ref : FEMS Microbiology Reviews , 29 :263 , 2005
Abstract : In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with "known" function are mis-annotated. Several global approaches are routinely employed to predict function, including sophisticated sequence analysis, gene expression, protein interaction, and protein structure. In the first coupling of genomics and enzymology, Phizicky and colleagues undertook a screen for specific enzymes using large pools of partially purified proteins and specific enzymatic assays. Here we present an overview of the further developments of this approach, which involve the use of general enzymatic assays to screen individually purified proteins for enzymatic activity. The assays have relaxed substrate specificity and are designed to identify the subclass or sub-subclasses of enzymes (phosphatase, phosphodiesterase/nuclease, protease, esterase, dehydrogenase, and oxidase) to which the unknown protein belongs. Further biochemical characterization of proteins can be facilitated by the application of secondary screens with natural substrates (substrate profiling). We demonstrate here the feasibility and merits of this approach for hydrolases and oxidoreductases, two very broad and important classes of enzymes. Application of general enzymatic screens and substrate profiling can greatly speed up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches.
ESTHER : Kuznetsova_2005_FEMS.Microbiol.Rev_29_263
PubMedSearch : Kuznetsova_2005_FEMS.Microbiol.Rev_29_263
PubMedID: 15808744
Gene_locus related to this paper: ecoli-yafa , ecoli-ybff , ecoli-ycjy , ecoli-yeiG , ecoli-YFBB , ecoli-yjfp , ecoli-ypfh , ecoli-yqia , ecoli-yuar

Title : Integrating structure, bioinformatics, and enzymology to discover function: BioH, a new carboxylesterase from Escherichia coli - Sanishvili_2003_J.Biol.Chem_278_26039
Author(s) : Sanishvili R , Yakunin AF , Laskowski RA , Skarina T , Evdokimova E , Doherty-Kirby A , Lajoie GA , Thornton JM , Arrowsmith CH , Savchenko A , Joachimiak A , Edwards AM
Ref : Journal of Biological Chemistry , 278 :26039 , 2003
Abstract : Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 A resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure.
ESTHER : Sanishvili_2003_J.Biol.Chem_278_26039
PubMedSearch : Sanishvili_2003_J.Biol.Chem_278_26039
PubMedID: 12732651
Gene_locus related to this paper: ecoli-bioh