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A modular research data management toolbox based on the programming language Python, the widely used computing platform Jupyter Notebook, the standardized data exchange format for analytical data (AnIML) and the generic repository Dataverse has been established and applied to analyze small‐angle X‐ray scattering (SAXS) data according to the FAIR data principles (findable, accessible, interoperable and reusable). The SAS‐tools library is a community‐driven effort to develop tools for data acquisition, analysis, visualization and publishing of SAXS data. Metadata from the experiment and the results of data analysis are stored as an AnIML document using the novel Python‐native pyAnIML API. The AnIML document, measured raw data and plots resulting from the analysis are combined into an archive in OMEX format and uploaded to Dataverse using the novel easyDataverse API, which makes each data set accessible via a unique DOI and searchable via a structured metadata block. SAS‐tools is applied to study the effects of alkyl chain length and counterions on the phase diagrams of alkyltrimethylammonium surfactants in order to demonstrate the feasibility and usefulness of a scalable data management workflow for experiments in physical chemistry. [ABSTRACT FROM AUTHOR]

EnzymeML is an XML‐based data exchange format that supports the comprehensive documentation of enzymatic data by describing reaction conditions, time courses of substrate and product concentrations, the kinetic model, and the estimated kinetic constants. EnzymeML is based on the Systems Biology Markup Language, which was extended by implementing the STRENDA Guidelines. An EnzymeML document serves as a container to transfer data between experimental platforms, modeling tools, and databases. EnzymeML supports the scientific community by introducing a standardized data exchange format to make enzymatic data findable, accessible, interoperable, and reusable according to the FAIR data principles. An application programming interface in Python supports the integration of software tools for data acquisition, data analysis, and publication. The feasibility of a seamless data flow using EnzymeML is demonstrated by creating an EnzymeML document from a structured spreadsheet or from a STRENDA DB database entry, by kinetic modeling using the modeling platform COPASI, and by uploading to the enzymatic reaction kinetics database SABIO‐RK. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)‐selective IRED from Myxococcus stipitatus (NADH‐IRED‐Ms) yielding a NADH‐dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)‐selectivity in asymmetric reductions has yet been reported. Herein, we applied semi‐rational enzyme engineering to switch the selectivity of NADH‐IRED‐Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2‐methylpyrroline compared to the wild‐type and afforded the (S)‐amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal‐structures of the NADPH‐dependent (R)‐IRED‐Ms wild‐type enzyme and the NADH‐dependent NADH‐IRED‐Ms variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant. [ABSTRACT FROM AUTHOR]

When studying protein‐ligand interactions, many different factors can influence the behaviour of the protein as well as the ligands. Molecular visualisation tools typically concentrate on the movement of single ligand molecules; however, viewing only one molecule can merely provide a hint of the overall behaviour of the system. To tackle this issue, we do not focus on the visualisation of the local actions of individual ligand molecules but on the influence of a protein and their overall movement. Since the simulations required to study these problems can have millions of time steps, our presented system decouples visualisation and data preprocessing: our preprocessing pipeline aggregates the movement of ligand molecules relative to a receptor protein. For data analysis, we present a web‐based visualisation application that combines multiple linked 2D and 3D views that display the previously calculated data The central view, a novel enhanced sequence diagram that shows the calculated values, is linked to a traditional surface visualisation of the protein. This results in an interactive visualisation that is independent of the size of the underlying data, since the memory footprint of the aggregated data for visualisation is constant and very low, even if the raw input consisted of several terabytes. [ABSTRACT FROM AUTHOR]

Expansins have the remarkable ability to loosen plant cell walls and cellulose material without showing catalytic activity and therefore have potential applications in biomass degradation. To support the study of sequence‐structure‐function relationships and the search for novel expansins, the Expansin Engineering Database (ExED, https://exed.biocatnet.de) collected sequence and structure data on expansins from Bacteria, Fungi, and Viridiplantae, and expansin‐like homologues such as carbohydrate binding modules, glycoside hydrolases, loosenins, swollenins, cerato‐platanins, and EXPNs. Based on global sequence alignment and protein sequence network analysis, the sequences are highly diverse. However, many similarities were found between the expansin domains. Newly created profile hidden Markov models of the two expansin domains enable standard numbering schemes, comprehensive conservation analyses, and genome annotation. Conserved key amino acids in the expansin domains were identified, a refined classification of expansins and carbohydrate binding modules was proposed, and new sequence motifs facilitate the search of novel candidate genes and the engineering of expansins. [ABSTRACT FROM AUTHOR]

Multicopper oxidases (MCOs) use copper ions as cofactors to oxidize a variety of substrates while reducing oxygen to water. MCOs have been identified in various taxa, with notable occurrences in fungi. The role of these fungal MCOs in lignin degradation sparked an interest due to their potential for application in biofuel production and various other industries. MCOs consist of different protein domains, which led to their classification into two‐, three‐, and six‐domain MCOs. The previously established Laccase and Multicopper Oxidase Engineering Database (https://lcced.biocatnet.de) was updated and now includes 51 058 sequences and 229 structures of MCOs. Sequences and structures of all MCOs were systematically compared. All MCOs consist of cupredoxin‐like domains. Two‐domain MCOs are formed by the N‐ and C‐terminal domain (domain N and C), while three‐domain MCOs have an additional domain (M) in between, homologous to domain C. The six‐domain MCOs consist of alternating domains N and C, each three times. Two standard numbering schemes were developed for the copper‐binding domains N and C, which facilitated the identification of conserved positions and a comparison to previously reported results from mutagenesis studies. Two sequence motifs for the copper binding sites were identified per domain. Their modularity, depending on the placement of the T1‐copper binding site, was demonstrated. Protein sequence networks showed relationships between two‐ and three‐domain MCOs, allowing for family‐specific annotation and inference of evolutionary relationships. [ABSTRACT FROM AUTHOR]

We present a workflow for kinetic modeling of biocatalytic reactions which combines methods from Bayesian learning and uncertainty quantification for model calibration, model selection, evaluation, and model reduction in a consistent statistical framework. Our workflow is particularly tailored to sparse data settings in which a considerable variability of the parameters remains after the models have been adapted to available data, a ubiquitous problem in many real-world applications. Our workflow is exemplified on an enzyme-catalyzed two-substrate reaction mechanism describing the symmetric carboligation of 3,5-dimethoxy-benzaldehyde to (R)-3,3',5,5' - tetramethoxybenzoin catalyzed by benzaldehyde lyase from Pseudomonas fluorescens. Results indicate a substrate-dependent inactivation of enzyme, which is in accordance with other recent studies. [ABSTRACT FROM AUTHOR]

The α/β‐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 α/β‐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 α/β‐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 β‐propeller fold. The C‐terminal domains show a β‐sandwich fold. The N‐terminal β‐propeller domain and the C‐terminal β‐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 α/β‐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 γ = 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 τ = 2.6, which strongly supports the connectedness of the sequence space of α/β‐hydrolases. Database : Supporting data about domains from other proteins with structural similarity to the N‐ or C‐terminal domains of α/β‐hydrolases are available in Data Repository of the University of Stuttgart (DaRUS) under doi: https://doi.org/10.18419/darus-458. [ABSTRACT FROM AUTHOR]

Thiamine diphosphate‐dependent decarboxylases catalyze both cleavage and formation of CC bonds in various reactions, which have been assigned to different homologous sequence families. This work compares 53 ThDP‐dependent decarboxylases with known crystal structures. Both sequence and structural information were analyzed synergistically and data were analyzed for global and local properties by means of statistical approaches (principle component analysis and principal coordinate analysis) enabling complexity reduction. The different results obtained both locally and globally, that is, individual positions compared with the overall protein sequence or structure, revealed challenges in the assignment of separated homologous families. The methods applied herein support the comparison of enzyme families and the identification of functionally relevant positions. The findings for the family of ThDP‐dependent decarboxylases underline that global sequence identity alone is not sufficient to distinguish enzyme function. Instead, local sequence similarity, defined by comparisons of structurally equivalent positions, allows for a better navigation within several groups of homologous enzymes. The differentiation between homologous sequences is further enhanced by taking structural information into account, such as BioGPS analysis of the active site properties or pairwise structural superimpositions. The methods applied herein are expected to be transferrable to other enzyme families, to facilitate family assignments for homologous protein sequences. [ABSTRACT FROM AUTHOR]

The Short‐chain Dehydrogenases/Reductases Engineering Database (SDRED) covers one of the largest known protein families (168 150 proteins). Assignment to the superfamilies of Classical and Extended SDRs was achieved by global sequence similarity and by identification of family‐specific sequence motifs. Two standard numbering schemes were established for Classical and Extended SDRs that allow for the determination of conserved amino acid residues, such as cofactor specificity determining positions or superfamily specific sequence motifs. The comprehensive sequence dataset of the SDRED facilitates the refinement of family‐specific sequence motifs. The glycine‐rich motifs for Classical and Extended SDRs were refined to improve the precision of superfamily classification. In each superfamily, the majority of sequences formed a tightly connected sequence network and belonged to a large homologous family. Despite their different sequence motifs and their different sequence length, the two sequence networks of Classical and Extended SDRs are not separate, but connected by edges at a threshold of 40% sequence similarity, indicating that all SDRs belong to a large, connected network. The SDRED is accessible at https://sdred.biocatnet.de/. [ABSTRACT FROM AUTHOR]

The direct enantioselective addition of water to unactivated alkenes could simplify the synthesis of chiral alcohols and solve a long‐standing challenge in catalysis. Here we report that an engineered fatty acid hydratase can catalyze the asymmetric hydration of various terminal and internal alkenes. In the presence of a carboxylic acid decoy molecule for activation of the oleate hydratase from E. meningoseptica, asymmetric hydration of unactivated alkenes was achieved with up to 93 % conversion, excellent selectivity (>99 % ee, >95 % regioselectivity), and on a preparative scale. Engineered for success: Asymmetric hydration of unactivated, aliphatic alkenes was achieved with a fatty acid hydratase generating chiral alcohols with high conversion, excellent regio‐ and enantioselectivity, and on a preparative scale. Key for high activity is a carboxylic acid decoy molecule that activates the wildtype and engineered enzymes for alkene hydration on alternative substrates. [ABSTRACT FROM AUTHOR]

Abstract: A structural model for thiamine‐diphosphate (ThDP)‐dependent transketolase (TK) was developed to analyse the effect of amino acid exchanges on the stereoselectivity of this synthetically important class of enzymes. In this study the carboligation of 3‐hydroxypyruvate as a donor and propanal, as well as pentanal, was studied. Based on literature data and additional mutagenesis studies using E. coli TK, a four‐state model was developed to explain the stereoselectivity of TKs by the relative orientation of donor and acceptor substrates in the active site prior to C−C‐bond formation. To enable a functional comparison of relevant amino acids of TKs from different species, a standard numbering scheme was developed. Using this concept, H26, H261, and F434 were identified as the key residues which mediate stereoselectivity, where two main factors influenced the arrangement of ThDP‐bound donor and acceptor prior to carboligation: the relative orientation of the substrate side chains and the orientation of the acceptor carbonyl group towards the donor hydroxy group. This model provides a first framework to understand the structure‐function relationships of TKs with respect to their stereoselectivity. [ABSTRACT FROM AUTHOR]

Abstract: The ω‐Transaminase Engineering Database (oTAED) was established as a publicly accessible resource on sequences and structures of the biotechnologically relevant ω‐transaminases (ω‐TAs) from Fold types I and IV. The oTAED integrates sequence and structure data, provides a classification based on fold type and sequence similarity, and applies a standard numbering scheme to identify equivalent positions in homologous proteins. The oTAED includes 67 210 proteins (114 655 sequences) which are divided into 169 homologous families based on global sequence similarity. The 44 and 39 highly conserved positions which were identified in Fold type I and IV, respectively, include the known catalytic residues and a large fraction of glycines and prolines in loop regions, which might have a role in protein folding and stability. However, for most of the conserved positions the function is still unknown. Literature information on positions that mediate substrate specificity and stereoselectivity was systematically examined. The standard numbering schemes revealed that many positions which have been described in different enzymes are structurally equivalent. For some positions, multiple functional roles have been suggested based on experimental data in different enzymes. The proposed standard numbering schemes for Fold type I and IV ω‐TAs assist with analysis of literature data, facilitate annotation of ω‐TAs, support prediction of promising mutation sites, and enable navigation in ω‐TA sequence space. Thus, it is a useful tool for enzyme engineering and the selection of novel ω‐TA candidates with desired biochemical properties. [ABSTRACT FROM AUTHOR]

ABSTRACT Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database () was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED- Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx5[ATS]x4Gx4[VIL]WNR[TS]x2[KR] and the active site motif Gx[DE]x[GDA]x[APS]x3{K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes. Proteins 2016; 84:600-610. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Three different reductases have been fused to CYP153 monooxygenase from Marinobacter aquaeolei. The most promising candidate has been analysed in terms of its linker part, which connects the reductase with the haem domain through sequence alignment of the corresponding reductase family CYP116B. To improve the artificial fusion construct, the linker length has been varied, thereby only altering the non-conserved middle part of the linker. This way seven artificial fusion constructs have been engineered, which varied in linker length between 11 and 32 amino acids ('natural' is 16). These variations showed a substantial impact on the fusion construct. The best mutant, extended by two amino acids, showed an improved activity (67 %), higher stability (67 % more active haem domain after 2 h) and a coupling efficiency of 94 % (55 % higher than before). Presented in this paper is an approach to find and optimise artificial fusion constructs for P450 monooxygenases. [ABSTRACT FROM AUTHOR]

ABSTRACT Cytochrome P450 monooxygenases (CYPs) are a large, highly diverse protein family with a common fold. The sequences, structures, and functions of CYPs have been extensively studied resulting in more than 53,000 scientific articles. A sequence-based literature mining algorithm was designed to systematically analyze this wealth of information on SNPs, designed mutations, structural interactions, or functional roles of individual residues. Structurally corresponding positions in different CYPs were compared and universal selectivity-determining positions were identified. Based on the Cytochrome P450 Engineering Database ( ) and a standard numbering scheme for all CYPs, 4000 residues in 168 CYPs mentioned in 2400 articles could be assigned to 440 structurally corresponding standard positions of the CYP fold, covering 96% of all standard positions. Seventeen individual standard positions were mentioned in the context of more than 32 different CYPs. The majority of these most frequently mentioned positions are located on the six substrate recognition sites and are involved in control of selectivity, such as the well-studied position 87 in CYP102A1 (P450BM-3) which was mentioned in the articles on 63 different CYPs. The recurrent citation of the 17 frequently mentioned positions for different CYPs suggests their universal functional relevance. Proteins 2015; 83:1593-1603. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Thiamine diphosphate (ThDP)-dependent enzymes form a diverse protein family which was classified into nine superfamilies. The cofactor ThDP is bound at the interface between two catalytic domains, the PYR and the PP domain. The nine superfamilies were assigned to five different structural architectures. Two superfamilies, the sulfopyruvate decarboxylases and α-ketoacid dehydrogenases 2, consist of separate PYR and PP domains. The oxidoreductase superfamily is of the intra-monomer/PYR-PP type with an N-terminal PYR and a subsequent PP domain. The active enzymes form homodimers with the ThDP cofactor bound at the interface between a PYR and a PP domain of the same monomer. Decarboxylases are of the inter-monomer/PYR-PP type with the cofactor bound between domains from different monomers. 1-Deoxy- d-xylulose-5-phosphate synthases are of the intra-monomer/PP-PYR type. The transketolases, α-ketoglutarate dehydrogenases, and α-ketoacid dehydrogenases 1 are of the inter-monomer/PP-PYR type. For the phosphonopyruvate decarboxylases, definitive assessment of the structural architecture is not possible due to lack of structure information. By applying a structure-based domain alignment method, sequences of more than 62,000 PYR and PP domains were identified and aligned. Although the sequence similarity of the catalytic domains is low between different superfamilies, seven positions were identified to be highly conserved, including the cofactor binding GDGX24,27N motif, the cofactor-activating glutamic acid, and two structurally equivalent glycines in both the PYR and the PP domain. An evolutionary pathway of ThDP-dependent enzymes is proposed which explains the sequence and structure diversity of this family by three basic evolutionary events: domain recruitment, domain linkage, and structural rearrangement of catalytic domains. Proteins 2014; 82:2523-2537. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to ( S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various ( S)-benzoins with excellent enantiomeric excess (>99 %) and very good conversion. [ABSTRACT FROM AUTHOR]

A general strategy is described that integrates data mining of enzyme families and molecular modeling of enzyme-substrate interactions to identify selectivity hotspots and to design focused variant libraries for changed regio- and stereoselectivity. This strategy is demonstrated for two case studies; the design of cytochrome P450 monooxygenases with improved regioselectivity and of thiamine diphosphate dependent enzymes with improved stereoselectivity. In both families, two selectivity hotspots are found in almost all sequences, and simple, generic rules are established to predict the effect of mutations at these positions on selectivity. The crucial role of the hotspot positions is validated for an increasing number of enzymes by designing variants with improved or switched selectivity. [ABSTRACT FROM AUTHOR]

The thiamine diphosphate-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) catalyzes a Stetter-like 1,4-addition of α-ketoglutarate to isochorismate in the biosynthesis of menaquinone (vitamin K). Here, we describe the carboligation potential of MenD from Bacillus subtilis ( BsMenD) for the nonphysiological 1,2-addition of decarboxylated α-ketoglutarate (succinylsemialdehyde) and various benzaldehyde derivatives. Furthermore, we engineer BsMenD variants for the enantiocomplementary asymmetric synthesis of functionalized α-hydroxy ketones. Wild type BsMenD shows an excellent chemo- as well as high ( R)-selectivity for the carboligation of α-ketoglutarate as the donor, and different benzaldehyde derivatives as acceptor yielding ( R)-α-hydroxy ketones with up to >99 % ee. By engineering ( S)-selective BsMenD variants, based on the recently developed S-pocket concept, we provide access to most of the corresponding ( S)-α-hydroxy ketones with up to 98 % ee. In particular, benzaldehyde and meta-substituted derivatives were converted with high enantioselectivities ( ee of 91-98 % ( S)). The significantly higher ( S)-selectivity of BsMenD variants than recently published MenD variants from Escherichia coli, could be attributed to a second-shell residue next to the S-pocket. A glycine residue, adjacent to the major S-pocket residues I476 and F477 (standard numbering), is assumed to result in higher structural flexibility in the S-pocket region of BsMenD, which in turn could result in improved stabilization of the antiparallel orientation of the acceptor. [ABSTRACT FROM AUTHOR]

ABSTRACT Cytochrome P450 monooxygenases (CYPs) constitute a ubiquitous, highly divergent protein family. Nevertheless, all CYPs share a common fold and conserved catalytic machinery. Based on the electron donor system, 10 classes of CYPs have been described, but most CYPs are members of class I accepting electrons from ferredoxin which is being reduced by FAD-containing reductase, or class II accepting electrons from FAD- and FMN-containing CPR-type reductase. Because of the low sequence conservation inside the two classes, the conserved class-specific positions are expected to be involved in aspects of electron transfer that are specific to the two types of reductases. In this work we present results from a conservation analysis of 16,732 CYP sequences derived from an updated version of the Cytochrome P450 Engineering Database (CYPED), using two class-specific numbering schemes. While no position was conserved on the distal, substrate-binding surface of the CYPs, several class-specific residues were found on the proximal, reductase-interacting surface; two class I-specific residues that were negatively charged, and three class II-specific residues that were aromatic or charged. The class-specific conservation of glycine and proline residues in the cysteine pocket indicates that there are class-specific differences in the flexibility of this element. Four heme-interacting arginines were conserved differently in each class, and a class-specific substitution of a heme-interacting tyrosine by histidine was found, pointing to a link between heme stabilization and the reductase type. Proteins 2014; 82:491-504. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Thiamine diphosphate-dependent enzymes are broadly distributed in all organisms, and they catalyse a broad range of C-C bond forming and breaking reactions. Enzymes belonging to the structural families of decarboxylases and transketolases have been particularly well investigated concerning their substrate range, mechanism of stereoselective carboligation and carbolyase reaction. Both structurally different enzyme families differ also in stereoselectivity: enzymes from the decarboxylase family are predominantly R-selective, whereas those from the transketolase family are S-selective. In recent years a key focus of our studies has been on stereoselective benzoin condensation-like 1,2-additions. Meanwhile, several S-selective variants of pyruvate decarboxylase, benzoylformate decarboxylase and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate ( SEPHCHC) synthase as well as R-selective transketolase variants were created that allow access to a broad range of enantiocomplementary α-hydroxyketones and α,α′-dihydroxyketones. This review covers recent studies and the mechanistic understanding of stereoselective C-C bond forming thiamine diphosphate-dependent enzymes, which has been guided by structure-function analyses based on mutagenesis studies and from influences of different substrates and organic co-solvents on stereoselectivity. [ABSTRACT FROM AUTHOR]

The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase from Escherichia coli ( EcMenD, E.C. 2.2.1.9) catalyzes the carboligation of α-ketoglutarate (α-KG) and various benzaldehyde derivatives with excellent chemo- as well as high R-selectivity (enantiomeric excess ( ee) >93 %) to yield chiral α-hydroxy ketones. Based on the recently developed S-pocket concept, we engineered S-selective EcMenD variants by optimizing the steric properties and stabilization of the acceptor substrate in the S-pocket. Moreover, the moderate S-selectivity of the EcMenD variant I474A/F475G described recently for the carboligation of α-KG and benzaldehyde ( ee=75 %) could be improved by selective destabilization of the R-pathway, which resulted in the variant I474A/F475G/R395Y ( ee=85 % S). Subsequent investigation of the acceptor substrate range of this new variant revealed high S-selectivity especially with meta-substituted benzaldehydes, which gave access to 5-hydroxy-4-oxo-5-arylpentanoates with excellent enantioselectivities of up to 99 % ee S. Thus, opening the S-pocket and simultaneous destabilization of the R-pathway provides a potential general new strategy to enhance the S-selectivity of ThDP-dependent enzymes. [ABSTRACT FROM AUTHOR]

Although catalytic reductions, cross-couplings, metathesis, and oxidation of CC double bonds are well established, the corresponding catalytic hydroxylations of CH bonds in alkanes, arenes, or benzylic (allylic) positions, particularly with O2, the cheapest, 'greenest', and most abundant oxidant, are severely lacking. Certainly, some promising examples in homogenous and heterogenous catalysis exist, as well as enzymes that can perform catalytic aerobic oxidations on various substrates, but these have never achieved an industrial-scale, owing to a low space-time-yield and poor stability. This review illustrates recent advances in aerobic oxidation catalysis by discussing selected examples, and aims to stimulate further exciting work in this area. Theoretical work on catalyst precursors, resting states, and elementary steps, as well as model reactions complemented by spectroscopic studies provide detailed insight into the molecular mechanisms of oxidation catalyses and pave the way for preparative applications. However, O2 also poses a safety hazard, especially when used for large scale reactions, therefore sophisticated methodologies have been developed to minimize these risks and to allow convenient transfer onto industrial scale. [ABSTRACT FROM AUTHOR]

Triterpene cyclases catalyze a broad range of cyclization reactions to form polycyclic triterpenes. Triterpene cyclases that convert squalene to hopene are named squalene-hopene cyclases (SHC) and triterpene cyclases that convert oxidosqualene are named oxidosqualene cyclases (OSC). Many sequences have been published, but there is only one structure available for each of SHCs and OSCs. Although they catalyze a similar reaction, the sequence similarity between SHCs and OSCs is low. A family classification based on phylogenetic analysis revealed 20 homologous families which are grouped into two superfamilies, SHCs and OSCs. Based on this family assignment, the Triterpene Cyclase Engineering Database (TTCED) was established. It integrates available information on sequence and structure of 639 triterpene cyclases as well as on structurally and functionally relevant amino acids. Family specific multiple sequence alignments were generated to identify the functionally relevant residues. Based on sequence alignments, conserved residues in SHCs and OSCs were analyzed and compared to experimentally confirmed mutational data. Functional schematic models of the central cavities of OSCs and SHCs were derived from structure comparison and sequence conservation analysis. These models demonstrate the high similarity of the substrate binding cavity of SHCs and OSCs and the equivalences of the respective residues. The TTCED is a novel source for comprehensive information on the triterpene cyclase family, including a compilation of previously described mutational data. The schematic models present the conservation analysis in a readily available fashion and facilitate the correlation of residues to a specific function or substrate interaction. Proteins 2012; © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Dirigent proteins impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of coniferyl alcohol. By an unknown mechanism, they direct the coupling of two phenoxy radicals toward the formation of optically active (+)- or (−)-pinoresinol. We show here that the dirigent protein AtDIR6 from Arabidopsis thaliana is a homodimeric all-beta protein in the superfamily of calycins. Based on its homology with calycins, the structure of AtDIR6 was modeled using allene oxide cyclase as template. The structural model of AtDIR6 was supported experimentally by confirmation of a predicted disulfide bridge and by the characterization of two N-linked glycans at the solvent-exposed protein surface. The model shows AtDIR6 as an eight-stranded antiparallel β-barrel with a central hydrophobic cavity for substrate binding, suggesting that dirigent proteins evolved from hydrophobic ligand-binding proteins. The data are fully consistent with the current view of the dirigent protein mode of action, according to which each subunit of the homodimer captures one of the substrate radicals and orients them in a way that precludes undesired reaction channels, thus favoring the formation of the optically pure coupling product. Structured digital abstract and by (), and by () [ABSTRACT FROM AUTHOR]

The thiamine diphosphate (ThDP)-dependent pyruvate decarboxylase from Acetobacter pasteurianus ( ApPDC) catalyzes the carboligation of aldehydes that yields ( R)-2-hydroxy ketones with high chemoselectivity in mixed carboligations of aliphatic donor and aromatic acceptor aldehydes. On the basis of the crystal structure of ApPDC, which was determined to a resolution of 2.75 Å, and biochemical data, we mapped the active site. This enabled us to design variants with tailor-made catalytic activities by modifications of the residues E469 and W388. Although the exchange of W388 by smaller amino acids yields variants with higher carboligase activity due to an increased access to the active site, the exchange of E469 to glycine opens the so-called S-pocket in ApPDC for aromatic aldehydes and thus alters the stereoselectivity. The variant ApPDC-E469G provides access to ( S)-phenylacetylcarbinol derivatives by enzymatic carboligation with a good stereoselectivity of up to 89 % enantiomeric excess. The variant nicely complements the toolbox of ThDP-dependent enzymes, which now gives access to all stereo- and regioisomers of the asymmetric aliphatic-aromatic cross-benzoin-like condensation. We prove that optimal stabilization of both aldehydes in the active site is essential to gain high yields and high selectivities. [ABSTRACT FROM AUTHOR]

The influence of the total number of cores, the number of cores dedicated to Particle mesh Ewald (PME) calculation and the choice of single vs. double precision on the performance of molecular dynamic (MD) simulations in the size of 70,000 to 1.7 million of atoms was analyzed on three different high-performance computing facilities employing GROMACS 4 by running about 6000 benchmark simulations. Small and medium sized systems scaled linear up to 64 and 128 cores, respectively. Systems with half a million to 1.2 million atoms scaled linear up to 256 cores. The best performance was achieved by dedicating 25% of the total number of cores to PME calculation. Double precision calculations lowered the performance by 30-50%. A database for collecting information about MD simulations and the achieved performance was created and is freely available online and allows the fast estimation of the performance that can be expected in similar environments. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011 [ABSTRACT FROM AUTHOR]

The large and diverse family of cytochrome P450 monooxygenases (CYPs) was systematically analyzed to identify selectivity- and specificity-determining residues in the substrate recognition site 5, which is located in close vicinity to the heme center. A positively charged heme-interacting residue was identified in the structures of 29 monooxygenases and in 97.7% of the 6379 CYP sequences investigated here. This heme-interacting residue restricts the conformation of the substrate recognition site 5 and is preferentially located at position 10 or 11 after the conserved ExxR motif (in 94.4% of the sequences), in 3.3% of the sequences at position 9 or 12. As a result, a classification by the position of the heme-interacting residue allows to predict residues that are closest to the heme center and restrict its accessibility. In 98.4% of all CYP sequences a preferentially hydrophobic residue is located at position 5 after the ExxR motif that is predicted to point close to the heme center. Replacing this residue by hydrophobic residues of different size has been shown to change substrate specificity and regioselectivity for CYPs of different superfamilies. Twenty-seven percent of all CYPs are predicted to contain a second selectivity-determining residue at position 9 after the ExxR motif that can be identified by the pattern EXXR-X(7)-{P}- x-P-[HKR]. Proteins 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYP102A1) with its flexible and widely opened active site is still not well understood. In the present work (−)-α-pinene bound complexes with two triple mutants were modeled to elucidate the contribution of the three major factors that mediate selectivity: active site shape, protein flexibility, and chemical reactivity of the substrate. For the triple mutant A74G F87V L188Q (GVQ), one stable, productive conformation of the substrate (conformation I) was identified by multiple molecular dynamics simulations. The model predicts pinene epoxide as a major product (42% pinene oxide, 23% verbenol) which is in agreement with the experimental product profile (70% pinene oxide, 20% verbenol). In contrast, for the triple mutant A74G F87G L188Q (GGQ) two stable productive substrate conformations were identified (conformations IIa and IIb), and verbenol was predicted as major product (81% verbenol, 16% myrtenol), which is in agreement with experimental results (77% verbenol, 10% myrtenol). The effect of chemical reactivity of the substrate was demonstrated by comparison of (−)-α-pinene to its regioisomer (−)-β-pinene, where the product profile is shifted from 68% pinocarveol and 32% myrtanal in mutant GVQ, to 40% pinocarveol and 60% myrtanal in mutant GGQ. Our results strongly suggest a major role of residue 87 in anchoring (−)-α-pinene during substrate binding which provides a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex. Proteins 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The Medium-Chain Dehydrogenase/Reductase Engineering Database (MDRED, ) has been established to serve as an analysis tool for a systematic investigation of sequence-structure-function relationships. It includes sequence and structure information of 2684 and 42 medium-chain dehydrogenases/reductases (MDRs), respectively. Although MDRs are very diverse in sequence, they have a conserved tertiary structure. MDRs are assigned to 199 homologous families and 29 superfamilies. For each family, annotated multiple sequence alignments are provided, and functionally relevant residues are annotated. Twenty-five superfamilies were classified as zinc-containing MDRs, four as non-zinc-containing MDRs. For the zinc-containing MDRs, three subclasses were identified by systematic analysis of a variable loop region, the quaternary structure determining loop (QSDL): the class of short, medium, and long QSDL, which include 11, 3, and 5 superfamilies, respectively. The length of the QSDL is predictive for tetramer (short QSDL) and dimer (long QSDL) formation. The class of medium QSDL includes both tetrameric and dimeric MDRs. The shape of the substrate-binding site is highly conserved in all zinc-containing MDRs with the exception of two variable regions, the substrate recognition sites (SRS): two residues located on the QSDL (SRS1) and, for the class of long QSDL, one residue located in the catalytic domain (SRS2). The MDRED is the first online-accessible resource of MDRs that integrates information on sequence, structure, and function. Annotation of functionally relevant residues assist the understanding of sequence-structure-function relationships. Thus, the MDRED serves as a valuable tool to identify potential hotspots for engineering properties such as substrate specificity. [ABSTRACT FROM AUTHOR]

Water is known to play a crucial role in protein structure, flexibility and activity. The use of molecular dynamics simulations allows detailed studies of complex protein-solvent interactions. Cluster analysis and density-based approaches have been successfully used for the identification and analysis of conserved water molecules and hydration patterns of proteins. However, appropriate tools for analysing long-time molecular dynamics simulations with respect to tracking and visualising the paths of solvent molecules are lacking. Our method focuses on visualising the solvent paths entering and leaving cavities of the protein and allows to study the route and dynamics of the exchange of tightly bound internal water molecules with the bulk solvent. The proposed visualisation also represents dynamic properties such as direction and velocity in the solvent. Especially, by clustering similar path-lines with respect to designated properties the visualisation can be abstracted to represent the principal paths of solvent molecules through the cavities. Its application in the analysis of long-time scale molecular dynamics simulations not only confirmed conjectures based on previous manual observations made by chance, but also led to novel insights into the dynamical and structural role of water molecules and its interplay with protein structure. [ABSTRACT FROM AUTHOR]