Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase

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 dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of L-threonine (L-Thr) to alpha-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of L-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium 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, beta-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 alpha 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.