ENZYMES OF CELL WALL BIOSYNTHESIS

细胞壁生物合成酶

基本信息

批准号：
2887042
负责人：
STEVEN L RODERICK
金额：
$ 22.99万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 2001-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2887042
关键词：
N acetylglucosamine X ray crystallography acyl carrier protein acyl coA acyltransferase apoenzymes chemical binding chemical kinetics chemical models enzyme substrate analog enzyme substrate complex fatty acylation microcalorimetry molecular site myristates uridine diphosphate

项目摘要

Lipopolysaccharide (LPS) is the major lipid component of the outermost monolayer of most gram-negative bacteria. This monolayer forms a diffusion barrier to hydrophobic and large hydrophilic agents to their periplasmic and cytosolic targets. The first step of LPS biosynthesis in E. coil is catalyzed by UDP-GIcNAc O-acyltransferase (lpxA). This enzyme catalyzes transfer of (R)-3-hydroxymyristic acid from the Acyl Carrier Protein (ACP) to UDP-GlcNAc. Cell lines with antibiotic supersusceptible phenotypes have been identified which are defective in lpxA as well as other early steps in LPS biosynthesis, establishing the validity of the LPS biosynthetic pathway and this enzyme in particular as targets for inhibitor design. The specific aims of this proposal are: 1. To solve the x-ray crystal structure of the apoenzyme. 2. To determine binding constants of sugar-nucleotide and acyl-ACP ligands to the enzyme. 3. To solve the x-ray crystal structure of enzyme/sugar-nucleotide complexes. 4. To crystallize tight binding enzyme/acyl-ACP complexes. Good, well-ordered single crystals of the apoenzyme are available. These crystals diffract to 2.5 Angstroms resolution and will support an x-ray crystal structure investigation. Fatty acyl-ACP substrate analogs will be prepared and the binding constants to the enzyme of these analogs and sugar nucleotides will be determined. This binding data will be used to investigate the substrate specificity due to fatty acyl chain length and acyl chain substituents as well as that due to the ACP polypeptide. Sugar nucleotides which form tight complexes with the enzyme will be investigated by soaking apoenzyme crystals in solutions of these compounds and determining their crystal structures. These results will be used to determine the interactions of the enzyme with its sugar nucleotide substrate. Tight binding acyl-ACP complexes identified in the binding studies will be used in crystallization experiments with the goal of preparing enzyme/acyl-ACP co-crystals. The information gained from the crystal structures of the apoenzyme and enzyme-ligand forms of the enzyme will provide a detailed description of the active site for use in the design of inhibitors directed against gram-negative pathogens.

脂多糖（LPS）是最外层脂质的主要成分大多数革兰氏阴性菌为单层。该单层形成对疏水剂和大亲水剂的扩散屏障周质和细胞质靶标。 LPS生物合成的第一步 E.coil 由 UDP-GlcNAc O-酰基转移酶 (lpxA) 催化。这种酶催化 (R)-3-羟基肉豆蔻酸从酰基载体转移蛋白质 (ACP) 转化为 UDP-GlcNAc。对抗生素超级敏感的细胞系已鉴定出 lpxA 和 LPS 生物合成的其他早期步骤，确定了该方法的有效性 LPS 生物合成途径和这种酶特别是作为目标抑制剂设计。该提案的具体目标是： 1. 解析脱辅基酶的X射线晶体结构。 2. 测定糖-核苷酸和酰基-ACP的结合常数酶的配体。 3. 解析酶/糖核苷酸的X射线晶体结构复合物。 4.使紧密结合的酶/酰基-ACP复合物结晶。可以获得良好的、有序的脱辅基酶单晶。这些晶体衍射分辨率为 2.5 埃，支持 X 射线晶体结构研究。脂肪酰基-ACP底物类似物将制备这些类似物与酶的结合常数和糖核苷酸将被测定。该绑定数据将用于研究由于脂肪酰基链长度而产生的底物特异性酰基链取代基以及ACP多肽的酰基链取代基。糖与酶形成紧密复合物的核苷酸将是通过将脱辅基酶晶体浸泡在这些溶液中进行研究化合物并确定其晶体结构。这些结果将用于确定酶与其糖的相互作用核苷酸底物。紧密结合的酰基-ACP 复合物在结合研究将用于结晶实验，其目标是制备酶/酰基-ACP共晶体。从获得的信息脱辅基酶的晶体结构和酶的酶配体形式将提供活动站点的详细描述，以供在针对革兰氏阴性病原体的抑制剂的设计。