|
type |
Journal Article |
authors |
Favrot L, Amorim Franco T, Blanchard JS. |
title |
Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase |
journal |
Biochemistry |
Activity |
4.3.1.19 |
Family |
4.3.1.19 |
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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