Author Report for: Pleiss JContact information:Pleiss JurgenInstitute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 StuttgartGermanyPhone : Fax :
Buchholz PCF, Feuerriegel G, Zhang H, Perez-Garcia P, Nover LL, Chow J, Streit WR, Pleiss J Ref: Proteins, 90:1443, 2022 : PubMed ESTHER: Buchholz_2022_Proteins_90_1443 PubMedSearch: Buchholz 2022 Proteins 90 1443 PubMedID: 35175626 Abstract Petroleum-based plastics are durable and accumulate in all ecological niches. Knowledge on enzymatic degradation is sparse. Today, less than 50 verified plastics-active enzymes are known. First examples of enzymes acting on the polymers polyethylene terephthalate (PET) and polyurethane (PUR) have been reported together with a detailed biochemical and structural description. Furthermore, very few polyamide (PA) oligomer active enzymes are known. In this article, the current known enzymes acting on the synthetic polymers PET and PUR are briefly summarized, their published activity data were collected and integrated into a comprehensive open access database. The Plastics-Active Enzymes Database (PAZy) represents an inventory of known and experimentally verified enzymes that act on synthetic fossil fuel-based polymers. Almost 3000 homologs of PET-active enzymes were identified by profile hidden Markov models. Over 2000 homologs of PUR-active enzymes were identified by BLAST. Based on multiple sequence alignments, conservation analysis identified the most conserved amino acids, and sequence motifs for PET- and PUR-active enzymes were derived. Title: The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity Zhang H, Perez-Garcia P, Dierkes RF, Applegate V, Schumacher J, Chibani CM, Sternagel S, Preuss L, Weigert S and Streit WR <9 more author(s)> Zhang H, Perez-Garcia P, Dierkes RF, Applegate V, Schumacher J, Chibani CM, Sternagel S, Preuss L, Weigert S, Schmeisser C, Danso D, Pleiss J, Almeida A, Hocker B, Hallam SJ, Schmitz RA, Smits SHJ, Chow J, Streit WR (- 9) Ref: Front Microbiol, 12:803896, 2021 : PubMed ESTHER: Zhang_2022_Front.Microbiol_12_803896 PubMedSearch: Zhang 2022 Front.Microbiol 12 803896 PubMedID: 35069509 Gene_locus related to this paper: flutr-f2ie04, 9flao-a0a0c1f4u8, 9flao-kjj39608, 9flao-a0a330mq60 Abstract Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil((a)) DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30 degreesC from a 7 mg PET foil platelet in a 200 microl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30deltaPorC) was solved at 2.1 A and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation. Title: Plastics degradation by hydrolytic enzymes: the Plastics-Active Enzymes Database - PAZy Buchholz PCF, Zhang H, Perez-Garcia P, Nover LL, Chow J, Streit WR, Pleiss J Ref: Authorea, :, 2021 : PubMed ESTHER: Buchholz_2021_Authorea__ PubMedSearch: Buchholz 2021 Authorea Abstract Petroleum based plastics are durable and accumulate in all ecological niches. Knowledge on enzymatic degradation is sparse. Today, less than 50 verified plastics-active enzymes are known. First examples of enzymes acting on the polymers polyethylene terephthalate (PET) and polyurethane (PUR) have been reported together with a detailed biochemical and structural description. Further, very few polyamide (PA) oligomer active enzymes are known. In this paper, the current known enzymes acting on the synthetic polymers PET and PUR are briefly summarized, their published activity data were collected and integrated into a comprehensive open access database. The Plastics-Active Enzymes Database (PAZy) represents an inventory of known and experimentally verified plastics-active enzymes. Almost 3000 homologues of PET-active enzymes were identified by profile hidden Markov models. Over 2000 homologues of PUR-active enzymes were identified by BLAST. Based on multiple sequence alignments, conservation analysis identified the most conserved amino acids, and sequence motifs for PET- and PUR-active enzymes were derived. Title: Molecular Mechanism of Methanol Inhibition in CALB-Catalyzed Alcoholysis: Analyzing Molecular Dynamics Simulations by a Markov State Model Carvalho HF, Ferrario V, Pleiss J Ref: J Chem Theory Comput, :, 2021 : PubMed ESTHER: Carvalho_2021_J.Chem.Theory.Comput__ PubMedSearch: Carvalho 2021 J.Chem.Theory.Comput PubMedID: 34494846 Abstract Lipases are widely used enzymes that catalyze hydrolysis and alcoholysis of fatty acid esters. At high concentrations of small alcohols such as methanol or ethanol, many lipases are inhibited by the substrate. The molecular basis of the inhibition of Candida antarctica lipase B (CALB) by methanol was investigated by unbiased molecular dynamics (MD) simulations, and the substrate binding kinetics was analyzed by Markov state models (MSMs). The modeled fluxes of productive methanol binding at concentrations between 50 mM and 5.5 M were in good agreement with the experimental activity profile of CALB, with a peak at 300 mM. The kinetic and structural analysis uncovered the molecular basis of CALB inhibition. Beyond 300 mM, the kinetic bottleneck results from crowding of methanol in the substrate access channel, which is caused by the gradual formation of methanol patches close to Leu140 (helix alpha5), Leu278, and Ile285 (helix alpha10) at a distance of 4-5 A from the active site. Our findings demonstrate the usefulness of unbiased MD simulations to study enzyme-substrate interactions at realistic substrate concentrations and the feasibility of scale-bridging by an MSM analysis to derive kinetic information. Title: Standardized Data, Scalable Documentation, Sustainable Storage - EnzymeML As A Basis For FAIR Data Management In Biocatalysis Pleiss J Ref: ChemCatChem, 13:3909, 2021 : PubMed ESTHER: Pleiss_2021_ChemCatChem_13_3909 PubMedSearch: Pleiss 2021 ChemCatChem 13 3909 Abstract The often reported reproducibility crisis in the biomedical sciences also applies to enzymology and biocatalysis, and mainly results from incomplete reporting of reaction conditions. In this Concept article, an infrastructure based on EnzymeML is sketched, which enables reporting, exchange, and storage of enzymatic data according to the FAIR data principles. EnzymeML is a novel data exchange format for enzymology and biocatalysis, which facilitates the application of the STRENDA Guidelines and thus makes data on enzyme-catalyzed reactions findable, accessible, interoperable, and reusable. EnzymeML enables the comprehensive documentation of metadata, thus fostering reproducibility and replicability in enzymology and biocatalysis. An EnzymeML Application Programming Interface integrates electronic lab notebooks with modelling platforms and databases on enzymatic reactions, and thus enables the seamless flow of enzymatic data from measurement to modelling to publication, without the need for manual intervention such as reformatting or editing. EnzymeML serves as a valuable tool for the design of biocatalytic experiments and contributes to the vision of a unified research data infrastructure for catalysis research. Title: The abundance of mRNA transcripts of bacteroidetal polyethylene terephthalate (PET) esterase genes may indicate a role in marine plastic degradation Zhang H, Dierkes R, Perez-Garcia P, Weigert S, Sternagel S, Hallam S, Schott T, Juergens K, Vollstedt C and Streit WR <8 more author(s)> Zhang H, Dierkes R, Perez-Garcia P, Weigert S, Sternagel S, Hallam S, Schott T, Juergens K, Vollstedt C, Chibani C, Danso D, Buchholz PCF, Pleiss J, Almeida A, Hocker B, Schmitz R, Chow J, Streit WR (- 8) Ref: ResearchSquare, :, 2021 : PubMed ESTHER: Zhang_2021_ResearchSquare__ PubMedSearch: Zhang 2021 ResearchSquare Gene_locus related to this paper: flutr-f2ie04, 9flao-a0a0c1f4u8, 9flao-kjj39608, 9flao-a0a1m6f5v0, 9flao-a0a330mq60 Abstract https://www.researchsquare.com/article/rs-567691/v2 Polyethylene terephthalate (PET) is an important synthetic polymer accumulating in nature 2 and recent studies have identified microorganisms capable of degrading PET. While the majority of 3 known PET hydrolases originate from the Actinobacteria and Proteobacteria, here we describe the 4 first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific 5 Hidden-Markov-Model (HMM)-based search algorithm we identified two promiscuous and cold6 active esterases derived from Aequorivita sp. (PET27) and Chryseobacterium jeonii (PET30) acting 7 on PET foil and powder. Notably, one of the enzymes (PET30) was able to hydrolyze PET at 8 temperatures between 4 - 30 C with a similar turnover rate compared to the well-known Ideonella 9 sakaiensis enzyme (IsPETase). 10 PET27 and PET30 homologues were detected in metagenomes encompassing a wide range 11 of different global climate zones. Additional transcript abundance mapping of marine samples imply 12 that these promiscuous enzymes and source organisms may play a role in the long-term 13 degradation of microplastic particles and fibers. Title: The modular structure of alpha/beta-hydrolases Bauer TL, Buchholz PCF, Pleiss J Ref: Febs J, 287:1035, 2020 : PubMed ESTHER: Bauer_2020_FEBS.J_287_1035 PubMedSearch: Bauer 2020 FEBS.J 287 1035 PubMedID: 31545554 Abstract The alpha/beta-hydrolase fold family is highly diverse in sequence, structure and biochemical function. To investigate the sequence-structure-function relationships, the Lipase Engineering Database (https://led.biocatnet.de) was updated. Overall, 280 638 protein sequences and 1557 protein structures were analysed. All alpha/beta-hydrolases consist of the catalytically active core domain, but they might also contain additional structural modules, resulting in 12 different architectures: core domain only, additional lids at three different positions, three different caps, additional N- or C-terminal domains and combinations of N- and C-terminal domains with caps and lids respectively. In addition, the alpha/beta-hydrolases were distinguished by their oxyanion hole signature (GX-, GGGX- and Y-types). The N-terminal domains show two different folds, the Rossmann fold or the beta-propeller fold. The C-terminal domains show a beta-sandwich fold. The N-terminal beta-propeller domain and the C-terminal beta-sandwich domain are structurally similar to carbohydrate-binding proteins such as lectins. The classification was applied to the newly discovered polyethylene terephthalate (PET)-degrading PETases and MHETases, which are core domain alpha/beta-hydrolases of the GX- and the GGGX-type respectively. To investigate evolutionary relationships, sequence networks were analysed. The degree distribution followed a power law with a scaling exponent gamma = 1.4, indicating a highly inhomogeneous network which consists of a few hubs and a large number of less connected sequences. The hub sequences have many functional neighbours and therefore are expected to be robust toward possible deleterious effects of mutations. The cluster size distribution followed a power law with an extrapolated scaling exponent tau = 2.6, which strongly supports the connectedness of the sequence space of alpha/beta-hydrolases. DATABASE: Supporting data about domains from other proteins with structural similarity to the N- or C-terminal domains of alpha/beta-hydrolases are available in Data Repository of the University of Stuttgart (DaRUS) under doi: https://doi.org/10.18419/darus-458. Title: Diverse effects of aqueous polar co-solvents on Candida antarctica lipase B Mangiagalli M, Carvalho H, Natalello A, Ferrario V, Pennati ML, Barbiroli A, Lotti M, Pleiss J, Brocca S Ref: Int J Biol Macromol, 150:930, 2020 : PubMed ESTHER: Mangiagalli_2020_Int.J.Biol.Macromol_150_930 PubMedSearch: Mangiagalli 2020 Int.J.Biol.Macromol 150 930 PubMedID: 32068052 Abstract Biocatalysis in mixtures of water and co-solvents represents an opportunity to expand the application of enzymes. However, in the presence of organic solvents, enzymes can undergo reversible inhibition, inactivation, or aggregation. In this work, we studied the effects of three co-solvents (methanol, acetone, and dimethyl sulfoxide - DMSO) on the function and structure of the recombinant Candida antarctica lipase B (rCALB), a widely used enzyme in biotechnological applications. The effects of co-solvents on rCALB were investigated by steady-state kinetics experiments, biophysical assays and by molecular dynamics simulations in the presence and upon incubation with the three co-solvents. Methanol and acetone were found to act as competitive inhibitors of rCALB and to promote its aggregation, whereas DMSO is a non-essential activator of rCALB. Title: Determinants and prediction of esterase substrate promiscuity patterns Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A and Ferrer M <32 more 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 (- 32) Ref: ACS Chemical Biology, 13:225, 2018 : PubMed 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 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. Title: Self-Assembly Nanostructures of Triglyceride-Water Interfaces Determine Functional Conformations of Candida antarctica Lipase B Benson SP, Pleiss J Ref: Langmuir, 33:3151, 2017 : PubMed ESTHER: Benson_2017_Langmuir_33_3151 PubMedSearch: Benson 2017 Langmuir 33 3151 PubMedID: 28274117 Abstract Candida antarctica lipase B (CalB) acts as a lipase when adsorbed to an acylglyceride interface and as an esterase when exposed to an aqueous environment. The effect of the molecular self-assembly nanostructure of triglyceride-water interfaces on structural conformations of adsorbed CalB and the implications to its catalytic function were studied by molecular dynamics simulations. Systems of CalB adsorbed to interfaces and solvated in water were compared. The two environments induced relative motions of helices alpha5 and alpha10 that resulted in open and closed conformations. The open conformation was stabilized by interactions between the polar and nonpolar amino acids of alpha5 and alpha10 and the nanostructure of triglyceride aggregates, which self-assembled into crystalline-like patterns of alternating polar and nonpolar lamellae. Thus, the structure of CalB has been adapted by evolution to the geometric constraints imposed by the interface nanostructure for optimized catalytic activity. Helices alpha5 and alpha10 have two functions. As mobile elements, they ensure access of bulky substrates to the active site in the open conformation. As a part of the active site pocket, they ensure binding of substrate molecules in a productive orientation near the active site. In water, access to the binding site is limited, and the smaller substrate binding site is beneficial for the binding of small, water-soluble substrates. The CalB crystal structure commonly used for protein engineering studies represents an intermediate state between open and closed, and may thus not be adequate to assess the function of CalB, neither as lipase nor as esterase. Title: BioCatNet: A Database System for the Integration of Enzyme Sequences and Biocatalytic Experiments Buchholz PCF, Vogel C, Reusch W, Pohl M, Rother D, Spiess AC, Pleiss J Ref: Chembiochem, 17:2093, 2016 : PubMed ESTHER: Buchholz_2016_Chembiochem_17_2093 PubMedSearch: Buchholz 2016 Chembiochem 17 2093 PubMedID: 27562499 Abstract The development of novel enzymes for biocatalytic processes requires knowledge on substrate profile and selectivity; this can be derived from databases and from publications. Often, these sources lack time-course data for the substrate or product, and an unambiguous link between experiment and enzyme sequence. The lack of integrated, original data hampers the comprehensive analysis of enzyme kinetics and the evaluation of sequence-function relationships. In order to accelerate enzyme engineering, BioCatNet integrates protein sequence, protein structure, and experimental data for a given enzyme family. BioCatNet explicitly assigns the enzyme sequence to the experimental data, which consists of information on reaction conditions and time-course data. BioCatNet facilitates the consistent documentation of reaction conditions, the archiving of time-course data, and the efficient exchange of experimental data among collaborators. Data integration is demonstrated for three case studies by using the TEED (Thiamine diphosphate-dependent Enzymes Engineering Database). Title: Binding of Solvent Molecules to a Protein Surface in Binary Mixtures Follows a Competitive Langmuir Model Kulschewski T, Pleiss J Ref: Langmuir, 32:8960, 2016 : PubMed ESTHER: Kulschewski_2016_Langmuir_32_8960 PubMedSearch: Kulschewski 2016 Langmuir 32 8960 PubMedID: 27523916 Abstract The binding of solvent molecules to a protein surface was modeled by molecular dynamics simulations of of Candida antarctica (C. antarctica) lipase B in binary mixtures of water, methanol, and toluene. Two models were analyzed: a competitive Langmuir model which assumes identical solvent binding sites with a different affinity toward water (KWat), methanol (KMet), and toluene (KTol) and a competitive Langmuir model with an additional interaction between free water and already bound water (KWatWat). The numbers of protein-bound molecules of both components of a binary mixture were determined for different compositions as a function of their thermodynamic activities in the bulk phase, and the binding constants were simultaneously fitted to the six binding curves (two components of three different mixtures). For both Langmuir models, the values of KWat, KMet, and KTol were highly correlated. The highest binding affinity was found for methanol, which was almost 4-fold higher than the binding affinities of water and toluene (KMet >> KWat approximately KTol). Binding of water was dominated by the water-water interaction (KWatWat). Even for the three protein surface patches of highest water affinity, the binding affinity of methanol was 2-fold higher than water and 8-fold higher than toluene (KMet > KWat > KTol). The Langmuir model provides insights into the protein destabilizing mechanism of methanol which has a high binding affinity toward the protein surface. Thus, destabilizing solvents compete with intraprotein interactions and disrupt the tertiary structure. In contrast, benign solvents such as water or toluene have a low affinity toward the protein surface. Water is a special solvent: only few water molecules bind directly to the protein; most water molecules bind to already bound water molecules thus forming water patches. A quantitative mechanistic model of protein-solvent interactions that includes competition and miscibility of the components contributes a robust basis for solvent and protein engineering. Title: Effects of methanol on lipases: molecular, kinetic and process issues in the production of biodiesel Lotti Marina, Pleiss J, Valero F, Ferrer P Ref: Biotechnol J, 10:22, 2015 : PubMed ESTHER: Lotti_2015_Biotechnol.J_10_22 PubMedSearch: Lotti 2015 Biotechnol.J 10 22 PubMedID: 25046365 Abstract The biotechnological production of biodiesel is based on transesterification/esterification reactions between a source of fatty acids and a short-chain alcohol, usually methanol, catalysed by enzymes belonging to the class known as lipases. Several lipases used in industrial processes, although stable in the presence of other organic solvents, are inactivated by methanol at or below the concentration optimal for biodiesel production, making it necessary to use stepwise methanol feeding or pre-treatment of the enzyme. In this review article we focus on what is currently know about methanol inactivation of lipases, a phenomenon which is not common to all lipase enzymes, with the goal of improving the biocatalytic process. We suggest that different mechanisms can lead to inactivation of different lipases, in particular substrate inhibition and protein unfolding. Attempts to improve the performances of methanol sensitive lipases by mutagenesis as well as process engineering approaches are also summarized. Title: The effect of thermodynamic properties of solvent mixtures explains the difference between methanol and ethanol in C.antarctica lipase B catalyzed alcoholysis Sasso F, Kulschewski T, Secundo F, Lotti Marina, Pleiss J Ref: J Biotechnol, 214:1, 2015 : PubMed ESTHER: Sasso_2015_J.Biotechnol_214_1 PubMedSearch: Sasso 2015 J.Biotechnol 214 1 PubMedID: 26325200 Abstract Kinetic modelling, molecular modelling, and experimental determination of the initial reaction velocity of lipase-catalyzed alcoholysis were combined to study the effect of the alcohol substrate to catalytic activity. The model system consisted of methanol or ethanol at varying concentrations, vinyl acetate as ester substrate 15.2% (v/v), toluene as organic solvent, water at a controlled thermodynamic activity of 0.09, and C. antarctica lipase B as enzyme. For both alcohol substrates, the initial reaction velocity increased sharply at low concentrations and reached a maximum at 0.7% (v/v) for methanol and 2% (v/v) for ethanol. For higher concentrations, the reaction rate decreased to a level of 74% and 60% of the peak value, respectively, due to substrate inhibition. The concentration dependency was described by a kinetic model, including a ping-pong bi-bi mechanism and competitive inhibition by the alcohol, and confirmed previous observations that methanol is more efficiently inhibiting the enzyme than ethanol. However, if the initial reaction velocity was expressed in terms of thermodynamic activity of the two alcohol substrates, the maximum of initial reaction velocity was similar for methanol (aMeOHmax=0.19) and ethanol (aEtOHmax=0.21). This was confirmed by molecular modelling which resulted in similar KM (0.22 and 0.19) and Ki values (0.44 and 0.49) for methanol and ethanol, respectively, if expressed in thermodynamic activities. Thus, the experimentally observed difference between methanol and ethanol is not due to differences in interaction with the enzyme but is a consequence of the thermodynamics of the substrate-solvent mixture. For low concentrations in toluene, the activity coefficient of methanol is 40% higher than the activity coefficient of ethanol (gammaMeOH=8.5, gammaEtOH=6.1). Title: Solvent Flux Method (SFM): A Case Study of Water Access to Candida antarctica Lipase B Benson SP, Pleiss J Ref: J Chem Theory Comput, 10:5206, 2014 : PubMed ESTHER: Benson_2014_J.Chem.Theory.Comput_10_5206 PubMedSearch: Benson 2014 J.Chem.Theory.Comput 10 5206 PubMedID: 26584392 Abstract The solvent flux method (SFM) was developed to comprehensively characterize the influx of solvent molecules from the solvent environment into the active site of a protein in the framework of molecular dynamics simulations. This was achieved by introducing a solvent concentration gradient as well as partially reorienting and rescaling the velocity vector of all solvent molecules contained within a spherical volume enclosing the protein, thus inducing an accelerated solvent influx toward the active site. In addition to the detection of solvent access pathway within the protein structure, it is hereby possible to identify potential amino acid positions relevant to solvent-related enzyme engineering with high statistical significance. The method is particularly aimed at improving the reverse hydrolysis reaction rates in nonaqueous media. Candida antarctica lipase B (CALB) binds to a triglyceride-water interface with its substrate entrance channel oriented toward the hydrophobic substrate interface. The lipase-triglyceride-water system served as a model system for SFM to evaluate the influx of water molecules to the active site. As a proof of principle for SFM, a previously known water access pathway in CALB was identified as the primary water channel. In addition, a secondary water channel and two pathways for water access which contribute to water leakage between the protein and the triglyceride-water interface were identified. Title: Molecular mechanism of deactivation of C. antarctica lipase B by methanol Kulschewski T, Sasso F, Secundo F, Lotti Marina, Pleiss J Ref: J Biotechnol, 168:462, 2013 : PubMed ESTHER: Kulschewski_2013_J.Biotechnol_168_462 PubMedSearch: Kulschewski 2013 J.Biotechnol 168 462 PubMedID: 24144811 Abstract The catalytic activity of Candida antarctica lipase B upon alcoholysis of a constant concentration of 15.2% vinyl acetate (vol/vol) and varying concentrations of methanol (0.7-60%) in toluene was determined experimentally by measuring the initial reaction velocity. The molecular mechanism of the deactivation of the enzyme by methanol was investigated by fitting the experimental data to a kinetic model and by molecular dynamics simulations of C. antarctica lipase B in toluene-methanol-water mixtures. The highest catalytic activity (280 U/mg) was observed at methanol concentrations as low as 0.7% methanol (vol/vol), followed by a sharp decrease at higher methanol concentrations. For methanol concentrations above 10% (vol/vol), catalytic activity was at 30% of the maximum activity. A variation of water activity in the range 0.02-0.09 had only minor effects. These experimental observations are described by a simple kinetic model using three assumptions: (1) a ping-pong bi-bi mechanism of the enzyme, (2) competitive inhibition by the substrate methanol, and (3) by describing enzyme kinetics by the thermodynamic activities of the substrates rather than by their concentrations. Two equilibrium constants of methanol (KM,MeOH=0.05 and Ki,MeOH=0.23) were derived by modeling methanol binding to the substrate binding site of the lipase in molecular dynamics simulations of protein-solvent systems at atomic resolution. Thus, the sharp maximum of catalytic activity of C. antarctica lipase B at 0.7% methanol is a direct consequence of the fact that methanol-toluene mixtures are far from ideal. Understanding the thermodynamics of solvent mixtures is prerequisite to a quantitative model of enzymatic activity in organic solvents. Title: Molecular modeling of lipase binding to a substrate-water interface Gruber CC, Pleiss J Ref: Methods Mol Biol, 861:313, 2012 : PubMed ESTHER: Gruber_2012_Methods.Mol.Biol_861_313 PubMedSearch: Gruber 2012 Methods.Mol.Biol 861 313 PubMedID: 22426727 Abstract Interactions of lipases with hydrophobic substrate-water interfaces are of great interest to design improved lipase variants and engineer reaction conditions. This chapter describes the necessary steps to carry out molecular dynamics simulations of Candida antarctica lipase B at tributyrin-water interface using the GROMACS simulation software. Special attention is drawn to the preparation of the protein and the substrate-water interface and to the analysis of the obtained trajectory. Title: Evolution of stability in a cold-active enzyme elicits specificity relaxation and highlights substrate-related effects on temperature adaptation Gatti-Lafranconi P, Natalello A, Rehm S, Doglia SM, Pleiss J, Lotti M Ref: Journal of Molecular Biology, 395:155, 2010 : PubMed ESTHER: Gatti-Lafranconi_2010_J.Mol.Biol_395_155 PubMedSearch: Gatti-Lafranconi 2010 J.Mol.Biol 395 155 PubMedID: 19850050 Abstract Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50 degrees C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity. Title: Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters Juhl PB, Doderer K, Hollmann F, Thum O, Pleiss J Ref: J Biotechnol, 150:474, 2010 : PubMed ESTHER: Juhl_2010_J.Biotechnol_150_474 PubMedSearch: Juhl 2010 J.Biotechnol 150 474 PubMedID: 20887757 Abstract Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester. Title: Rational design of Pseudozyma antarctica lipase B yielding a general esterification catalyst Liu D, Trodler P, Eiben S, Koschorreck K, Muller M, Pleiss J, Maurer SC, Branneby C, Schmid RD, Hauer B Ref: Chembiochem, 11:789, 2010 : PubMed ESTHER: Liu_2010_Chembiochem_11_789 PubMedSearch: Liu 2010 Chembiochem 11 789 PubMedID: 20209560 Abstract Pseudozyma antarctica lipase B (CALB) shows activity in the acrylation of hydroxypropylcarbamate, a racemic mixture of enantiomers of primary and secondary alcohols. However, full conversion is hampered by the slowly reacting S enantiomer of the secondary alcohol. The same is true for a wide range of secondary alcohols, for example, octan-2- and -3-ol. In order to get high conversion in these reactions in a short time, the stereospecificity pocket of CALB was redesigned by using predictions from molecular modeling. Positions 278, 104, and 47 were targeted, and a library for two-site saturation mutagenesis at positions 104 and 278 was constructed. The library was then screened for hydrolysis of acrylated hydroxypropylcarbamates. The best mutants L278A, L278V, L278A/W104F, and L278A/W104F/S47A showed an increased conversion in hydrolysis and transesterification of more than 30 %. While the wild-type showed only 73 % conversion in the acrylation of hydroxypropylcarbamate after 6 h, 97 % conversion was achieved by L278A in this time. Besides this, L278A/W104F reached >96 % conversion in the acrylation of octan-2- and -3-ol within 48 h and showed a significant decrease in stereoselectivity, while the wild-type reached only 68 and 59 % conversion, respectively. Thus the new biocatalysts can be used for efficient transformation of racemic alcohols and esters with high activity when the high stereoselectivity of the wild-type hampers complete conversion of racemic substrates in a short time. Title: Solvent-induced lid opening in lipases: a molecular dynamics study Rehm S, Trodler P, Pleiss J Ref: Protein Science, 19:2122, 2010 : PubMed ESTHER: Rehm_2010_Protein.Sci_19_2122 PubMedSearch: Rehm 2010 Protein.Sci 19 2122 PubMedID: 20812327 Abstract In most lipases, a mobile lid covers the substrate binding site. In this closed structure, the lipase is assumed to be inactive. Upon activation of the lipase by contact with a hydrophobic solvent or at a hydrophobic interface, the lid opens. In its open structure, the substrate binding site is accessible and the lipase is active. The molecular mechanism of this interfacial activation was studied for three lipases (from Candida rugosa, Rhizomucor miehei, and Thermomyces lanuginosa) by multiple molecular dynamics simulations for 25 ns without applying restraints or external forces. As initial structures of the simulations, the closed and open structures of the lipases were used. Both the closed and the open structure were simulated in water and in an organic solvent, toluene. In simulations of the closed lipases in water, no conformational transition was observed. However, in three independent simulations of the closed lipases in toluene the lid gradually opened. Thus, pathways of the conformational transitions were investigated and possible kinetic bottlenecks were suggested. The open structures in toluene were stable, but in water the lid of all three lipases moved towards the closed structure and partially unfolded. Thus, in all three lipases opening and closing was driven by the solvent and independent of a bound substrate molecule. Title: Structural classification by the Lipase Engineering Database: a case study of Candida antarctica lipase A Widmann M, Juhl PB, Pleiss J Ref: BMC Genomics, 11:123, 2010 : PubMed ESTHER: Widmann_2010_BMC.Genomics_11_123 PubMedSearch: Widmann 2010 BMC.Genomics 11 123 PubMedID: 20170513 Abstract BACKGROUND: The Lipase Engineering Database (LED) integrates information on sequence, structure and function of lipases, esterases and related proteins with the alpha/beta hydrolase fold. A new superfamily for Candida antarctica lipase A (CALA) was introduced including the recently published crystal structure of CALA. Since CALA has a highly divergent sequence in comparison to other alpha/beta hydrolases, the Lipase Engineering Database was used to classify CALA in the frame of the already established classification system. This involved the comparison of CALA to similar structures as well as sequence-based comparisons against the content of the LED. RESULTS: The new release 3.0 (December 2009) of the Lipase Engineering Database contains 24783 sequence entries for 18585 proteins as well as 656 experimentally determined protein structures, including the structure of CALA. In comparison to the previous release 1 with 4322 protein and 167 structure entries this update represents a significant increase in data volume. By comparing CALA to representative structures from all superfamilies, a structure from the deacetylase superfamily was found to be most similar to the structure of CALA. While the alpha/beta hydrolase fold is conserved in both proteins, the major difference is found in the cap region. Sequence alignments between both proteins show a sequence similarity of only 15%. A multisequence alignment of both protein families was used to create hidden Markov models for the cap region of CALA and showed that the cap region of CALA is unique among all other proteins of the alpha/beta hydrolase fold. By specifically comparing the substrate binding pocket of CALA to other binding pockets of alpha/beta hydrolases, the binding pocket of Candida rugosa lipase was identified as being highly similar. This similarity also applied to the lid of Candida rugosa lipase in comparison to the potential lid of CALA. CONCLUSION: The LED serves as a valuable tool for the systematic analysis of single proteins or protein families. The updated release 3.0 was used for the evaluation of alpha/beta hydrolases. The HTML version of the database with new features is available at http://www.led.uni-stuttgart.de and provides sequences, structures and a set of analysis tools including phylogenetic trees and HMM profiles. Title: Molecular mechanism of the hydration of Candida antarctica lipase B in the gas phase: Water adsorption isotherms and molecular dynamics simulations Branco RJ, Graber M, Denis V, Pleiss J Ref: Chembiochem, 10:2913, 2009 : PubMed ESTHER: Branco_2009_Chembiochem_10_2913 PubMedSearch: Branco 2009 Chembiochem 10 2913 PubMedID: 19847841 Abstract Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (a(w)) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein-bound water molecules increased linearly with a(w), until at a(w)=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate-binding site. At higher water activity, the thickness of the hydration shell increased up to 10 A close to a(w)=1. Above a limit of 1600 protein-bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate-binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive. Title: Morphing activity between structurally similar enzymes: from heme-free bromoperoxidase to lipase Chen B, Cai Z, Wu W, Huang Y, Pleiss J, Lin Z Ref: Biochemistry, 48:11496, 2009 : PubMed ESTHER: Chen_2009_Biochemistry_48_11496 PubMedSearch: Chen 2009 Biochemistry 48 11496 PubMedID: 19883129 Abstract In this study, to explore the plasticity of the alpha/beta-hydrolase fold family, we converted bromoperoxidase A2 (BPO-A2) from Streptomyces aureofaciens to a lipase by structure comparison with lipase A (LipA) from Bacillus subtilis. These two enzymes have similar structures (2.1 A rmsd) and a very low level of sequence identity ( approximately 18%). A variant BL1 was constructed by deleting the caplike domain of BPO-A2 and further fine-tuning the newly formed substrate binding site. The lipase activity was successfully transplanted on BL1, while the halogenation activity was totally lost. BL1 also showed higher hydrolytic activities toward long chain p-nitrophenyl esters, such as p-nitrophenyl caprylate (3.7-fold) and p-nitrophenyl palmitate (7.0-fold), while its activity toward a short chain ester (p-nitrophenyl acetate) decreased dramatically, to only 1.2% of that of BPO-A2. After two rounds of directed evolution and site-directed mutagenesis on selected residues, several mutants with both improved hydrolytic activities and substrate preferences toward long chain substrates were obtained. The highest hydrolytic activity toward p-nitrophenyl palmitate of the best mutant BL1-2-E8-plusI was improved by 40-fold compared with that of BL1. These results demonstrate the possibility of manipulating the caplike domain of alpha/beta-hydrolase fold enzymes and provide further understanding of the structure-function relationship of the alpha/beta-hydrolase fold enzymes. The design strategy used in this study could serve as a useful approach for constructing variants with targeted catalytic properties using the alpha/beta-hydrolase fold. Title: Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking Juhl PB, Trodler P, Tyagi S, Pleiss J Ref: BMC Struct Biol, 9:39, 2009 : PubMed ESTHER: Juhl_2009_BMC.Struct.Biol_9_39 PubMedSearch: Juhl 2009 BMC.Struct.Biol 9 39 PubMedID: 19493341 Abstract BACKGROUND: Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. RESULTS: Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i) enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii) enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii) substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. CONCLUSION: The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand), although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria. Title: The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases Knoll M, Hamm TM, Wagner F, Martinez V, Pleiss J Ref: BMC Bioinformatics, 10:89, 2009 : PubMed ESTHER: Knoll_2009_BMC.Bioinformatics_10_89 PubMedSearch: Knoll 2009 BMC.Bioinformatics 10 89 PubMedID: 19296857 Abstract BACKGROUND: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common alpha/beta-hydrolase fold and a catalytic triad, which is also found in other alpha/beta-hydrolases. Title: Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase Trodler P, Schmid RD, Pleiss J Ref: BMC Struct Biol, 9:38, 2009 : PubMed ESTHER: Trodler_2009_BMC.Struct.Biol_9_38 PubMedSearch: Trodler 2009 BMC.Struct.Biol 9 38 PubMedID: 19476626 Abstract BACKGROUND: The characteristic of most lipases is the interfacial activation at a lipid interface or in non-polar solvents. Interfacial activation is linked to a large conformational change of a lid, from a closed to an open conformation which makes the active site accessible for substrates. While for many lipases crystal structures of the closed and open conformation have been determined, the pathway of the conformational transition and possible bottlenecks are unknown. Therefore, molecular dynamics simulations of a closed homology model and an open crystal structure of Burkholderia cepacia lipase in water and toluene were performed to investigate the influence of solvents on structure, dynamics, and the conformational transition of the lid. RESULTS: The conformational transition of B. cepacia lipase was dependent on the solvent. In simulations of closed B. cepacia lipase in water no conformational transition was observed, while in three independent simulations of the closed lipase in toluene the lid gradually opened during the first 10-15 ns. The pathway of conformational transition was accessible and a barrier was identified, where a helix prevented the lid from opening to the completely open conformation. The open structure in toluene was stabilized by the formation of hydrogen bonds.In simulations of open lipase in water, the lid closed slowly during 30 ns nearly reaching its position in the closed crystal structure, while a further lid opening compared to the crystal structure was observed in toluene. While the helical structure of the lid was intact during opening in toluene, it partially unfolded upon closing in water. The closing of the lid in water was also observed, when with eight intermediate structures between the closed and the open conformation as derived from the simulations in toluene were taken as starting structures. A hydrophobic beta-hairpin was moving away from the lid in all simulations in water, which was not observed in simulations in toluene. The conformational transition of the lid was not correlated to the motions of the beta-hairpin structure. CONCLUSION: Conformational transitions between the experimentally observed closed and open conformation of the lid were observed by multiple molecular dynamics simulations of B. cepacia lipase. Transitions in both directions occurred without applying restraints or external forces. The opening and closing were driven by the solvent and independent of a bound substrate molecule. Title: Rational design of a new one-step purification strategy for Candida antarctica lipase B by ion-exchange chromatography Trodler P, Nieveler J, Rusnak M, Schmid RD, Pleiss J Ref: Journal of Chromatography A, 1179:161, 2008 : PubMed ESTHER: Trodler_2008_J.Chromatogr.A_1179_161 PubMedSearch: Trodler 2008 J.Chromatogr.A 1179 161 PubMedID: 18154980 Abstract A fast and efficient one-step method for purification of lipase B from Candida antarctica by ion-exchange chromatography was developed by rational design. The electrostatic properties of the enzyme were calculated and validated by isoelectric focusing and measurement of the titration curve. C. antarctica lipase B shows an unusual pH profile with a broad isoelectric region from pH 4 to 8. At pH 3 C. antarctica lipase B can be bound to a cation-exchange chromatography column and was purified to homogeneity with a purification factor of 2.4. It was stable at pH 3, the residual activity was still 80% after 6 days incubation at 20 degrees C. The broad isoelectric region of C. antarctica lipase B is unique as compared to almost all other alpha/beta-hydrolases which have a well-defined isoelectric point. A search in the lipase engineering database resulted in only one further alpha/beta-hydrolase, the Fusarium solani cutinase, which also has a broad isoelectric region. Title: Modeling structure and flexibility of Candida antarctica lipase B in organic solvents Trodler P, Pleiss J Ref: BMC Struct Biol, 8:9, 2008 : PubMed ESTHER: Trodler_2008_BMC.Struct.Biol_8_9 PubMedSearch: Trodler 2008 BMC.Struct.Biol 8 9 PubMedID: 18254946 Abstract BACKGROUND: The structure and flexibility of Candida antarctica lipase B in water and five different organic solvent models was investigated using multiple molecular dynamics simulations to describe the effect of solvents on structure and dynamics. Interactions of the solvents with the protein and the distribution of water molecules at the protein surface were examined. Title: Analysis of the distribution of functionally relevant rare codons Widmann M, Clairo M, Dippon J, Pleiss J Ref: BMC Genomics, 9:207, 2008 : PubMed ESTHER: Widmann_2008_BMC.Genomics_9_207 PubMedSearch: Widmann 2008 BMC.Genomics 9 207 PubMedID: 18457591 Abstract BACKGROUND: The substitution of rare codons with more frequent codons is a commonly applied method in heterologous gene expression to increase protein yields. However, in some cases these substitutions lead to a decrease of protein solubility or activity. To predict these functionally relevant rare codons, a method was developed which is based on an analysis of multisequence alignments of homologous protein families. Title: DWARF--a data warehouse system for analyzing protein families Fischer M, Thai QK, Grieb M, Pleiss J Ref: BMC Bioinformatics, 7:495, 2006 : PubMed ESTHER: Fischer_2006_BMC.Bioinformatics_7_495 PubMedSearch: Fischer 2006 BMC.Bioinformatics 7 495 PubMedID: 17094801 Abstract BACKGROUND: The emerging field of integrative bioinformatics provides the tools to organize and systematically analyze vast amounts of highly diverse biological data and thus allows to gain a novel understanding of complex biological systems. The data warehouse DWARF applies integrative bioinformatics approaches to the analysis of large protein families. DESCRIPTION: The data warehouse system DWARF integrates data on sequence, structure, and functional annotation for protein fold families. The underlying relational data model consists of three major sections representing entities related to the protein (biochemical function, source organism, classification to homologous families and superfamilies), the protein sequence (position-specific annotation, mutant information), and the protein structure (secondary structure information, superimposed tertiary structure). Tools for extracting, transforming and loading data from public available resources (ExPDB, GenBank, DSSP) are provided to populate the database. The data can be accessed by an interface for searching and browsing, and by analysis tools that operate on annotation, sequence, or structure. We applied DWARF to the family of alpha/beta-hydrolases to host the Lipase Engineering database. Release 2.3 contains 6138 sequences and 167 experimentally determined protein structures, which are assigned to 37 superfamilies 103 homologous families. CONCLUSION: DWARF has been designed for constructing databases of large structurally related protein families and for evaluating their sequence-structure-function relationships by a systematic analysis of sequence, structure and functional annotation. It has been applied to predict biochemical properties from sequence, and serves as a valuable tool for protein engineering. Title: Biochemical profiling in silico--predicting substrate specificities of large enzyme families Tyagi S, Pleiss J Ref: J Biotechnol, 124:108, 2006 : PubMed ESTHER: Tyagi_2006_J.Biotechnol_124_108 PubMedSearch: Tyagi 2006 J.Biotechnol 124 108 PubMedID: 16519956 Abstract A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules. Title: How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli Koschorreck M, Fischer M, Barth S, Pleiss J Ref: BMC Genomics, 6:49, 2005 : PubMed ESTHER: Koschorreck_2005_BMC.Genomics_6_49 PubMedSearch: Koschorreck 2005 BMC.Genomics 6 49 PubMedID: 15804363 Abstract BACKGROUND: In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443-448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. RESULTS: The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. CONCLUSION: A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family-specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases. Title: The database of epoxide hydrolases and haloalkane dehalogenases: one structure, many functions Barth S, Fischer M, Schmid RD, Pleiss J Ref: Bioinformatics, 20:2845, 2004 : PubMed ESTHER: Barth_2004_Bioinformatics_20_2845 PubMedSearch: Barth 2004 Bioinformatics 20 2845 PubMedID: 15117755 Abstract The epoxide hydrolases and haloalkane dehalogenases database (EH/HD) integrates sequence and structure of a highly diverse protein family, including mainly the Asp-hydrolases of EHs and HDs but also proteins, such as Ser-hydrolases non-heme peroxidases, prolyl iminopetidases and 2-hydroxymuconic semialdehyde hydrolases. These proteins have a highly conserved structure, but display a remarkable diversity in sequence and function. A total of 305 protein entries were assigned to 14 homologous families, forming two superfamilies. Annotated multisequence alignments and phylogenetic trees are provided for each homologous family and superfamily. Experimentally derived structures of 19 proteins are superposed and consistently annotated. Sequence and structure of all 305 proteins were systematically analysed. Thus, deeper insight is gained into the role of a highly conserved sequence motifs and structural elements. AVAILABILITY: The EH/HD database is available at http://www.led.uni-stuttgart.de Title: Sequence and structure of epoxide hydrolases: a systematic analysis Barth S, Fischer M, Schmid RD, Pleiss J Ref: Proteins, 55:846, 2004 : PubMed ESTHER: Barth_2004_Proteins_55_846 PubMedSearch: Barth 2004 Proteins 55 846 PubMedID: 15146483 Abstract Epoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. More than 100 epoxide hydrolases (EH) have been identified or predicted, and 3 structures are available. Although they catalyze the same chemical reaction, sequence similarity is low. To identify conserved regions, all EHs were aligned. Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs. Bacterial EHs show a high sequence diversity. Based on structure comparison of three known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed. Although core and cap domain are highly conserved, the structural differences between the EHs are restricted to only two loops: the NC-loop connecting the core and the cap and the cap-loop, which is inserted into the cap domain. EHs were assigned to either of three clusters based on loop length. By using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled. Representative models are available from the Lipase Engineering Database, http://www.led.uni-stuttgart.de. Title: Structure and dynamics of Candida rugosa lipase: the role of organic solvent Tejo BA, Salleh AB, Pleiss J Ref: J Mol Model, 10:358, 2004 : PubMed ESTHER: Tejo_2004_J.Mol.Model_10_358 PubMedSearch: Tejo 2004 J.Mol.Model 10 358 PubMedID: 15597204 Abstract The effect of organic solvent on the structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (C(alpha) rms deviation 1-1.3 A). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as a rigid body about its average position. While in water the deviations were about 1.6 A, organic solvent reduced flexibility to 0.9 A. This increase rigidity was caused by two salt bridges (Lys85-Asp284, Lys75-Asp79) and a stable hydrogen bond (Lys75-Asn 292) in organic solvent. Thus, organic solvents stabilize the lid but render the side chains in the hydrophobic substrate-binding site more mobile. [figure: see text]. Superimposition of open (black, PDB entry 1CRL) and closed (gray, PDB entry 1TRH) conformers of C. rugosa lipase. The mobile lid is indicated. Title: A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases Henke E, Bornscheuer UT, Schmid RD, Pleiss J Ref: Chembiochem, 4:485, 2003 : PubMed ESTHER: Henke_2003_Chembiochem_4_485 PubMedSearch: Henke 2003 Chembiochem 4 485 PubMedID: 12794858 Abstract Carboxylesterases containing the sequence motif GGGX catalyze the hydrolysis of esters of chiral tertiary alcohols, albeit with only low to moderate enantioselectivity, for three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity for this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this amino acid for an alanine residue led to a sixfold increase of enantioselectivity (E = 19) towards 2-phenyl-3-butin-2-yl acetate. However, the effect of this mutation is specific: the same mutant had the opposite enantiopreference towards the substrate linalyl acetate. Thus, depending on the substrate structure, the same mutant has either increased enantioselectivity or opposite enantiopreference compared to the wild-type enzyme. Title: Activity of lipases and esterases towards tertiary alcohols: insights into structure-function relationships Henke E, Pleiss J, Bornscheuer UT Ref: Angew Chem Int Ed Engl, 41:3211, 2002 : PubMed ESTHER: Henke_2002_Angew.Chem.Int.Ed.Engl_41_3211 PubMedSearch: Henke 2002 Angew.Chem.Int.Ed.Engl 41 3211 PubMedID: 12207392 Title: Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity Schmitt J, Brocca S, Schmid RD, Pleiss J Ref: Protein Engineering, 15:595, 2002 : PubMed ESTHER: Schmitt_2002_Protein.Eng_15_595 PubMedSearch: Schmitt 2002 Protein.Eng 15 595 PubMedID: 12200542 Abstract The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8. Title: Probing the acyl binding site of acetylcholinesterase by protein engineering Pleiss J, Mionetto N, Schmid RD Ref: J Mol Catal B Enzym, 6:287, 1999 : PubMed ESTHER: Pleiss_1999_J.Mol.Catal.B.Enzym_6_287 PubMedSearch: Pleiss 1999 J.Mol.Catal.B.Enzym 6 287 Inhibitor(s) related to this paper: Furathiocarb Abstract Recombinant acetylcholinesterase from rat brain and two mutants were studied for their hydrolytic activity toward acetyl- and butyrylthiocholine substrates and for their sensitivity toward organophosphate and carbamate inhibitors. Both mutants, a point mutant where F295 was replaced by leucine, and a second mutant where loop PQES was replaced by SG, were designed for increased size of the acyl binding pocket. Wild type and mutant enzymes were expressed in baculovirus-infected insect cells and biochemically characterized. As expected, wild type rat brain acetylcholinesterase hydrolyzed acetylthiocholine, but not butyrylthiocholine. Sensitivity toward small- and medium-sized organophosphate inhibitors like paraoxon-methyl and paraoxon-ethyl was comparable, but bulky organophosphates like ethoprophos were less efficient inhibitors. This tendency applied to carbamates as well, since small carbamoyl moieties like carbofuran and aldicarb were stronger inhibitors than furathiocarb which features a bulky carbamoyl moiety. In contrast to wild type enzyme, both mutants were capable of hydrolyzing butyrylthiocholine. However, kcat/Km toward acetylthiocholine of the F295L mutant was reduced if compared to the wild type enzyme. All five organophosphate and three carbamate inhibitors inhibited mutant F295L more efficiently than the wild type enzyme. Title: Anatomy of lipase binding sites: the scissile fatty acid binding site Pleiss J, Fischer M, Schmid RD Ref: Chemistry & Physic of Lipids, 93:67, 1998 : PubMed ESTHER: Pleiss_1998_Chem.Phys.Lipids_93_67 PubMedSearch: Pleiss 1998 Chem.Phys.Lipids 93 67 PubMedID: 9720251 Abstract Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 A in cutinase and 22 A in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity. | |||||||
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