Michael Knoll

Factors mediating activity, selectivity, and substrate specificity for the thiamin diphosphate-dependent enzymes benzaldehyde lyase and benzoylformate decarboxylase.

Benzaldehyde lyase from Pseudomonas fluorescens and benzoylformate decarboxylase from Pseudomonas putida are homologous thiamin diphosphate‐dependent enzymes that catalyze carboligase and carbolyase reactions. Both enzymes catalyze the formation of chiral 2‐hydroxy ketones from aldehydes. However, the reverse reaction has only been observed with benzaldehyde lyase. Whereas benzaldehyde lyase is strictly R specific, the stereoselectivity of benzoylformate decarboxylase from P. putida is dependent on the structure and orientation of the substrate aldehydes. In this study, the binding sites of both enzymes were investigated by using molecular modelling studies to explain the experimentally observed differences in the activity, stereo‐ and enantioselectivity and substrate specificity of both enzymes. We designed a detailed illustration that describes the shape of the binding site of both enzymes and sufficiently explains the experimental effects observed with the wild‐type enzymes and different variants. These findings demonstrate that steric reasons are predominantly responsible for the differences observed in the (R)‐benzoin cleavage and in the formation of chiral 2‐hydroxy ketones.

Rational protein design of ThDP-dependent enzymes-engineering stereoselectivity.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is an exceptional thiamin diphosphate‐dependent enzyme, as it catalyzes the formation of (S)‐2‐hydroxy‐1‐phenylpropan‐1‐one from benzaldehyde and acetaldehyde. This is the only currently known S‐selective reaction (92 % ee) catalyzed by this otherwise R‐selective class of enzymes. Here we describe the molecular basis of the introduction of S selectivity into ThDP‐dependent decarboxylases. By shaping the active site of BFD through the use of rational protein design, structural analysis, and molecular modeling, optimal steric stabilization of the acceptor aldehyde in a structural element called the S pocket was identified as the predominant interaction for adjusting stereoselectivity. Our studies revealed Leu461 as a hot spot for stereoselectivity in BFD. Exchange to alanine and glycine resulted in variants that catalyze the S‐stereoselective addition of larger acceptor aldehydes, such as propanal with benzaldehyde and its derivatives—a reaction not catalyzed by the wild‐type enzyme. Crystal structure analysis of the variant BFDL461A supports the modeling studies.

The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases

BackgroundPolyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common α/β-hydrolase fold and a catalytic triad, which is also found in other α/β-hydrolases.ResultsThe PHA Depolymerase Engineering Database (DED, http://www.ded.uni-stuttgart.de) has been established as a tool for systematic analysis of this enzyme family. The DED contains sequence entries of 587 PHA depolymerases, which were assigned to 8 superfamilies and 38 homologous families based on their sequence similarity. For each family, multiple sequence alignments and profile hidden Markov models are provided, and functionally relevant residues are annotated.ConclusionThe DED is a valuable tool which can be applied to identify new PHA depolymerase sequences from complete genomes in silico, to classify PHA depolymerases, to predict their biochemical properties, and to design enzyme variants with improved properties.

The Cytochrome P450 Engineering Database

SUMMARY The Cytochrome P450 Engineering Database (CYPED) has been designed to serve as a tool for a comprehensive and systematic comparison of protein sequences and structures within the vast and diverse family of cytochrome P450 monooxygenases (CYPs). The CYPED currently integrates sequence and structure data of 3911 and 25 proteins, respectively. Proteins are grouped into homologous families and superfamilies according to Nelsons classification. Nonclassified CYP sequences are assigned by similarity. Functionally relevant residues are annotated. The web accessible version contains multisequence alignments, phylogenetic trees and HMM profiles. The CYPED is regularly updated and supplies all data for download. Thus, it provides a valuable data source for phylogenetic analysis, investigation of sequence-function relationships and the design of CYPs with improved biochemical properties. ABBREVIATIONS Cytochrome P450 Engineering Database, CYPED; cytochrome P450 monooxygenase, CYP; Hidden Markov Model, HMM. AVAILABILITY www.cyped.uni-stuttgart.de

S‐Selective Mixed Carboligation by Structure‐Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus

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.

The Medium-Chain Dehydrogenase/reductase Engineering Database: a systematic analysis of a diverse protein family to understand sequence-structure-function relationship.

The Medium‐Chain Dehydrogenase/Reductase Engineering Database (MDRED, http://www.mdred.uni‐stuttgart.de) 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.