Li Yongli, Florova Galina, Reynolds Kevin A
Department of Medicinal Chemistry and Institute of Structural Biology and Drug Discovery, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia 23219, USA.
J Bacteriol. 2005 Jun;187(11):3795-9. doi: 10.1128/JB.187.11.3795-3799.2005.
The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (approximately 70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a deltafabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.
II型解离脂肪酸合酶催化的脂肪酸生物合成的第一步延伸反应由3-酮酰基-酰基载体蛋白（ACP）合酶III（KASIII，FabH）催化。该酶由fabH基因编码，催化酰基辅酶A（CoA）引物与丙二酸单酰-ACP之间的脱羧缩合反应。在仅产生直链脂肪酸（SCFA）的生物体如大肠杆菌中，FabH对乙酰辅酶A具有底物偏好性。在产生SCFA和支链脂肪酸（BCFA）混合物的链霉菌和其他生物体中，FabH已被证明可利用直链和支链酰基辅酶A底物。我们在此报告了天蓝色链霉菌突变体（YL/ecFabH）的产生，其中fabH基因的染色体拷贝已被替换，脂肪酸生物合成的基本过程由基于质粒表达的大肠杆菌FabH（与链霉菌酶仅具有35%的氨基酸同一性）启动。YL/ecFabH突变体主要产生SCFA（86%）。相比之下，在天蓝色链霉菌亲本菌株和天蓝色链霉菌YL/sgFabH（携带表达浅青紫链霉菌FabH的质粒的ΔfabH突变体）中，BCFA占主导（约70%）。这些结果提供了首个明确证据，即体外观察到的FabH底物特异性是生物体中脂肪酸合成的决定因素。YL/ecFabH菌株在固体和液体培养基上的生长速度均显著较慢。YL/ecFabH细胞提取物中的FabH活性水平也显著低于YL/sgFabH细胞提取物中的活性水平，这表明脂肪酸合成速率降低可能是观察到的生长速率下降的原因。YL/ecFabH中低水平BCFA的产生表明，要么大肠杆菌FabH对不同酰基辅酶A底物的耐受性比之前认为的更高，要么在天蓝色链霉菌中存在另一条启动BCFA生物合成的途径。
