Skinner D D, Morgenstern M R, Fedechko R W, Denoya C D
Bioprocess Research, Central Research Division, Pfizer Inc., Groton, Connecticut 06340.
J Bacteriol. 1995 Jan;177(1):183-90. doi: 10.1128/jb.177.1.183-190.1995.
A cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1 alpha), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1 beta), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (alpha or beta) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1 [alpha beta] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1 alpha) and ORF2 (E1 beta) coexpressed under the control of the T7 promoter.
阿维链霉菌支链α-酮酸脱氢酶（BCDH）的E1α、E1β和E2亚基的一组编码基因已被克隆并测序。开放阅读框1（ORF1）（E1α）长1146个核苷酸，将编码一个40969Da（381个氨基酸）的多肽。ORF2（E1β）长1005个核苷酸，将编码一个35577Da（334个氨基酸）的多肽。ORF1和ORF2之间的基因间隔为73bp。不完整的ORF3（E2）的推定ATG起始密码子与ORF2的终止密码子重叠。计算机辅助搜索表明，ORF1和ORF2的推导产物类似于几种原核和真核BCDH复合物的相应E1亚基（α或β）。当这些ORF在大肠杆菌中过表达时，分别检测到约41kDa和34kDa的蛋白质，它们分别是预测的阿维链霉菌ORF1和ORF2产物的近似质量。此外，在携带在T7启动子控制下共表达的阿维链霉菌ORF1（E1α）和ORF2（E1β）的大肠杆菌细胞中检测到特异性E1[αβ]BCDH活性。
