Krüger N, Oppermann F B, Lorenzl H, Steinbüchel A
Institut für Mikrobiologie, Georg-August-Universität zu Göttingen, Germany.
J Bacteriol. 1994 Jun;176(12):3614-30. doi: 10.1128/jb.176.12.3614-3630.1994.
E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product, which is a regulatory protein required for expression of the A. eutrophus aco genes, and the A. eutrophus acoX gene product, which has an unknown function, respectively. The aco genes of C. magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.
巨大梭菌乙偶姻脱氢酶系统的E2（二氢硫辛酰胺乙酰转移酶）和E3（二氢硫辛酰胺脱氢酶）通过三步法从以乙偶姻为碳源生长的细胞中共同纯化得到。变性的E2 - E3制剂包含两条多肽，其相对分子质量分别为49,000和67,000。对这两种蛋白质进行微量测序，结果显示氨基酸序列相同。利用基于最近纯化的E1（乙偶姻脱氢酶，依赖胸腺嘧啶焦磷酸）的α和β亚基N端序列以及E2 - E3的寡核苷酸探针，在单个ClaI限制性片段上鉴定出结构基因acoA（编码E1α）、acoB（编码E1β）、acoC（编码E2）和acoL（编码E3），并在大肠杆菌中表达。测定了acoA（978 bp）、acoB（999 bp）、acoC（1,332 bp）和acoL（1,734 bp）以及acoX（996 bp）和acoR（1,956 bp）的核苷酸序列。从acoA、acoB、acoC和acoL推导得到的E1α（相对分子质量35,532）、E1β（相对分子质量35,541）、E2（相对分子质量48,149）和E3（相对分子质量61,255）的氨基酸序列分别与甲醇杆菌乙偶姻脱氢酶系统和真养产碱菌乙偶姻裂解系统相应组分的氨基酸序列具有显著相似性。还发现与各种2 - 氧代酸脱氢酶复合物的酶组分有明显同源性，表明这两个酶系统之间关系密切。由于acoC的5'编码区部分重复到acoL的相应部分，巨大梭菌乙偶姻脱氢酶系统的E3组分含有一个N端硫辛酰结构域，这在二氢硫辛酰胺脱氢酶中是独特的。我们发现AcoR和AcoX序列与真养产碱菌acoR基因产物（真养产碱菌aco基因表达所需的调节蛋白）和真养产碱菌acoX基因产物（功能未知）分别有很强的相似性。巨大梭菌的aco基因可能组织在一个单一操纵子（acoABXCL）中；acoR位于该操纵子的上游。
