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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.

Macrokinetic Michaelis-Menten models based on thermodynamic activity provide insights into enzyme kinetics because they separate substrate-enzyme from substrate-solvent interactions. Kinetic parameters are estimated from experimental progress curves of enzyme-catalyzed reactions. Three pitfalls are discussed: deviations between thermodynamic and concentration-based models, product effects on the substrate activity coefficient, and product inhibition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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)., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti-tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self-sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild-type enzyme converts (4R)-limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio-, stereo- and chemoselectivity, compared to wild-type. From this library the variant with the highest perillyl alcohol ratio was fine-tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)-limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The importance of biocatalysis for ecologically sustainable syntheses in the chemical industry and for applications in everyday life is increasing. To design efficient applications, it is important to know the related enzyme kinetics; however, the measurement is laborious and error-prone. Flow reactors are suitable for rapid reaction parameter screening; here, a novel workflow is proposed including digital image processing (DIP) for the quantification of product concentrations, and the use of structured data acquisition with EnzymeML spreadsheets combined with ontology-based semantic information, leading to rapid and smooth data integration into a simulation tool for kinetics evaluation. One of the major findings is that a flexibly adaptive ontology is essential for FAIR (findability, accessibility, interoperability, reusability) data handling. Further, Python interfaces enable consistent data transfer. [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]

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]

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 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]

We report the exploration of the evolutionary relationship between imine reductases (IREDs) and other dehydrogenases. This approach is informed by the sequence similarity between these enzyme families and the recently described promiscuous activity of IREDs for the highly reactive carbonyl compound 2,2,2-trifluoroacetophenone. Using the structure of the R-selective IRED from Streptosporangium roseum (R-IRED-Sr) as a model, β-hydroxyacid dehydrogenases (βHADs) were identified as the dehydrogenases most similar to IREDs. To understand how active site differences in IREDs and βHADs enable the reduction of predominantly C = N or C = O bonds respectively, we substituted amino acid residues in βHADs with the corresponding residues from the R-IRED-Sr and were able to increase the promiscuous activity of βHADs for C = N functions by a single amino acid substitution. Variants βHADAt_K170D and βHADAt_K170F lost mainly their keto acid reduction activity and gained the ability to catalyze the reduction of imines. Moreover, the product enantiomeric purity for a bulky imine substrate could be increased from 23% ee (R-IRED-Sr) to 97% ee (βHADAt_K170D/F_F231A) outcompeting already described IRED selectivity. [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]

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]

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]

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]

Stereoselective traffic engineering The cover picture shows how engineered S‐selective EcMenD variants catalyze the carboligation of α‐ketoglutarate and benzaldehyde derivatives with excellent enantioselectivities of up to 99 % ee S, in contrast to the wild‐type enzyme, which produces R‐enantiomers. In their Full Paper on p. 3587 ff., M. Pohl et al. describe how they engineer S‐selective EcMenD variants by optimizing steric properties and stabilization of the acceptor substrate in the S‐pocket. This method, inspired by the recently developed S‐pocket concept, involves opening the S‐pocket with simultaneous destabilization of the R‐pathway to enhance the S‐selectivity of thiamine diphosphate‐dependent enzymes. [ABSTRACT FROM AUTHOR]

The cover image, by Jürgen Pleiss et al., is based on the Article Identification of imine reductase‐specific sequence motifs, 10.1002/prot.25008. [ABSTRACT FROM AUTHOR]

A correction to the article "A Tailor-Made Chimeric Thiamine Diphosphate Dependent Enzyme for the Direct Asymmetric Synthesis of (S)-Benzoins" that was published in the 2014 issue is presented.