Methylmalonyl-CoA decarboxylase from Propionigenium modestum cloning and sequencing of the structural genes and purification of the enzyme complex
Methylmalonyl-CoA decarboxylase from Propionigenium modestum cloning and sequencing of the structural genes and purification of the enzyme complex
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DOI：
10.1111/j.1432-1033.1997.0590a.x
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发表时间：
1997-12-01
期刊：
影响因子：
--
通讯作者：
Dimroth, P
中科院分区：
文献类型：
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作者：
Bott, M;Pfister, K;Dimroth, P
丙酸丙酯发酵过程中，甲基丙二酰辅酶A脱羧酶催化唯一的能量守恒步骤：(S)-甲基丙二酰辅酶A脱羧基为丙酰辅酶A，与Na+跨细胞膜的矢量运输偶联，从而产生用于三磷酸腺苷合成的钠离子动力。利用其他Na+转运脱羧酶β亚基之间的序列相似性，用简并引物进行聚合酶链式反应，扩增出白念珠菌β亚基基因的一部分。然后将克隆的PCR产物作为同源探针，从基因组DNA中克隆合适的片段。对3.7kb区域的序列分析发现了四个基因，它们可能形成了一个转录单位MmdADCB。值得注意的是，微小韦洛氏菌中存在一个编码6-kDa epsilon亚基的同源MmdADECB基因，该基因在P.modemum中缺失。通过序列比较，可以将这些蛋白的功能归类为：MMDA(56.1 kDa；α-亚基)，羧基转移酶；MmdB(41.2 kDa；β-亚基)，羧基生物素载体-蛋白脱羧酶；MmdC(13.1 kDa，γ-亚基)，生物素载体蛋白。MmdD(14.2 kDa：Delta亚基)可能是组装复合体所必需的，如相应的微小弧菌蛋白所示。甲基丙二酰辅酶A脱羧酶以正十二烷基麦芽糖苷为底物，经亲和层析分离纯化15倍。纯化的蛋白质由4个亚基组成，N端序列分析表明其中3个亚基分别为MMDA、MmdD和MmdC。纯化的酶比活力高达25U/mg蛋白，对(S)-甲基丙二酰辅酶A的表观K-m值约为12 mU。与微小弧菌的5个亚基复合体相比，该酶的4个亚基似乎更不稳定，这可能是缺乏表观亚基的结果。
Methylmalonyl-CoA decarboxylase catalyses the only energy-conserving step during succinate fermentation by Propionigenium modestum: the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA is coupled to the vectorial transport of Na+ across the cytoplasmic membrane, thereby creating a sodium ion motive force that is used for ATP synthesis. By taking advantage of the sequence similarity between the beta-subunits of other Na+-transport decarboxylases, a portion of the P. modestum beta-subunit gene was amplified by PCR with degenerated primers. The cloned PCR product then served as homologous probe for cloning suitable fragments from genomic DNA. Sequence analysis of a 3.7-kb region identified four genes which probably form a transcriptional unit, mmdADCB. Remarkably a mmdE gene which is present in the homologous mmdADECB cluster from Veillonella parvula and encodes the 6-kDa epsilon-subunit, is missing in P. modestum. By sequence comparisons, the following functions could be assigned to the P. modestum proteins: MmdA (56.1 kDa; alpha-subunit), carboxyltransferase; MmdB (41.2 kDa; beta-subunit), carboxybiotin-carrier-protein decarboxylase; MmdC (13.1 kDa; gamma-subunit), biotin carrier protein. MmdD (14.2 kDa: delta-subunit) presumably is essential for the assembly of the complex, as shown for the corresponding V. parvula protein. Methylmalonyl-CoA decarboxylase was solubilized from membranes of P. modestum with n-dodecylmaltoside and enriched 15-fold by affinity chromatography on monomeric avidin resin. The purified protein was composed of four subunits, three of which were identified by N-terminal sequence analysis as MmdA, MmdD, and MmdC. The purified enzyme exhibited a specific activity of up to 25 U/mg protein and an apparent K-m value for (S)-methylmalonyl-CoA of approximate to 12 mu M. Compared to the five-subunit complex of V. parvula, the four-subunit enzyme of P. modestum appeared to be more labile, presumably a consequence of the lack of the epsilon-subunit.