How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme–substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor–acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor–substrate complex.
Background: Toluene dioxygenase reductase converts the redox equivalents of NADH under oxic conditions. Results: Reduced reductase forms a stable complex with NAD ϩ that shows slower electron transfer to dioxygen.
The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent ␣-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic ␣-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by ␣-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.Over the past years numerous thiamine diphosphatedependent (ThDP) 5 ␣-keto acid decarboxylases have been identified. These enzymes catalyze the non-oxidative decarboxylation of ␣-keto acids to aldehydes. The elementary steps of the catalytic cycle of these enzymes are identical. Initially, the cofactor is activated by deprotonation of its C2 atom. Subsequently, the generated cofactor ylide attacks the ␣-carbonyl atom of the substrate, yielding a tetrahedral pre-decarboxylation intermediate. This intermediate is decarboxylated, resulting in the second resonance-stabilized carbanion/enamine intermediate. Finally, the enamine intermediate is protonated, and the reaction product (aldehyde) is released (1).
DatabaseStructural data for holo-EcODC (ThDP-EcODC) in the absence of additional ligands and in complex with either ADP or acetyl CoA have been submitted to the Protein Data Bank under the accession numbers 2q27, 2q28 and 2q29, respectively. The gene yfdU from Escherichia coli encodes a putative oxalyl coenzyme A decarboxylase, a thiamine diphosphate-dependent enzyme that is potentially involved in the degradation of oxalate. The enzyme has been purified to homogeneity. The kinetic constants for conversion of the substrate oxalyl coenzyme A by the enzyme in the absence and presence of the inhibitor coenzyme A, as well as in the absence and presence of the activator adenosine 5¢-diphosphate, were determined using a novel continuous optical assay. The effects of these ligands on the solution and crystal structure of the enzyme were studied using small-angle X-ray scattering and X-ray crystal diffraction. Analyses of the obtained crystal structures of the enzyme in complex with the cofactor thiamine diphosphate, the activator adenosine 5¢-diphosphate and the inhibitor acetyl coenzyme A, as well as the corresponding solution scattering patterns, allow comparison of the oligomer structures of the enzyme complexes under various experimental conditions, and provide insights into the architecture of substrate and effector binding sites. Abbreviations EcODC, oxalyl CoA decarboxylase from Escherichia coli; OfODC, oxalyl CoA decarboxylase from Oxalobacter formigenes; PADP, 3¢-phosphoadenosine 5¢-diphosphate; ThDP, thiamine diphosphate.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.