Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, Tennessee, USA.
Department of Infectious Diseases, St Jude Children's Research Hospital, Memphis, Tennessee, USA.
J Biol Chem. 2021 Dec;297(6):101434. doi: 10.1016/j.jbc.2021.101434. Epub 2021 Nov 18.
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 (MadA) and a carboxy-terminal hot dog dimerization domain (MadA) 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, MadA, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadA 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.
大肠杆菌中的细菌脂肪酸合成是由β-酮酰-ACP 合酶 III 酶 FabH 催化乙酰-CoA 与丙二酰-ACP 酰基辅酶蛋白（ACP）缩合启动的。由于 yiiD 基因的存在，大肠杆菌 ΔfabH 敲除株具有生存能力，该基因允许 FabH 独立的脂肪酸合成起始。然而，yiiD 基因产物的分子功能尚不清楚。在这里，我们证明 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（MadA）和羧基末端热狗二聚化结构域（MadA），后者编码丙二酰-ACP 脱羧酶功能。变形菌 Mad 蛋白家族的成员要么是两个结构域的 MadA（GNAT-热狗），要么是独立的 MadB（热狗）脱羧酶。使用结构导向的、定点突变 Shewanella oneidensis 的 MadB，我们鉴定出保守催化环上的天冬酰胺 45 对脱羧酶活性至关重要。我们还发现，MadA、MadA 或 MadB 的表达都能恢复大肠杆菌 ΔfabH 菌株的正常细胞大小和生长速率，而 MadA 的表达则不能。最后，我们验证了 GlmU，一种双功能葡萄糖胺-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，合成关键中间产物 UDP-GlcNAc，是 ACP 结合蛋白。乙酰-ACP 是葡萄糖胺-1-磷酸 N-乙酰转移酶/N-乙酰-葡萄糖胺-1-磷酸尿苷酰转移酶的首选底物，除了是延伸缩合酶 FabB 和 FabF 的底物外。因此，我们得出结论，Mad 家族的丙二酰-ACP 脱羧酶为脂肪酸、脂多糖、肽聚糖和肠杆菌共同抗原生物合成提供乙酰-ACP，以支持其起始。
