Braune A, Bendrat K, Rospert S, Buckel W
Laboratorium für Mikrobiologie, Philipps-Universität, Marburg, Germany.
Mol Microbiol. 1999 Jan;31(2):473-87. doi: 10.1046/j.1365-2958.1999.01189.x.
Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans (clostridal cluster IX), a strict anaerobic inhabitant of animal intestines, uses the free energy of decarboxylation (delta G(o) approximately -30 kJ mol-1) in order to translocate Na+ from the inside through the cytoplasmic membrane. The proton, which is required for decarboxylation, most probably comes from the outside. The enzyme consists of four different subunits. The largest subunit, alpha or GcdA (65 kDa), catalyses the transfer of CO2 from glutaconyl-CoA to biotin covalently attached to the gamma-subunit, GcdC. The beta-subunit, GcdB, is responsible for the decarboxylation of carboxybiotin, which drives the Na+ translocation (approximate K(m) for Na+ 1 mM), whereas the function of the smallest subunit, delta or GcdD, is unclear. The gene gcdA is part of the 'hydroxyglutarate operon', which does not contain genes coding for the other three subunits. This paper describes that the genes, gcdDCB, are transcribed in this order from a distinct operon. The delta-subunit (GcdD, 12 kDa), with one potential transmembrane helix, probably serves as an anchor for GcdA. The biotin carrier (GcdC, 14 kDa) contains a flexible stretch of 50 amino acid residues (A26-A75), which consists of 34 alanines, 14 prolines, one valine and one lysine. The beta-subunit (GcdB, 39 kDa) comprising 11 putative transmembrane helices shares high amino acid sequence identities with corresponding deduced gene products from Veillonella parvula (80%, clostridial cluster IX), Archaeoglobus fulgidus (61%, Euryarchaeota), Propionigenium modestum (60%, clostridial cluster XIX), Salmonella typhimurium (51%, enterobacteria) and Klebsiella pneumoniae (50%, enterobacteria). Directly upstream of the promoter region of the gcdDCB operon, the 3' end of gctM was detected. It encodes a protein fragment with 73% sequence identity to the C-terminus of the alpha-subunit of methylmalonyl-CoA decarboxylase from V. parvula (MmdA). Hence, it appears that A. fermentans should be able to synthesize this enzyme by expression of gctM together with gdcDCB, but methylmalonyl-CoA decarboxylase activity could not be detected in cell-free extracts. Earlier observations of a second, lower affinity binding site for Na+ of glutaconyl-CoA decarboxylase (apparent K(m) 30 mM) were confirmed by identification of the cysteine residue 243 of GcdB between the putative hellces VII and VIII, which could be specifically protected from alkylation by Na+. The alpha-subunit was purified from an overproducing Escherichia coli strain and was characterized as a putative homotrimer able to catalyse the carboxylation of free biotin.
来自发酵氨基酸球菌（梭菌属第九簇）的戊二酰辅酶A脱羧酶是动物肠道中的严格厌氧菌，它利用脱羧的自由能（ΔG°约为 -30 kJ/mol）将Na⁺从细胞内转运穿过细胞质膜。脱羧所需的质子很可能来自细胞外。该酶由四个不同的亚基组成。最大的亚基α或GcdA（65 kDa）催化CO₂从戊二酰辅酶A转移到与γ亚基GcdC共价连接的生物素上。β亚基GcdB负责羧基生物素的脱羧反应，驱动Na⁺转运（Na⁺的近似Kₘ为1 mM），而最小的亚基δ或GcdD的功能尚不清楚。基因gcdA是 “羟基戊二酸操纵子” 的一部分，该操纵子不包含编码其他三个亚基的基因。本文描述了基因gcdDCB从一个独特的操纵子按此顺序转录。δ亚基（GcdD，12 kDa）有一个潜在的跨膜螺旋，可能作为GcdA的锚定物。生物素载体（GcdC，14 kDa）包含一段由50个氨基酸残基组成的柔性片段（A26 - A75），其中包括34个丙氨酸、14个脯氨酸、1个缬氨酸和1个赖氨酸。包含11个推定跨膜螺旋的β亚基（GcdB，39 kDa）与细小韦荣球菌（80%，梭菌属第九簇）、嗜热栖热放线菌（61%，广古菌门）、适度丙酸杆菌（60%，梭菌属第十九簇）、鼠伤寒沙门氏菌（51%，肠杆菌科）和肺炎克雷伯菌（50%，肠杆菌科）相应推导基因产物具有高度的氨基酸序列同一性。在gcdDCB操纵子启动子区域的直接上游，检测到gctM的3' 端。它编码一个与细小韦荣球菌甲基丙二酰辅酶A脱羧酶α亚基C端具有73% 序列同一性的蛋白质片段（MmdA）。因此，似乎发酵氨基酸球菌应该能够通过gctM与gdcDCB的共同表达来合成这种酶，但在无细胞提取物中未检测到甲基丙二酰辅酶A脱羧酶活性。早期关于戊二酰辅酶A脱羧酶第二个低亲和力Na⁺结合位点（表观Kₘ为30 mM）的观察结果，通过鉴定GcdB在推定螺旋VII和VIII之间的半胱氨酸残基243得到证实，该残基可被Na⁺特异性保护不被烷基化。α亚基从过量表达的大肠杆菌菌株中纯化出来，并被表征为一个推定的同三聚体，能够催化游离生物素的羧化反应。
