Structural and functional characterization of tautomerase and aspartase/fumarase superfamily enzymes

Biocatalysis is a branch of biochemistry that exploits the ability of an enzyme to convert a substrate into a compound of economic value in an environmentally friendly manner. Enzymes are known to catalyze complex chemical reactions in their active site pockets and it is important to understand the intricacies of the reaction mechanism at the molecular level. X-ray crystallography, a powerful technique, is used to calculate the position of individual atoms comprising an enzyme in the three-dimensional space. Solving structures of enzymes in complex with a ligand tells us how a compound binds in the enzyme active site and the role of the different amino acid residues during catalysis. This knowledge further enables the engineering of enzymes for efficient and selective catalysis. The thesis of Harshwardhan Poddar describes the structural, functional and mechanistic characterization of three enzymes belonging to the tautomerase and aspartase/fumarase superfamily of enzymes. 4-Oxalocrotonate tautomerase (4-OT) is a versatile enzyme capable of promiscuously catalyzing synthetically useful C-C bond-forming reactions. The mechanism of these important reactions has been elucidated by solving structures of the wild-type and engineered 4-OT variants in complex with substrates. In addition, a new member of the tautomerase superfamily, RhCC, was identified and shown to catalyze a cofactor-independent oxygenation reaction, previously unseen in any other member of this superfamily. Finally, a new enzyme capable of catalyzing C-N bond forming reactions, called ethylenediamine-N,N’-disuccinic acid lyase (EDDS lyase), was identified in the aspartase/fumarase superfamily of enzymes. Crystal structures in complex with substrates and product gave important insights into the mechanism and synthetic potential of this robust enzyme. These three enzymes have shown their potential in the production of valuable compounds such as precursors for pharmaceuticals and various food additives, and the work described in this thesis brings forth new opportunities for further research into the optimization of the properties of these enzymes for industrial applications

Means and methods for synthesizing precursors of y-aminobutyric acid (gaba) analogs

The invention relates to the fields of drug development and biocatalysis, more specifically to a biocatalytic route for asymmetric synthesis of precursors of y-aminobutyric acid (GABA) analogs. Provided is an isolated mutant 4-oxalocrototonate tautomerase (4-OT) enzyme comprising the following mutations (i) leucine at position 8 substituted with a tyrosine (L8Y) or a phenylalanine (L8F); (ii) methionine at position 45 substituted with a tyrosine (M45Y); and (iii) phenylalanine at position 50 substituted with an alanine (F50A), wherein the positions are numbered according to the amino acid sequence of 4-OT of Pseudomonas putida. Also provided is a method for the synthesis of a precursor for the pharmaceutically relevant enantiomer of a GABA analog, comprising (i) providing a y-nitroaldehyde using the 4-OT mutant enzyme, followed by (ii) subjecting the thus obtained y-nitroaldehyde to an enzymatic oxidation reaction catalyzed by an aldehyde dehydrogenase (EC 1.2.1.3)

Structural basis for the catalytic mechanism of ethylenediamine-N,N′-disuccinic acid lyase, a carbon-nitrogen bond-forming enzyme with broad substrate scope

The natural aminocarboxylic acid product ethylenediamine-N,N′-disuccinic acid [(S,S)-EDDS] is able to form a stable complex with metal ions, making it an attractive biodegradable alternative for the synthetic metal chelator ethylenediamine tetraacetic acid (EDTA), which is currently used at large scale in numerous applications. Previous studies have demonstrated that biodegradation of (S,S)-EDDS may be initiated by an EDDS lyase, converting (S,S)-EDDS via the intermediate N-(2-aminoethyl)aspartic acid (AEAA) into ethylenediamine and two molecules of fumarate. However, current knowledge of this enzyme is limited due to the absence of structural data. Here, we describe the identification and characterization of an EDDS lyase from Chelativorans sp. BNC1, which has a broad substrate scope, accepting various mono- and diamines for addition to fumarate. We report crystal structures of the enzyme in an unliganded state and in complex with formate, succinate, fumarate, AEAA and (S,S)-EDDS. The structures reveal a tertiary and quaternary fold that is characteristic of the aspartase/fumarase superfamily and support a mechanism that involves general base-catalyzed, sequential two-step deamination of (S,S)-EDDS. This work broadens our understanding of mechanistic diversity within the aspartase/fumarase superfamily and will aid in the optimization of EDDS lyase for asymmetric synthesis of valuable (metal-chelating) aminocarboxylic acids

Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases

The Michael-type addition reaction is widely used in organic synthesis for carbon-carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon-carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon-carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence-function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary 'Michaelases'. These 'Michaelases' catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives

Evidence for the Formation of an Enamine Species during Aldol and Michael-type Addition Reactions Promiscuously Catalyzed by 4-Oxalocrotonate Tautomerase

The enzyme 4-oxalocrotonate tautomerase (4-OT), which has a catalytic N-terminal proline residue (Pro1), can promiscuously catalyze various carbon–carbon bond-forming reactions, including aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde, and Michael-type addition of acetaldehyde to a wide variety of nitroalkenes to yield valuable γ-nitroaldehydes. To gain insight into how 4-OT catalyzes these unnatural reactions, we carried out exchange studies in D2O, and X-ray crystallography studies. The former established that H–D exchange within acetaldehyde is catalyzed by 4-OT and that the Pro1 residue is crucial for this activity. The latter showed that Pro1 of 4-OT had reacted with acetaldehyde to give an enamine species. These results provide evidence of the mechanism of the 4-OT-catalyzed aldol and Michael-type addition reactions in which acetaldehyde is activated for nucleophilic addition by Pro1-dependent formation of an enamine intermediate

A guide to time‐resolved structural analysis of light‐activated proteins

International audienceDynamical changes in protein structures are essential for protein function and occur over femtoseconds to seconds timescales. X-ray free electron lasers have facilitated investigations of structural dynamics in proteins with unprecedented temporal and spatial resolution. Light-activated proteins are attractive targets for time-resolved structural studies, as the reaction chemistry and associated protein structural changes can be triggered by short laser pulses. Proteins with different light-absorbing centres have evolved to detect light and harness photon energy to bring about downstream chemical and biological output responses. Following light absorption, rapid chemical/small-scale structural changes are typically localised around the chromophore. These localised changes are followed by larger structural changes propagated throughout the photoreceptor/photocatalyst that enables the desired chemical and/or biological output response. Time-resolved serial femtosecond crystallography (SFX) and solution scattering techniques enable direct visualisation of early chemical change in light-activated proteins on timescales previously inaccessible, whereas scattering gives access to slower timescales associated with more global structural change. Here, we review how advances in time-resolved SFX and solution scattering techniques have uncovered mechanisms of photochemistry and its coupling to output responses. We also provide a prospective on how these time-resolved structural approaches might impact on other photoreceptors/photoenzymes that have not yet been studied by these methods