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B6db references: 30226377

type Journal Article
authors Favrot L, Amorim Franco T, Blanchard JS.
title Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase
journal Biochemistry
sel selected
ui 30226377
year (2018)
volume 57
number 41
pages 6003-6012
keywords DOI: 10.1021/acs.biochem.8b00871
abstract The biosynthesis of branched-chain amino acids or BCAAs (L-isoleucine, L-leucine, and L-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of L-isoleucine, L-leucine, and L-valine is very efficient, requiring only eight enzymes. Threonine deaminase (TD), a PLP-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of L-threonine (L-Thr) to -ketobutyrate, ammonia and water and is the first step in the biosynthesis of L-isoleucine. We have cloned, expressed and biochemically characterized the reaction catalyzed by M. smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to L-threonine, L-allo-threonine and L-serine are also used as substrates by TD and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, -chloro-L-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator L-valine or the end-product feedback inhibitor of the BCAA pathway in which TD is involved, L-isoleucine. Primary deuterium kinetic isotopes are small, suggesting C proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that L-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.
last changed 2018/11/27 08:57

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