The X-ray structure of L-threonine dehydrogenase from the common hospital pathogen Clostridium difficile

In many prokaryotes, the first step of threonine metabolism is catalysed by the enzyme threonine dehydrogenase (TDH), which uses NAD+ to oxidize its substrate to 2-amino-3-ketobutyrate. The absence of a functional TDH gene in humans suggests that inhibitors of this enzyme may have therapeutic potential against pathogens which are reliant on this enzyme. Here, TDH from Clostridium difficile has been cloned and overexpressed, and the X-ray structure of the apoenzyme form has been determined at 2.6 Å resolution.

Threonine in mammals: indispensability and transamination

As is known, amino acid threonine is not synthesized in the vertebrates when it does not come with food and the decomposition of threonine under the action of threonine dehydratase is irreversible process. Some facts point to the presence of insignificant threonine synthesis in animals. The question arises about the possibility of biosynthesis of threonine in animals in the absence of it in food, that is, its interchangeability. Research on this issue is important for compiling the diet of animals. The article shows that the threonine cannot be synthesized by reversibility of the reaction of its decomposition as well why threonine dehydrogenase in the tissues of mammals cannot be used in threonine biosynthesis. It is concluded that some quantity of threonine is involved in transamination.

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase fromClostridium beijerinckiiNCIMB 8052

ABSTRACTAcetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase fromClostridium beijerinckiiNCIMB 8052. Anin silicoscreen of theC. beijerinckiigenome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression inEscherichia coli. The purified enzyme (C. beijerinckiiacetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn2+atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR inC. beijerinckiiis discussed.