Lai Chiou-Yan, Cronan John E
Department of Microbiology and Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 2003 Dec 19;278(51):51494-503. doi: 10.1074/jbc.M308638200. Epub 2003 Sep 30.
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 [1-14C]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.
由fabH基因编码的β-酮脂酰-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型脂肪酸合成的首个延伸反应（克莱森缩合反应）。然而，缺乏KAS III催化关键反应的直接体内证据，因为尚未分离出缺乏该活性的突变生物体。我们报道了首个缺乏KAS III的细菌菌株，这是在革兰氏阳性细菌乳酸乳球菌乳酸亚种IL1403中构建的fabH突变体。该突变菌株在KAS III活性位点区域存在框内缺失，通过使用补充了饱和和不饱和长链脂肪酸来源的培养基进行基因置换而分离得到。该突变菌株缺乏KAS III活性，在不补充外源长链脂肪酸的情况下无法生长，这表明KAS III在细胞代谢中起关键作用。然而，乳酸乳球菌fabH缺失突变体生长仅需要长链不饱和脂肪酸，不需要长链饱和脂肪酸来源。当脂肪酸合成因生物素饥饿（这会阻止丙二酰辅酶A的合成）而受阻时，生长需要饱和脂肪酸和不饱和脂肪酸，因此fabH缺失菌株中必定存在另一条饱和脂肪酸合成途径。实际上，体内[1-14C]乙酸掺入脂肪酸的实验表明，fabH突变体保留了野生型菌株约10%的脂肪酸合成能力，并且这种残余合成能力优先转向该途径的饱和分支。此外，质谱分析表明，fabH突变体在脂肪酸饥饿时保留了低水平的棕榈酸。分离得到了fabH缺失突变体菌株的衍生物，它们是辛酸营养缺陷型，这与生化研究结果一致，表明FabH的主要作用是产生短链脂肪酸引物。我们还通过基于质粒的基因插入/缺失系统证实了FabH在大肠杆菌中的必要性。这些结果共同提供了首个遗传学证据，证明FabH在革兰氏阳性和革兰氏阴性细菌的II型脂肪酸生物合成起始过程中进行主要的缩合反应。
