Enzymology of Homoserine Acylation in Methionine Biosynt

描述（由申请人提供）：甲硫氨酸生物合成途径，在哺乳动物中不存在，产生细菌生存所需的两种化合物，蛋氨酸和s -腺苷蛋氨酸。除非从环境中获得足够的蛋氨酸，否则这种途径的破坏会阻止细菌生长。因此，这一途径中的酶可能成为新型抗菌化合物的潜在靶点。蛋氨酸生物合成的第一个独特步骤是同型丝氨酸的g-羟基酰化，控制同型丝氨酸进入该途径的通量。这种酰化是由两种酶之一催化的，即同丝氨酸转乙酰化酶（HTA）或同丝氨酸转琥珀酰化酶（HTS）。该项目的长期目标是确定这些酶是否是抗菌剂的潜在靶点，并设计出作为先导化合物的抑制剂。这两种酶的初始动力学表征已被报道。这项建议将实现三个具体目标。首先，确定HTA的Ser-Asp-His催化三元组的氨基酸。这将通过序列比对、定点诱变和稳态动力学表征的结合来完成。其次，识别HTS与HTA不同的活性位点残基。这将通过序列比对、定点诱变、化学修饰和稳态动力学表征的结合来完成。第三，将对HTA、HTS和选择突变体进行结构分析，努力将功能与结构联系起来。所有这三个目标都直接支持这个项目的长期目标。这些实验将用于培养生物化学领域的本科生和硕士生

DESCRIPTION (provided by applicant): The methionine biosynthetic pathway, absent in mammals, produces two compounds required for bacterial survival, methionine and S-adenosylmethionine. Disruption of this pathway prevents bacterial growth unless sufficient methionine is obtained from the environment. Therefore, enzymes in this pathway may potentially be targets for novel antibacterial compounds. The first unique step in methionine biosynthesis, acylation of the g-hydroxyl of homoserine, controls flux of homoserine into the pathway. This acylation is catalyzed by one of two enzymes, homoserine transacetylase (HTA) or homoserine transsuccinylase (HTS). The long-term goals of this project are to ascertain whether these enzymes are potential targets for antibacterial agents and to design inhibitors that will function as lead compounds. Initial kinetic characterizations of both enzymes have been reported. In this proposal three specific aims will be pursued. First, the amino acids comprising the Ser-Asp-His catalytic triad of HTA will be identified. This will be accomplished through the combination of sequence alignments, site-directed mutagenesis and steady-state kinetic characterization. Second, the active site residues of HTS, which are different from those of HTA, will be identified. This will be accomplished through a combination of sequence alignment, site-directed mutagenesis, chemical modification, and steady-state kinetic characterization. Third, structural analysis of HTA, HTS, and select mutants will be pursued in an effort to correlate function with structure. All three of these aims directly support the long-term goals of this project. The proposed experiments will be used to train both undergraduate and Master's level students in the area of biochemistry