Purification, characterization, and identification of novel inhibitors of the β-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus
Purification, characterization, and identification of novel inhibitors of the β-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus
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DOI：
10.1128/aac.46.5.1310-1318.2002
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发表时间：
2002-05-01
影响因子：
4.9
通讯作者：
Reynolds, KA
中科院分区：
文献类型：
--
作者：
He, X;Reynolds, KA
金黄色葡萄球菌是一种用途广泛且危险的病原体，是社区获得性和医院获得性感染的主要原因之一。金黄色葡萄球菌多重耐药菌株的增加需要开发具有以前未开发作用机制的新型抗生素，例如抑制β -酮酰 - 酰基载体蛋白（ACP）合酶III（FabH）。这种酶在细菌II型脂肪酸合酶中启动脂肪酸生物合成，催化丙二酰 - ACP和酰基辅酶A（CoA）底物之间的脱羧缩合反应，对细菌的生存能力至关重要。我们在金黄色葡萄球菌基因组中仅鉴定出一个fabH，并表明它编码的蛋白质与枯草芽孢杆菌（bFabH1）、大肠杆菌（ecFabH）和结核分枝杆菌（mtFabH）的FabH蛋白质分别具有57%、40%和34%的氨基酸序列同一性。额外的基因组序列分析显示，这种金黄色葡萄球菌FabH（saFabH）在某些耐甲氧西林金黄色葡萄球菌（MRSA）和耐万古霉素金黄色葡萄球菌（VRSA）菌株中没有突变。saFabH在大肠杆菌中表达，带有一个N - 末端多聚组氨酸标签，随后通过金属螯合和尺寸排阻色谱法进行纯化。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析显示其分子量为37 kDa，而凝胶过滤显示分子量为66.7 kDa，这表明saFabH具有非共价同源二聚体结构。丙二酰 - ACP的表观Km值为1.76 ± 0.40 μM，该酶对乙酰 - CoA（kcat，16.18 min⁻¹；Km，6.18 ± 0.9 μM）、丁酰 - CoA（kcat，42.90 min⁻¹；Km，2.32 ± 0.12 μM）和异丁酰 - CoA（kcat，98.0 min⁻¹；Km，0.32 ± 0.04 μM）有活性。saFabH被硫乳霉素弱抑制（50%抑制浓度[IC50]，> 100 μM），但被两种新的FabH抑制剂，5 - 氯 - 4 - 苯基 - [1,2] - 二硫醇 - 3 - 酮（IC50，1.87 ± 0.10 μM）和4 - 苯基 - 5 - 苯基亚氨基 - [1,2,4]二噻唑烷 - 3 - 酮（IC50，0.775 ± 0.08 μM）有效抑制。
Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the P-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent K-m for malonyl-ACP was 1.76 +/- 0.40 muM, and the enzyme was active with acetyl-CoA (k(cat), 16.18 min(-1); K-m, 6.18 +/- 0.9 muM), butyryl-CoA (k(cat), 42.90 min(-1); K-m, 2.32 +/- 0.12 muM), and isobutyryl-CoA (k(cat), 98.0 min(-1); K-m, 0.32 +/- 0.04 muM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], > 100 muM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 +/- 0.10 muM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one(IC50, 0.775 +/- 0.08 muM).