Jiang Yanfang, Chan Chi Ho, Cronan John E
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
Biochemistry. 2006 Aug 22;45(33):10008-19. doi: 10.1021/bi060842w.
The gene encoding the unique soluble acyl-acyl carrier protein synthetase (AasS) of the bioluminescent Vibrio harveyi strain B392 has been isolated by expression cloning in Escherichia coli. This enzyme catalyzes the ATP-dependent acylation of the thiol of acyl carrier protein (ACP) with fatty acids with chain lengths from C4 to C18. The gene (called aasS) encodes a protein of 60 kDa, a hexahistidine-tagged version of which was readily expressed in E. coli and purified in large quantities. Surprisingly, the sequence of the encoded protein was significantly more similar to that of an acyl-CoA synthetase of the distantly related bacterium, Thermus thermophilus, than to that of the membrane-bound acyl-acyl carrier protein synthetase of E. coli, an enzyme that catalyzes the same reaction from a more closely related organism. Indeed, the AasS sequence can readily be modeled on the known crystal structures of the T. thermophilus acyl-CoA synthetase with remarkably high levels of conservation of the catalytic site residues. To test the possible role of AasS in the fatty aldehyde-dependent bioluminescence pathway of V. harveyi, the chromosomal aasS gene of the organism was disrupted by insertion of a kanamycin cassette by homologous recombination. The resulting aasS::kan strains retained low levels of acyl-acyl carrier protein synthetase consistent with prior indications of a second such activity in this bacterium. The mutant strains grew normally and had normal levels of bioluminescence but were deficient in the incorporation of exogenous octanoic acid into the cellular phospholipids of V. harveyi, particularly at low octanoate concentrations. These data indicate that AasS is responsible for a high-affinity and high-capacity uptake system that efficiently converts exogenous fatty acids into acyl-ACP species competent to enter the fatty acid biosynthetic cycle.
通过在大肠杆菌中进行表达克隆，已分离出编码发光哈维氏弧菌菌株B392独特的可溶性酰基 - 酰基载体蛋白合成酶（AasS）的基因。该酶催化酰基载体蛋白（ACP）的硫醇与链长为C4至C18的脂肪酸进行ATP依赖性酰化反应。该基因（称为aasS）编码一种60 kDa的蛋白质，其六聚组氨酸标签版本在大肠杆菌中易于表达并大量纯化。令人惊讶的是，与大肠杆菌中膜结合的酰基 - 酰基载体蛋白合成酶（一种在亲缘关系更近的生物体中催化相同反应的酶）相比，编码蛋白的序列与远缘细菌嗜热栖热菌的酰基辅酶A合成酶的序列更为相似。实际上，AasS序列可以很容易地根据嗜热栖热菌酰基辅酶A合成酶的已知晶体结构进行建模，催化位点残基具有非常高的保守水平。为了测试AasS在哈维氏弧菌脂肪酸醛依赖性生物发光途径中的可能作用，通过同源重组插入卡那霉素盒破坏了该生物体的染色体aasS基因。所得的aasS::kan菌株保留了低水平的酰基 - 酰基载体蛋白合成酶，这与该细菌中第二种此类活性的先前迹象一致。突变菌株生长正常，生物发光水平正常，但在将外源辛酸掺入哈维氏弧菌的细胞磷脂中存在缺陷，特别是在低辛酸浓度下。这些数据表明，AasS负责一个高亲和力和高容量的摄取系统，该系统有效地将外源脂肪酸转化为能够进入脂肪酸生物合成循环的酰基 - ACP物种。
