Search

Biocatalysis is becoming a data science. High-throughput experimentation generates a rapidly increasing stream of biocatalytic data, which is the raw material for mechanistic and novel data-driven modeling approaches for the predictive design of improved biocatalysts and novel bioprocesses. The holistic and molecular understanding of enzymatic reaction systems will enable us to identify and overcome kinetic bottlenecks and shift the thermodynamics of a reaction. The full characterization and modeling of reaction systems is a community effort; therefore, published methods and results should be findable, accessible, interoperable, and reusable (FAIR), which is achieved by developing standardized data exchange formats, by a complete and reproducible documentation of experimentation, by collaborative platforms for developing sustainable software and for analyzing data, and by repositories for publishing results together with raw data. The FAIRification of biocatalysis is a prerequisite to developing highly automated laboratory infrastructures that improve the reproducibility of scientific results and reduce the time and costs required to develop novel synthesis routes.

The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://enzymeml.org.

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 antarcticalipase 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 α5), Leu278, and Ile285 (helix α10) at a distance of 4–5 Å 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.

Experimental data on thermophysical properties of solvent mixtures such as aqueous deep eutectic solvents (DES) are scattered in scientific publications. It is desirable to integrate thermophysical properties with parameters that describe a DES in a human and machine readable format. On the basis of the Chemical Markup Language (CML), a standardized exchange format for density, viscosity, conductivity, and water activity of solvent mixtures was established and applied to represent published data on choline chloride/glycerol/water mixtures. In total, 300 different data sets served as a basis for data analysis by machine learning. Gradient tree boosting (GB) was used to predict thermophysical properties from the collected parameters, resulting in an excellent correlation between predicted, experimental, and simulation data. The experimental viscosity data was modeled assuming an Arrhenius dependency on temperature and by determining two parameters (η0and Eη). Integration of experimental and simulation data into a standardized exchange format makes data findable, accessible, interoperable, and reusable and enables machine learning methods. To facilitate data exchange, we recommend researchers publish experimental and simulated data on thermophysical properties as a CML-formatted Supporting Information with an associated digital object identifier (DOI).

Calculating free energies of binding (ΔGbind) between ligands and their target protein is of major interest to drug discovery and safety, yet it is still associated with several challenges and difficulties. Linear interaction energy (LIE) is an efficient in silico method for ΔGbindcomputation. LIE models can be trained and used to directly calculate binding affinities from interaction energies involving ligands in the bound and unbound states only, and LIE can be combined with statistical weighting to calculate ΔGbindfor flexible proteins that may bind their ligands in multiple orientations. Here, we investigate if LIE predictions can be effectively improved by explicitly including the entropy of (de)solvation into our free-energy calculations. For that purpose, we combine LIE calculations for the protein–ligand-bound state with explicit free-energy perturbation to rigorously compute the unbound ligand’s solvation free energy. We show that for 28 Cytochrome P450 2A6 (CYP2A6) ligands, coupling LIE with alchemical solvation free-energy calculation helps to improve obtained correlation between computed and reference (experimental) binding data.

The tail-to-head terpene cyclization is arguably one of the most complex reactions found in nature. The hydrogen-bond-based resorcinarene capsule represents the first man-made enzyme-like catalyst that is capable of catalyzing this reaction. Based on noncovalent interactions between the capsule and the substrate, the product selectivity can be tuned by using different leaving groups. A detailed mechanistic investigation was performed to elucidate the reaction mechanism. For the cyclization of geranyl acetate, it was found that the cleavage of the leaving group is the rate-determining step. Furthermore, the studies revealed that trace amounts of acid are required as cocatalyst. A series of control experiments demonstrate that a synergistic interplay between the supramolecular capsule and the acid traces is required for catalytic activity.

Aqueous solutions of Candida antarcticalipase B (CALB) were simulated considering three different water models (SPC/E, TIP3P, TIP4P) by a series of molecular dynamics (MD) simulations of three different box sizes (L = 9, 14, and 19 nm) to determine the diffusion coefficient, the water viscosity and the protein density. The protein–water systems were equilibrated for 500 ns, followed by 100 ns production runs which were analysed. The diffusional properties of CALB were characterized by the Stokes radius (RS), which was derived from the diffusion coefficient and the viscosity. RSwas compared to the geometric radius (RG) of CALB, which was derived from the protein density. RSand RGdiffered by 0.27 nm for SPC/E and by 0.40 and 0.39 nm for TIP3P and TIP4P, respectively, which characterizes the thickness of the diffusive hydration layer on the protein surface. The simulated hydration layer of CALB resulted in agreement with those experimentally determined for other seven different proteins of comparable size. By avoiding the most common pitfalls, protein diffusion can be reliably simulated: simulating different box sizes to account for the finite size effect, equilibrating the protein–water system sufficiently, and using the complete production run for the determination of the diffusion coefficient.

Esterases receive special attention because of 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 rank (classify) the 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 data sets.

ABSTRACTThe Lactamase Engineering Database (www.LacED.uni-stuttgart.de) was developed to facilitate the classification and analysis of TEM β-lactamases. The current version contains 474 TEM variants. Two hundred fifty-nine variants form a large scale-free network of highly connected point mutants. The network was divided into three subnetworks which were enriched by single phenotypes: one network with predominantly 2be and two networks with 2br phenotypes. Fifteen positions were found to be highly variable, contributing to the majority of the observed variants. Since it is expected that a considerable fraction of the theoretical sequence space is functional, the currently sequenced 474 variants represent only the tip of the iceberg of functional TEM β-lactamase variants which form a huge natural reservoir of highly interconnected variants. Almost 50% of the variants are part of a quartet. Thus, two single mutations that result in functional enzymes can be combined into a functional protein. Most of these quartets consist of the same phenotype, or the mutations are additive with respect to the phenotype. By predicting quartets from triplets, 3,916 unknown variants were constructed. Eighty-seven variants complement multiple quartets and therefore have a high probability of being functional. The construction of a TEM β-lactamase network and subsequent analyses by clustering and quartet prediction are valuable tools to gain new insights into the viable sequence space of TEM β-lactamases and to predict their phenotype. The highly connected sequence space of TEM β-lactamases is ideally suited to network analysis and demonstrates the strengths of network analysis over tree reconstruction methods.

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 antarcticalipase 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.

Using molecular dynamics (MD) simulations, the density of single proteins and its temperature dependence was modelled starting from the experimentally determined protein structure and a generic, transferable force field, without the need of prior parameterization. Although all proteins consist of the same 20 amino acids, their density in aqueous solution varies up to 10% and the thermal expansion coefficient up to twofold. To model the protein density, systematic MD simulations were carried out for 10 proteins with a broad range of densities (1.32–1.43 g/cm3) and molecular weights (7–97 kDa). The simulated densities deviated by less than 1.4% from their experimental values that were available for four proteins. Further analyses of protein density showed that it can be essentially described as a consequence of amino acid composition. For five proteins, the density was simulated at different temperatures. The simulated thermal expansion coefficients ranged between 4.3 and 7.1 × 10−4 K−1and were similar to the experimentally determined values of ribonuclease-A and lysozyme (deviations of 2.4 and 14.6%, respectively). Further analyses indicated that the thermal expansion coefficient is linked to the temperature dependence of atomic fluctuations: proteins with a high thermal expansion coefficient show a low increase in flexibility at increasing temperature. A low increase in atomic fluctuations with temperature has been previously described as a possible mechanism of thermostability. Thus, a high thermal expansion coefficient might contribute to protein thermostability.

ABSTRACTMetallo-β-lactamases (MBLs) are enzymes that hydrolyze β-lactam antibiotics, resulting in bacterial resistance to these drugs. These proteins have caused concerns due to their facile transference, broad substrate spectra, and the absence of clinically useful inhibitors. To facilitate the classification, nomenclature, and analysis of MBLs, an automated database system was developed, the Metallo-β-Lactamase Engineering Database (MBLED) (http://www.mbled.uni-stuttgart.de). It contains information on MBLs retrieved from the NCBI peptide database while strictly following the nomenclature by Jacoby and Bush (http://www.lahey.org/Studies/) and the generally accepted class B β-lactamase (BBL) standard numbering scheme for MBLs. The database comprises 597 MBL protein sequences and enables systematic analyses of these sequences. A systematic analysis employing the database resulted in the generation of mutation profiles of assigned IMP- and VIM-type MBLs, the identification of five MBL protein entries from the NCBI peptide database that were inconsistent with the Jacoby and Bush nomenclature, and the identification of 15 new IMP candidates and 9 new VIM candidates. Furthermore, the database was used to identify residues with high mutation frequencies and variability (mutation hot spots) that were unexpectedly distant from the active site located in the ββ sandwich: positions 208 and 266 in the IMP family and positions 215 and 258 in the VIM family. We expect that the MBLED will be a valuable tool for systematically cataloguing and analyzing the increasing number of MBLs being reported.

Different approaches were made to predict the adsorbed orientation based on rigid, flexible, or a mixture of both models. To determine the role of flexibility during adsorption, the orientation of lysozyme adsorbed to a negatively charged ligand surface was predicted by a rigid and a flexible model based on two differing protein structures at atomic resolution. For the rigid model, the protein structures were placed at different distances from the ligand surface and the electrostatic interaction energy was calculated for all possible orientations. The results were compared to a flexible model where the binding to the ligand surface was modeled by multiple molecular dynamics simulations starting with 14 initial orientations. Different aspects of the adsorption process were not covered by the rigid model and only detectable by the flexible model. Whereas the results of the rigid model depended sensitively on the protein-surface distance and the protein structure, the preferred orientation obtained by the flexible model was closer to a previous experimental determined orientation, robust toward the initial orientation and independent of the initial protein structure. Additionally, it was possible to obtain insights into the preferred binding process of lysozyme on a negatively charged surface by the flexible model.

The Ω-loop of TEM β-lactamase is involved in substrate recognition and catalysis. Its dynamical properties and interaction with water molecules were investigated by performing multiple molecular dynamics simulations of up to 50 ns. Protein flexibility was assessed by calculating the root mean-square fluctuations and the generalized order parameter, S2. The residues in secondary structure elements are highly ordered, whereas loop regions are more flexible, which is in agreement with previous experimental observations. Interestingly, the Ω-loop (residues 161–179) is rigid with order parameters similar to secondary structure elements, with the exception of the tip of the loop (residues 173–177) that has a considerably higher flexibility and performs an opening and closing motion on the 50-ns timescale. The rigidity of the main part of the Ω-loop is mediated by stabilizing and highly conserved water bridges inside a cavity lined by the Ω-loop and residues 65–69 of the protein core. In contrast, the flexible tip of the Ω-loop lacks these interactions. Hydration of the cavity and exchange of the water molecules with the bulk solvent occurs via two pathways: the flexible tip that serves as a door to the cavity, and a temporary water channel involving the side chain of Arg164.

In order to make thermophysical properties of complex liquid mixtures available to a comprehensive analysis, we developed a data management and analysis platform based on the standard data exchange format ThermoML. The practicability of integrating thermophysical data from experiments and simulations was demonstrated for two binary mixtures, methanol–water and glycerol–water, by systematically studying the dependence of densities and diffusion coefficients from water content over the whole composition range and temperatures between 278.15 and 318.15 K. Experimental data were extracted manually from the literature. The same parameter space was explored by comprehensive molecular dynamics simulations, whose results were directly transferred to the analysis platform. The benefit of data integration was illustrated by assessing the transferability of the force fields, which had been developed for pure compounds to different compositions and temperatures, and by analyzing the excess mixing properties as a measure of nonideality of methanol–water and glycerol–water mixtures. The core of the data management and analysis platform is the newly developed Python library pyThermoML, which represents metadata, the parameters, and the experimentally determined or simulated properties as Python data classes. The feasibility of a seamless data flow from data acquisition to a comprehensive data analysis was demonstrated. PyThermoML enables interoperability and reusability of the datasets. The publication of ThermoML documents on the Dataverse installation of the University of Stuttgart (DaRUS) makes thermophysical data findable and accessible and thus FAIR.

ABSTRACTA set of 49 high-resolution (≤2.2 Å) structures of the TEM, SHV, and CTX-M class A β-lactamase families was systematically analyzed to investigate the role of conserved water molecules in the stabilization of the Ω-loop. Overall, 13 water molecules were found to be conserved in at least 45 structures, including two water positions which were found to be conserved in all structures. Of the 13 conserved water molecules, 6 are located at the Ω-loop, forming a dense cluster with hydrogen bonds to residues at the Ω-loop as well as to the rest of the protein. This layer of conserved water molecules is packed between the Ω-loop and the rest of the protein and acts as structural glue, which could reduce the flexibility of the Ω-loop. A correlation between conserved water molecules and conserved protein residues could in general not be detected, with the exception of the conserved water molecules at the Ω-loop. Furthermore, the evolutionary relationship between the three families, derived from the number of conserved water molecules, is similar to the relationship derived from phylogenetic analysis.

A new variant allele CYP2D6*62 (g.4044C>T; R441C) of the drug-metabolizing cytochrome P450 (P450) CYP2D6 was identified in a person with reduced sparteine oxidation phenotype, which was unexpected based on a genetic CYP2D6*1A/*41 background. The recombinantly expressed variant protein had no activity toward propafenone as a result of missing heme incorporation. Sequence alignment revealed that the positively charged R441 residue is part of the heme-binding signature but not strictly conserved among all the P450s. A compilation of described P450 monooxygenase variants revealed that other enzymes can functionally tolerate even nonconservative amino acid changes at the corresponding position (i.e., the invariant cysteine 2). This suggests that heme binding in mammalian P450s depends not only on the integrity of the heme-binding signature but also on other family- and subfamily-specific sequence determinants.

Gerichtete Evolution und Metabolic Engineering sind die Werkzeuge, mit denen sich Mikroorganismen und Enzyme für die Synthese von Spezialchemikalien und Zwischenprodukten optimieren lassen. Mehr und mehr biokatalytische Umsetzungen finden den Weg aus dem Labor in die Pilotanlage. Bei der Selektivoxidation mit Sauerstoff erwächst der heterogenen und homogenen Katalyse in der Biokatalyse eine immer stärkere Konkurrenz. Gerichtete Evolution und Metabolic Engineering sind die Werkzeuge, mit denen sich Mikroorganismen und Enzyme für die Synthese von Spezialchemikalien und Zwischenprodukten optimieren lassen. Mehr und mehr biokatalytische Umsetzungen finden den Weg aus dem Labor in die Pilotanlage. Bei der Selektivoxidation mit Sauerstoff erwächst der heterogenen und homogenen Katalyse in der Biokatalyse eine immer stärkere Konkurrenz.

Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc‐containing IMP‐1 metallo‐β‐lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal‐containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc–zinc distance increased by ∼1 Å in the substrate complex compared with the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum‐mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc‐containing metallo‐β‐lactamases as free proteins, in complex with inhibitors and in complex with substrates.

Abstract. Human cytochrome P450 (CYP) 2B6 activates the anticancer prodrug cyclophosphamide (CPA) by 4-hydroxylation. In contrast, the same enzyme catalyzes N-deethylation of a structural isomer, the prodrug ifosfamide (IFA), thus causing severe adverse drug effects. To model the molecular interactions leading to a switch in regioselectivity, the structure of CYP2B6 was modeled based on the structure of rabbit CYP2C5. We modeled the missing 22-residue loop in CYP2C5 between helices F and G (the F-G loop), which is not resolved in the X-ray structure, by molecular dynamics (MD) simulations using a simulated annealing protocol. The modeled conformation of the loop was validated by unconstrained MD simulations of the complete enzymes (CYP2C5 and CYP2B6) in water for 70 and 120 ps, respectively. The simulations were stable and led to a backbone r.m.s. deviation of 1.7 Å between the two CYPs. The shape of the substrate binding site of CYP2B6 was further analyzed. It consists of three well-defined hydrophobic binding pockets adjacent to the catalytic heme. Size, shape and hydrophobicity of these pockets were compared to the shapes of the two structurally isomeric substrates. In their preferred orientation in the binding site, both substrates fill all three binding pockets without repulsive interactions. The distance to the heme iron is short enough for 4-hydroxylation and N-deethylation to occur for CPA and IFA, respectively. However, if the substrates are docked in the non-preferred orientation (such that 4-hydroxylation and N-deethylation would occur for IFA and CPA, respectively), one pocket is left empty, and clashes were observed between the substrates and the enzyme. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00894-002-0104-y.

No Abstract

No 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.

A three-dimensional model of the variable domain of the atrazine-specific Fab fragment K411B was constructed by molecular modeling using known structures of highly homologous immunoglobulins as templates. Molecular dynamic simulations and cross-reactivity data were used to predict residues responsible for the binding of the hapten 4-chloro-6-(isopropylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (iPr/Cl/C6) instead of atrazine. Specific binding pockets could be defined for the chlorine, the isopropylamino group and the C6-spacer of the hapten. The influence of various amino acids on hapten binding was investigated by site-directed mutagenesis, and the effect of these mutations was analyzed by capture ELISA using the hapten iPr/Cl/C6 and 4-amino-6-chloro-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (H/Cl/C6). GlyH100a seems to be important in determining the conformation of the heavy-chain complementarity determining region H3; replacing it with any other residue prevented the binding of the hapten. Altering residues responsible for the binding of the chlorine atom (TrpH33, GluH50 and TyrL96) decreased the affinity significantly. Hapten-spacer recognition can be attributed to the interaction with PheL32; replacing PheL32 by leucine reduced the affinity towards iPr/Cl/C6. A triple mutant Fab fragment (GlnL89Glu, ValH37Ile and GluL3Val) showed an affinity 5-fold greater towards iPr/Cl/C6 compared to the wild-type K411B, as a result of better recognition of the isopropylamino group of iPr/Cl/C6.

Abstract. Lipases are widely used catalysts for highly enantioselective resolution of chiral secondary alcohols. While stereopreference is determined predominantly by the substrate structure, stereoselectivity (enantioselectivity and diastereoselectivity) depends on the atomic details of interactions between substrate and lipase. Experimentally obtained stereoselectivity and activity in the hydrolysis of butanoic acid esters of two secondary alcohols with two neighboring stereocenters by Candida rugosa lipase have been investigated by computer-aided molecular modeling of tetrahedral substrate intermediates in complex with the lipase. Breakdown of these intermediates is considered to be the rate-limiting step. Steric interactions of stereoisomers with the side chain of catalytic histidine led to different orientations of the imidazole. The distance d(H

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megateriumwas engineered by rational evolutionto achieve improved hydroxylation activity for medium chain length substrates (C8–C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on ω-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards ω-p-nitrophenoxydecanoic acid (10-pNCA) and ω-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

Lipases exhibit specific catalytic properties that make them attractive to biotechnological applications. Most important are the broad substrate specificity and the regio‐ and stereoselectivity of lipases. Despite mechanistic and structural similarities lipases differ significantly with respect to stereoselectivity toward natural and synthetic substrates. Models developed to describe and predict stereoselectivity toward certain types of synthetic substrates, e. g., secondary alcohols cannot be applied to natural acylglycerols, that are hydrolyzed by several animal and microbial lipases in a regioselective or stereoselective manner. Therefore, computer‐aided molecular modeling studies were used in order to predict the stereopreference of lipases toward triradylglycerols. Lipase variants with modified stereoselectivity properties toward triacylglycerols were engineered by re‐designing the recombinant enzyme. To understand the interactions governing lipase stereoselectivity towards natural substrates, knowledge of the structure of enzyme‐substrate complexes at the atomic level is essential. Such information can be obtained by X‐ray or NMR analysis of covalent enzyme‐inhibitor complexes. The crystal structures of enzymes complexed with triacylglycerol analog inhibitors allowed the identification of distinct binding sites for the three hydrophobic chains of the inhibitor.

Evolutionary relationships of protein families can be characterized either by networks or by trees. Whereas trees allow for hierarchical grouping and reconstruction of the most likely ancestral sequences, networks lack a time axis but allow for thresholds of pairwise sequence identity to be chosen and, therefore, the clustering of family members with presumably more similar functions. Here, we use the large family of arylsulfatases and phosphonate monoester hydrolases to investigate similarities, strengths and weaknesses in tree and network representations. For varying thresholds of pairwise sequence identity, values of betweenness centrality and clustering coefficients were derived for nodes of the reconstructed ancestors to measure the propensity to act as a bridge in a network. Based on these properties, ancestral protein sequences emerge as bridges in protein sequence networks. Interestingly, many ancestral protein sequences appear close to extant sequences. Therefore, reconstructed ancestor sequences might also be interpreted as yet-to-be-identified homologues. The concept of ancestor reconstruction is compared to consensus sequences, too. It was found that hub sequences in a network, e.g. reconstructed ancestral sequences that are connected to many neighbouring sequences, share closer similarity with derived consensus sequences. Therefore, some reconstructed ancestor sequences can also be interpreted as consensus sequences.

We created a toolbox of recombinant, microbial lipases, which allows us in combination with a lipase database to choose among the overexpressed lipases the most appropriate for a specific application and to improve it further via mutagenesis. By systematic comparison of geometry and properties of the scissile fatty acid binding site of five representative lipases of each family of structurally homologous lipases, three subgroups can be defined. Hence, efficient expression systems for the functional production of large amounts of microbial lipases, representing different lipase subgroups, were developed. In particular, recombinant lipases from Bacillus thermocatenulatusand Pseudomonas cepaciawere functionally overexpressed in E. coli. The lipase genes from Geotrichum candidumCMICC 335426 and Rhizopus oryzaewere overexpressed in Pichia pastoris. Due to an unusual codon usage that prevents heterologous expression, the LIP1gene (1647 nt) of Candida rugosawas completely synthesized and overexpressed in Pichia pastoris.

The synthesis of n-butanol and cinnamic alcohol esters of glucuronic acid and the esterification of ascorbic acid (vitamin C) with phenylbutyric acid was performed with lipase from Candida antarctica B (CAL-B, SP435) in a mainly solid-phase system. Products were obtained in 15 to 22 % yield. Computer modelling based on the structure of CAL-B was used to elucidate the access of glucuronic acid to the catalytic site of the lipase. A fixation of glucuronic acid via H-bonds to Q157, D134 and H224 during the transition state was observed. © Rapid Science Ltd. 1998

No Abstract

No Abstract

Summary: 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