Keating D H, Carey M R, Cronan J E
Department of Microbiology, University of Illinois at Urbana-Champaign 61801, USA.
J Biol Chem. 1995 Sep 22;270(38):22229-35. doi: 10.1074/jbc.270.38.22229.
Acyl carrier protein (ACP) is the carrier of fatty acids during their synthesis and utilization. ACPs (or ACP-like protein domains) have been found throughout biology and share significant amino acid sequence similarities. All ACPs undergo a post-translational modification in which 4'-phosphopantetheine is transferred from CoA to a specific serine of apo-ACP. This modification is essential for activity because fatty acids are bound in thioester linkage to the sulfhydryl of the prosthetic group. Overproduction of Escherichia coli ACP from multicopy plasmids strongly inhibits growth of E. coli. We report that upon overexpression of ACP in E. coli post-translational modification is inefficient and the apo protein accumulates and blocks cell growth by inhibition of lipid metabolism. Moreover, a mutant form of ACP that is unable to undergo post-translational modification is a potent inhibitor of growth. Finally, we observed that an increase in the efficiency of modification of overexpressed ACP results in decreased toxicity. The accumulated apo-ACP acts as a potent in vitro inhibitor of the sn-glycerol-3-phosphate acyltransferase resulting in an inability to transfer the completed fatty acid to sn-glycerol 3-phosphate. The degree of inhibition depended upon the species of donor acyl chain. Utilization of cis-vaccenoyl-ACP by the sn-glycerol-3-phosphate acyltransferase was inhibited to a much greater extent by apo-ACP than was utilization of palmitoyl-ACP. 1-Acyl glycerol-3-phosphate acyltransferase was also inhibited in vitro by apo-ACP, although not at physiologically relevant concentrations. These in vitro data are supported by in vivo labeling data, which showed a large decrease in cis-vaccenate incorporation into phospholipid during overproduction of ACP, but no decrease in the rate of synthesis of long chain acyl-ACPs. These data indicate that acylation of sn-glycerol 3-phosphate is the major site of inhibition by apo-ACP.
酰基载体蛋白（ACP）是脂肪酸合成和利用过程中的载体。在整个生物界都发现了ACP（或ACP样蛋白结构域），它们具有显著的氨基酸序列相似性。所有的ACP都经历一种翻译后修饰，即4'-磷酸泛酰巯基乙胺从辅酶A转移到脱辅基ACP的一个特定丝氨酸上。这种修饰对于活性至关重要，因为脂肪酸以硫酯键与辅基的巯基结合。从多拷贝质粒过量表达大肠杆菌ACP会强烈抑制大肠杆菌的生长。我们报告，在大肠杆菌中过量表达ACP时，翻译后修饰效率低下，脱辅基蛋白积累，并通过抑制脂质代谢来阻断细胞生长。此外，一种无法进行翻译后修饰的ACP突变形式是生长的有效抑制剂。最后，我们观察到过量表达的ACP修饰效率的提高会导致毒性降低。积累的脱辅基ACP作为sn-甘油-3-磷酸酰基转移酶的一种有效的体外抑制剂，导致无法将完整的脂肪酸转移到sn-甘油3-磷酸上。抑制程度取决于供体酰基链的种类。apo-ACP对sn-甘油-3-磷酸酰基转移酶利用顺式- vaccenoyl-ACP的抑制程度比对棕榈酰-ACP的利用抑制程度大得多。1-酰基甘油-3-磷酸酰基转移酶在体外也受到apo-ACP的抑制，尽管在生理相关浓度下没有受到抑制。这些体外数据得到了体内标记数据的支持，体内标记数据显示，在过量生产ACP期间，顺式- vaccenate掺入磷脂的量大幅下降，但长链酰基-ACP的合成速率没有下降。这些数据表明，sn-甘油3-磷酸的酰化是apo-ACP抑制的主要位点。
