Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.

中文翻译：

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丙二酰基载体蛋白脱羧酶活性促进变形菌中的脂肪酸和细胞被膜生物合成。

大肠杆菌中的细菌脂肪酸合成是通过 β-酮酰基-ACP 合酶 III 酶 FabH 将乙酰辅酶 A 与丙二酰基-酰基载体蛋白 (ACP) 缩合而启动的。大肠杆菌 ΔfabH 敲除菌株之所以可行，是因为 yiiD 基因允许启动不依赖于 FabH 的脂肪酸合成。然而，yiiD 基因产物的分子功能尚不清楚。在这里，我们展示了 yiiD 基因产物是丙二酰-ACP 脱羧酶 (MadA)。MadA 具有两个独立折叠的结构域：氨基末端 N-乙酰转移酶 (GNAT) 结构域 (MadAN) 和羧基末端热狗二聚化结构域 (MadAC)，编码丙二酰基-ACP 脱羧酶功能。变形菌 Mad 蛋白家族的成员是两个结构域 MadA（GNAT-热狗）或独立的 MadB（热狗）脱羧酶。通过对 Shewanella oneidensis 的 MadB 进行结构引导、定点诱变，我们确定了保守催化环上的 Asn45 对于脱羧酶活性至关重要。我们还发现，MadA、MadAC 或 MadB 表达均使大肠杆菌 ΔfabH 菌株恢复正常细胞大小和生长速率，而 MadAN 表达则不然。最后，我们验证了 GlmU（一种合成关键中间体 UDP-GlcNAc 的双功能葡萄糖胺-1-磷酸 N-乙酰转移酶/N-乙酰-葡萄糖胺-1-磷酸尿苷酰转移酶）是 ACP 结合蛋白。乙酰基-ACP 是优选的 1-磷酸葡糖胺 N-乙酰基转移酶/N-乙酰基-1-磷酸葡糖胺尿嘧啶基转移酶底物，此外也是延伸缩合酶 FabB 和 FabF 的底物。因此，我们得出结论，丙二酰 ACP 脱羧酶 Mad 家族提供乙酰 ACP，以支持变形菌中脂肪酸、脂多糖、肽聚糖和肠杆菌共同抗原生物合成的启动。