Ono M, Matsuzawa H, Ohta T
Department of Agricultural Chemistry, University of Tokyo.
J Biochem. 1990 Jan;107(1):21-6. doi: 10.1093/oxfordjournals.jbchem.a123004.
The gene for L-lactate dehydrogenase (LDH) from Thermus aquaticus YT-1 was cloned in Escherichia coli, using the Thermus caldophilus LDH gene as a hybridization probe, and its complete nucleotide sequence was determined. The LDH gene comprised 930 base pairs, starting with a GTG initiation codon. Its sequence had high homology (85.8% identity) with the LDH gene of T. caldophilus. The G + C content of the T. aquaticus gene was 70.9%, higher than that of the chromosomal DNA (67.4%). In particular, that in the third position of the codons used was 91.0%, similar to the T. caldophilus gene. The primary structure of T. aquaticus LDH was deduced from the nucleotide sequence of the LDH gene. It comprises 310 amino acid residues, as does T. caldophilus LDH, and its molecular mass was calculated to be 33,210 daltons. The amino acid sequence of the T. aquaticus LDH had 87.1% identity with that of the T. caldophilus LDH. At 23 positions, the respective residues differed in charge and polarity. These differences must be related to the differences in kinetic properties between the two enzymes. The constructed plasmid overproduced the T. aquaticus LDH in E. coli.
以嗜热栖热放线菌乳酸脱氢酶（LDH）基因作为杂交探针，将嗜热水栖菌YT-1的L-乳酸脱氢酶（LDH）基因克隆到大肠杆菌中，并测定了其完整的核苷酸序列。LDH基因由930个碱基对组成，起始密码子为GTG。其序列与嗜热栖热放线菌的LDH基因具有高度同源性（同一性为85.8%）。嗜热水栖菌基因的G + C含量为70.9%，高于染色体DNA（67.4%）。特别是，所使用密码子第三位的G + C含量为91.0%，与嗜热栖热放线菌基因相似。从LDH基因的核苷酸序列推导了嗜热水栖菌LDH的一级结构。它由310个氨基酸残基组成，嗜热栖热放线菌LDH也是如此，其分子量经计算为33210道尔顿。嗜热水栖菌LDH的氨基酸序列与嗜热栖热放线菌LDH的氨基酸序列同一性为87.1%。在23个位置上，各自的残基在电荷和极性方面存在差异。这些差异必定与这两种酶动力学性质的差异有关。构建的质粒在大肠杆菌中过量表达了嗜热水栖菌LDH。
