Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase.
Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase.
复制标题
DOI：
10.1021/acs.biochem.8b00871
复制
发表时间：
2018-09
期刊：
影响因子：
2.9
通讯作者：
L. Favrot;Tathyana M Amorim Franco;J. Blanchard
中科院分区：
文献类型：
--
作者：
L. Favrot;Tathyana M Amorim Franco;J. Blanchard
支链氨基酸或支链氨基酸（l-异亮氨酸，l-亮氨酸和l-缬氨酸）的生物合成在真细菌中是必不可少的，但哺乳动物是支链氨基酸营养缺陷型，使该途径中的酶成为抗菌药物开发的极好靶点。l-异亮氨酸、l-亮氨酸和l-缬氨酸的生物合成非常有效，仅需要八种酶。苏氨酸脱氢酶（TD）是由ilvA基因编码的吡哆醛5 '-磷酸（PLP）依赖性酶，是负责将l-苏氨酸（l-Thr）转化为α-酮丁酸、氨和水的酶，这是l-异亮氨酸生物合成的第一步。我们已经克隆，表达，并生化特征的反应催化的耻垢分枝杆菌TD（简称MsIlvA）使用稳态动力学和动力学同位素效应。我们在这里表明，除了L-苏氨酸，L-别苏氨酸和L-丝氨酸也被用作底物的TD，都表现出S形，非米氏动力学。奇怪的是，β-氯-l-丙氨酸也是一种底物，而不是预期的抑制剂。TD的酶活性对变构调节剂的存在敏感，所述变构调节剂包括激活剂l-缬氨酸或TD所涉及的BCAA途径的终产物反馈抑制剂l-异亮氨酸。初级氘动力学同位素很小，表明Cα质子提取仅部分限速。溶剂动力学同位素显着较大，表明在反应过程中发生的质子转移也是部分限速的。最后，我们证明，l-环丝氨酸，PLP依赖性酶的一般抑制剂，是一个很好的苏氨酸脱氨酶的抑制剂。
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 α-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, β-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 Cα 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.