β-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis
β-ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis
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DOI：
10.1074/jbc.m308638200
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发表时间：
2003-12-19
影响因子：
4.8
通讯作者：
Cronan, JE
中科院分区：
文献类型：
--
作者：
Lai, CY;Cronan, JE
由fabH基因编码的β-酮酰基-酰基载体蛋白（ACP）合酶III（KAS III，也称为乙酰乙酰-ACP合酶）被认为催化细菌和植物质体中II型脂肪酸合成的第一延伸反应（克莱森缩合）。然而，直接在体内的证据表明，KAS III催化一个必要的反应是缺乏的，因为没有突变体生物缺乏这种活动已被隔离。我们报告了第一个缺乏KAS III的细菌菌株，这是一种在革兰氏阳性菌乳酸乳球菌乳酸亚种IL 1403中构建的FabH突变体。突变株携带的KAS III活性位点区域的框内缺失，并通过使用补充有饱和和不饱和长链脂肪酸来源的培养基进行基因置换来分离。突变株缺乏KAS III活性，并且在不补充外源性长链脂肪酸的情况下不能生长，这表明KAS III在细胞代谢中起重要作用。然而，L.乳酸菌fabH缺失突变体的生长仅需要长链不饱和脂肪酸，不需要长链饱和脂肪酸的来源。因为当脂肪酸合成被生物素饥饿阻断（其阻止丙二酰-CoA的合成）时，生长需要饱和和不饱和脂肪酸两者，所以在fabH缺失菌株中必须保留另一种饱和脂肪酸合成途径。事实上，在体内将[1-C-14]乙酸酯掺入脂肪酸中表明，fabH突变体保留了野生型菌株的约10%的脂肪酸合成能力，并且这种剩余的合成能力优先转移到途径的饱和分支。此外，质谱分析表明，fabH突变体保留低水平的棕榈酸脂肪酸饥饿后。衍生物的fabH缺失突变株分离的辛酸营养缺陷型与生化研究表明，FabH的主要作用是生产短链脂肪酸引物一致。我们还证实了FabH在大肠杆菌中的必要性，通过使用基于质粒的基因插入/缺失系统。这些结果一起提供了第一个遗传证据，证明FabH在革兰氏阳性和革兰氏阴性细菌中II型脂肪酸生物合成的起始中进行主要的缩合反应。
beta-Ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called acetoacetyl-ACP synthase) encoded by the fabH gene is thought to catalyze the first elongation reaction (Claisen condensation) of type II fatty acid synthesis in bacteria and plant plastids. However, direct in vivo evidence that KAS III catalyzes an essential reaction is lacking, because no mutant organism deficient in this activity has been isolated. We report the first bacterial strain lacking KAS III, a fabH mutant constructed in the Gram-positive bacterium Lactococcus lactis subspecies lactis IL1403. The mutant strain carries an in-frame deletion of the KAS III active site region and was isolated by gene replacement using a medium supplemented with a source of saturated and unsaturated long-chain fatty acids. The mutant strain is devoid of KAS III activity and fails to grow in the absence of supplementation with exogenous long-chain fatty acids demonstrating that KAS III plays an essential role in cellular metabolism. However, the L. lactis fabH deletion mutant requires only long-chain unsaturated fatty acids for growth, a source of long-chain saturated fatty acids is not required. Because both saturated and unsaturated fatty acids are required for growth when fatty acid synthesis is blocked by biotin starvation ( which prevents the synthesis of malonyl-CoA), another pathway for saturated fatty acid synthesis must remain in the fabH deletion strain. Indeed, incorporation of [1-C-14] acetate into fatty acids in vivo showed that the fabH mutant retained about 10% of the fatty acid synthetic ability of the wild-type strain and that this residual synthetic capacity was preferentially diverted to the saturated branch of the pathway. Moreover, mass spectrometry showed that the fabH mutant retained low levels of palmitic acid upon fatty acid starvation. Derivatives of the fabH deletion mutant strain were isolated that were octanoic acid auxotrophs consistent with biochemical studies indicating that the major role of FabH is production of short-chain fatty acid primers. We also confirmed the essentiality of FabH in Escherichia coli by use of a plasmid-based gene insertion/deletion system. Together these results provide the first genetic evidence demonstrating that FabH conducts the major condensation reaction in the initiation of type II fatty acid biosynthesis in both Gram-positive and Gram-negative bacteria.