Hoang T T, Schweizer H P
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
J Bacteriol. 1999 Sep;181(17):5489-97. doi: 10.1128/JB.181.17.5489-5497.1999.
The Pseudomonas aeruginosa fabI structural gene, encoding enoyl-acyl carrier protein (ACP) reductase, was cloned and sequenced. Nucleotide sequence analysis revealed that fabI is probably the last gene in a transcriptional unit that includes a gene encoding an ATP-binding protein of an ABC transporter of unknown function. The FabI protein was similar in size and primary sequence to other bacterial enoyl-ACP reductases, and it contained signature motifs for the FAD-dependent pyridine nucleotide reductase and glucose/ribitol dehydrogenase families, respectively. The chromosomal fabI gene was disrupted, and the resulting mutant was viable but possessed only 62% of the total enoyl-ACP reductase activity found in wild-type cell extracts. The fabI-encoded enoyl-ACP reductase activity was NADH dependent and inhibited by triclosan; the residual activity in the fabI mutant was also NADH dependent but not inhibited by triclosan. An polyhistidine-tagged FabI protein was purified and characterized. Purified FabI (i) could use NADH but not NADPH as a cofactor; (ii) used both crotonyl-coenzyme A and crotonyl-ACP as substrates, although it was sixfold more active with crotonyl-ACP; and (iii) was efficiently inhibited by low concentrations of triclosan. A FabI Gly95-to-Val active-site amino acid substitution was generated by site-directed mutagenesis, and the mutant protein was purified. The mutant FabI protein retained normal enoyl-ACP reductase activity but was highly triclosan resistant. When coupled to FabI, purified P. aeruginosa N-butyryl-L-homoserine lactone (C4-HSL) synthase, RhlI, could synthesize C4-HSL from crotonyl-ACP and S-adenosylmethionine. This reaction was NADH dependent and inhibited by triclosan. The levels of C4-HSL and N-(3-oxo)-dodecanoyl-L-homoserine lactones were reduced 50% in a fabI mutant, corroborating the role of FabI in acylated homoserine lactone synthesis in vivo.
编码烯酰-酰基载体蛋白（ACP）还原酶的铜绿假单胞菌fabI结构基因被克隆并测序。核苷酸序列分析表明，fabI可能是一个转录单元中的最后一个基因，该转录单元包括一个编码功能未知的ABC转运蛋白的ATP结合蛋白的基因。FabI蛋白在大小和一级序列上与其他细菌烯酰-ACP还原酶相似，并且分别包含FAD依赖的吡啶核苷酸还原酶和葡萄糖/核糖醇脱氢酶家族的特征基序。染色体上的fabI基因被破坏，产生的突变体是存活的，但仅具有野生型细胞提取物中总烯酰-ACP还原酶活性的62%。fabI编码的烯酰-ACP还原酶活性依赖于NADH并被三氯生抑制；fabI突变体中的残余活性也依赖于NADH但不被三氯生抑制。一个带有多组氨酸标签的FabI蛋白被纯化并进行了表征。纯化的FabI（i）可以使用NADH而不是NADPH作为辅因子；（ii）使用巴豆酰辅酶A和巴豆酰-ACP作为底物，尽管它对巴豆酰-ACP的活性高六倍；（iii）被低浓度的三氯生有效抑制。通过定点诱变产生了FabI Gly95到Val的活性位点氨基酸取代，并纯化了突变蛋白。突变的FabI蛋白保留了正常的烯酰-ACP还原酶活性，但对三氯生具有高度抗性。当与FabI偶联时，纯化的铜绿假单胞菌N-丁酰-L-高丝氨酸内酯（C4-HSL）合酶RhlI可以从巴豆酰-ACP和S-腺苷甲硫氨酸合成C4-HSL。该反应依赖于NADH并被三氯生抑制。fabI突变体中C4-HSL和N-（3-氧代）-十二烷酰-L-高丝氨酸内酯的水平降低了50%，证实了FabI在体内酰化高丝氨酸内酯合成中的作用。
