Hawkins C F, Borges A, Perham R N
Department of Biochemistry, University of Cambridge, England.
Eur J Biochem. 1990 Jul 31;191(2):337-46. doi: 10.1111/j.1432-1033.1990.tb19128.x.
A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.
嗜热脂肪芽孢杆菌丙酮酸脱氢酶多酶复合体中编码丙酮酸脱氢酶（硫辛酰胺）组分（E1）的α和β链的一段4175bp的EcoRI DNA片段已在大肠杆菌中克隆。测定了其核苷酸序列。借助从纯化的多肽链直接测定的氨基酸序列，鉴定并确认了与E1α亚基（368个氨基酸，Mr 41,312，不包括起始甲硫氨酸残基）和E1β亚基（324个氨基酸，Mr 35,306，不包括起始甲硫氨酸残基）相对应的开放阅读框（pdhA、pdhB）。E1β基因起始于E1α终止密码子下游仅3bp处。在间隔73bp的较长间隙之后，是另一个但不完整的开放阅读框的起始，根据其已知氨基酸序列，该阅读框编码复合体的二氢硫辛酰乙酰转移酶（E2）组分。所有三个基因之前都有潜在的核糖体结合位点，并且基因簇位于一段显示出众多可能启动子序列的DNA区域的紧邻下游。嗜热脂肪芽孢杆菌丙酮酸脱氢酶复合体的E1α和E1β亚基与来自哺乳动物线粒体和恶臭假单胞菌的丙酮酸和支链2-氧代酸脱氢酶复合体的E1α和E1β亚基表现出显著的序列相似性。特别是，E1α链包含在所有以硫胺二磷酸为辅因子的酶中都发现的高度保守的序列基序。
