Structural and Functional Characterization of Mycobacterium tuberculosis Homoserine Transacetylase.
Structural and Functional Characterization of Mycobacterium tuberculosis Homoserine Transacetylase.
复制标题
结核分枝杆菌高丝氨酸转乙酰酶的结构和功能特征。
DOI：
10.1021/acsinfecdis.2c00541
复制
发表时间：
2023
影响因子：
5.3
通讯作者：
Aldrich,CourtneyC
中科院分区：
文献类型：
--
作者：
Sharma,Sachin;Jayasinghe,YahaniP;Mishra,NeerajKumar;Orimoloye,MoyosoreO;Wong,Tsung-Yun;Dalluge,JosephJ;Ronning,DonaldR;Aldrich,CourtneyC
结核分枝杆菌(Mtb)缺乏功能性高丝氨酸转乙酰酶(HTA)，在蛋氨酸的生物合成、蛋白质合成以及多种必需的腺苷-L-蛋氨酸依赖酶的活性方面受到影响。此外，由于代谢流重定向到赖氨酸生物合成途径，赖氨酸的毒性积累进一步解除了缺陷突变体的武装。缺失突变体的研究和脱辅酶的结晶学研究分别证实了MtbHTA是一种必需的酶，并揭示了一个可配基的结合部位。为了寻找这种酶的机制特征，我们报道了MtbHTA的关键结构细节和全面的功能特征。对乙酰化HTA中间速度和初始速度的结晶学和质谱学观察符合乒乓球动力学机理。对野生型hta及其定点突变体进行了一组天然底物和替代底物的动力学表征，以了解底物的特异性并确定催化的关键残基。荧光猝灭滴定实验表明，底物─乙酰辅酶A和l-高丝氨酸─均与羟色胺发生强、弱结合作用。此外，乙酰-辅酶A对底物的抑制和辅酶A-乙酰基-L-高丝氨酸对产物的抑制构成了反馈调控机制的基础。通过提供关键的机械和结构信息，这些研究为围绕这一有吸引力的Mtb目标进行基于结构的设计工作提供了基础。
Mycobacterium tuberculosis(Mtb) lacking functional homoserine transacetylase (HTA) is compromised in methionine biosynthesis, protein synthesis, and in the activity of multiple essentialS-adenosyl-l-methionine-dependent enzymes. Additionally, deficient mutants are further disarmed by the toxic accumulation of lysine due to a redirection of the metabolic flux toward the lysine biosynthetic pathway. Studies with deletion mutants and crystallographic studies of the apoenzyme have, respectively, validatedMtbHTA as an essential enzyme and revealed a ligandable binding site. Seeking a mechanistic characterization of this enzyme, we report crucial structural details and comprehensive functional characterization ofMtbHTA. Crystallographic and mass spectral observation of the acetylated HTA intermediate and initial velocity studies were consistent with a ping-pong kinetic mechanism. Wild-type HTA and its site-directed mutants were kinetically characterized with a panel of natural and alternative substrates to understand substrate specificity and identify critical residues for catalysis. Titration experiments using fluorescence quenching showed that both substrates─acetyl-CoA andl-homoserine─engage in a strong and weak binding interaction with HTA. Additionally, substrate inhibition by acetyl-CoA and product inhibition by CoA andO-acetyl-l-homoserine were proposed to form the basis of a feedback regulation mechanism. By furnishing key mechanistic and structural information, these studies provide a foundation for structure-based design efforts around this attractiveMtbtarget.
登录
查看更多内容
Dan Purich（作者）：酶动力学：催化
影响因子：
2.8
作者：
P. Tipton
通讯作者：
P. Tipton
影响因子：
56.9
作者：
STURGILLKOSZYCKI, S;SCHLESINGER, PH;RUSSELL, DG
通讯作者：
RUSSELL, DG
耻垢分枝杆菌 ATCC 19420 的 O-乙酰高丝氨酸乙酰转移酶的晶体结构和生化特征。
影响因子：
3.1
作者：
Hye;Jiyeon Hong;Kyung
通讯作者：
Kyung
Paul F. Cook 和 W. W. Cleland 的“酶动力学和机制”
影响因子：
5.6
作者：
Irene Lee
通讯作者：
Irene Lee
影响因子：
3.4
作者：
Zhang YJ;Rubin EJ
通讯作者：
Rubin EJ