Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.
Arch Biochem Biophys. 2011 Aug 15;512(2):167-74. doi: 10.1016/j.abb.2011.06.006. Epub 2011 Jun 16.
Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are examples of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA-DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K(m)=12μM) as a cofactor more effectively than NADH (K(m)=26μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA-DHDPR has ∼20-fold greater binding affinity for NADPH (K(d)=1.5μM) relative to NADH (K(d)=29μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism; with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP(+). This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA-DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.
鉴于抗生素耐药性（包括金黄色葡萄球菌中的耐甲氧西林金黄色葡萄球菌，MRSA）的迅速上升，迫切需要确定新的药物靶标。细菌赖氨酸生物合成途径中的酶就是此类靶标的示例，包括二氢二吡啶羧酸还原酶（DHDPR，E.C. 1.3.1.26），它是必需细菌基因的产物。DHDPR 在赖氨酸生物合成途径中催化 NAD(P)H 依赖性将二氢二吡啶羧酸（DHDP）还原为四氢二吡啶羧酸（THDP）。我们发现，MRSA-DHDPR 表现出独特的核苷酸特异性，更有效地利用 NADPH（K(m)=12μM）作为辅因子，而不是 NADH（K(m)=26μM）。然而，当使用 NADPH 作为辅因子时，该酶会被高浓度的 DHDP 抑制，但使用 NADH 时则不会。等温滴定量热法（ITC）研究表明，MRSA-DHDPR 对 NADPH 的结合亲和力约为 NADH 的 20 倍（K(d)=1.5μM 对 K(d)=29μM）。与 ITC 研究相结合的动力学研究表明，该酶遵循强制顺序三元复合物机制；DHDP 通过与 NADP(+) 形成非生产性三元复合物来抑制该酶。这项工作首次描述了 MRSA-DHDPR 的催化机制和辅因子偏好，并为抑制这一有效的抗菌药物靶标提供了合理的方法。
