Constantin Vogel

Engineering stereoselectivity of ThDP-dependent enzymes

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.

Conservation analysis of class-specific positions in cytochrome P450 monooxygenases: Functional and structural relevance

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.

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

BackgroundStandard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP) -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD) is still missing.Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult.ResultsWe developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM) was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8) was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the Sc PDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S)-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED) and was used to systematically analyze functionally and structurally relevant positions in the superfamily of ThDP-dependent decarboxylases.ConclusionsThe numbering scheme serves as a tool for the reliable sequence alignment of ThDP-dependent decarboxylases and the unambiguous identification and communication of corresponding positions. Thus, it is the basis for the systematic and automated analysis of sequence-encoded properties such as structural and functional relevance of amino acid positions, because the analysis of conserved positions, the identification of correlated mutations and the determination of subfamily specific amino acid distributions depend on reliable multisequence alignments and the unambiguous identification of the alignment columns. The method is reliable and robust and can easily be adapted to further protein families.

The modular structure of ThDP-dependent enzymes

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.

Tailoring the S‐Selectivity of 2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate Synthase (MenD) from Escherichia coli

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.

A Tailor-Made Chimeric Thiamine Diphosphate Dependent Enzyme for the Direct Asymmetric Synthesis of (S)-Benzoins

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.

Identification of universal selectivity-determining positions in cytochrome P450 monooxygenases by systematic sequence-based literature mining

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 (www.CYPED.BioCatNet.de) 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.

MenD from Bacillus subtilis: A Potent Catalyst for the Enantiocomplementary Asymmetric Synthesis of Functionalized α‐Hydroxy Ketones

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.

BioCatNet: a database system for the integration of enzyme sequences and biocatalytic experiments

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).

Network Analysis of Sequence-Function Relationships and Exploration of Sequence Space of TEM β-Lactamases

ABSTRACT The 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.