Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine.
Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine.
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DOI：
10.1021/acschembio.7b00142
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发表时间：
2017-05-19
影响因子：
4
通讯作者：
Blanchard JS
中科院分区：
文献类型：
--
作者：
Amorim Franco TM;Favrot L;Vergnolle O;Blanchard JS
支链转氨酶是一种依赖于吡哆醛5 ' -磷酸（PLP）的酶，在细菌中负责所有三种支链氨基酸（l -亮氨酸、l -异亮氨酸和l -缬氨酸）的生物合成的最后一步。我们研究了D-环丝氨酸和l -环丝氨酸对结核分枝杆菌（MtIlvE）支链转氨酶失活的机制。d -环丝氨酸目前仅用于治疗耐多药结核病。我们的研究结果表明，两种异构体对MtIlvE的失活具有时间和浓度依赖性，l -环丝氨酸是该酶的40倍抑制剂。最低抑制浓度（MIC）研究表明，l -环丝氨酸对结核分枝杆菌生长的抑制作用是d -环丝氨酸的10倍。此外，我们已经结晶了mtilve - d -环丝氨酸抑制酶，确定了1.7 Å的结构。与MtIlvE结合的共价d -环丝氨酸- pmp加合物的结构揭示了d -环丝氨酸环是平面的和芳香的，正如之前在其他酶系统中观察到的那样。质谱分析表明，d -环丝氨酸-和l -环丝氨酸- pmp配合物具有相同的质量，并且可能是相同的芳构化异唑产物。然而，MtIlvE d -环丝氨酸- pmp和MtIlvE l -环丝氨酸- pmp加合物的形成动力学有很大不同。虽然MtIlvE d -环丝氨酸- pmp复合物的形成动力学可以适合于单一指数，但MtIlvE l -环丝氨酸- pmp复合物的形成发生在两个步骤中。我们提出了D-环丝氨酸和l -环丝氨酸失活的化学机制，这表明立体化学决定了不同失活动力学的结构作用。这些结果表明，d -环丝氨酸对结核分枝杆菌活性的作用机制可能比以前认为的更为复杂，并且d -环丝氨酸可能损害多种plp依赖性酶的体内活性，包括MtIlvE。
The branched-chain aminotransferase is a pyridoxal 5′-phosphate (PLP)-dependent enzyme responsible for the final step in the biosynthesis of all three branched-chain amino acids, L-leucine, L-isoleucine, and L-valine, in bacteria. We have investigated the mechanism of inactivation of the branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE) by D- and L-cycloserine. D-Cycloserine is currently used only in the treatment of multidrug–drug-resistant tuberculosis. Our results show a time-and concentration-dependent inactivation of MtIlvE by both isomers, with L-cycloserine being a 40-fold better inhibitor of the enzyme. Minimum inhibitory concentration (MIC) studies revealed that L-cycloserine is a 10-fold better inhibitor of Mycobacterium tuberculosis growth than D-cycloserine. In addition, we have crystallized the MtIlvE-D-cycloserine inhibited enzyme, determining the structure to 1.7 Å. The structure of the covalent D-cycloserine-PMP adduct bound to MtIlvE reveals that the D-cycloserine ring is planar and aromatic, as previously observed for other enzyme systems. Mass spectrometry reveals that both the D-cycloserine- and L-cycloserine-PMP complexes have the same mass, and are likely to be the same aromatized, isoxazole product. However, the kinetics of formation of the MtIlvE D-cycloserine-PMP and MtIlvE L-cycloserine-PMP adducts are quite different. While the kinetics of the formation of the MtIlvE D-cycloserine-PMP complex can be fit to a single exponential, the formation of the MtIlvE L-cycloserine-PMP complex occurs in two steps. We propose a chemical mechanism for the inactivation of D- and L-cycloserine which suggests a stereochemically determined structural role for the differing kinetics of inactivation. These results demonstrate that the mechanism of action of D-cycloserine’s activity against M. tuberculosis may be more complicated than previously thought and that D-cycloserine may compromise the in vivo activity of multiple PLP-dependent enzymes, including MtIlvE.
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影响因子：
3.7
作者：
Bharath SR;Bisht S;Harijan RK;Savithri HS;Murthy MR
通讯作者：
Murthy MR
影响因子：
4
作者：
Lambert, RJW;Pearson, J
通讯作者：
Pearson, J
影响因子：
4.8
作者：
Hegde, SS;Blanchard, JS
通讯作者：
Blanchard, JS
影响因子：
30.8
作者：
Desjardins CA;Cohen KA;Munsamy V;Abeel T;Maharaj K;Walker BJ;Shea TP;Almeida DV;Manson AL;Salazar A;Padayatchi N;O'Donnell MR;Mlisana KP;Wortman J;Birren BW;Grosset J;Earl AM;Pym AS
通讯作者：
Pym AS
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH