Biosystems Science Course, Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan.
Arch Microbiol. 2010 Aug;192(8):639-50. doi: 10.1007/s00203-010-0595-3. Epub 2010 Jun 15.
The genes encoding monomer- and dimer-type isocitrate dehydrogenase (IDH) isozymes from a psychrotrophic bacterium, Pseudomonas psychrophila, were cloned and sequenced. Open reading frames of the genes were 2,226 and 1,257 bp in length and corresponded to polypeptides composed of 741 and 418 amino acids, respectively. The deduced amino acid sequences showed high sequence identity with those of psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea, (about 70% identity) and the respective types of the putative IDH genes from other bacteria of genus Pseudomonas (more than 80% identity). The two genes were located in opposite direction from each other with a spacer of 463 bases in the order of dimeric and monomeric IDH genes on the chromosomal DNA, but analyses of northern blotting and 5'-terminal regions of the mRNAs revealed that they are transcribed independently. The expression of monomer- and dimer-type IDH genes in C. maris are known to be cold- and acetate-inducible, respectively, while only slight inductions by low temperature and/or acetate were observed in the expression of the P. psychrophila monomer- and dimer-type IDH genes. Both of these IDH isozymes overproduced in Escherichia coli showed mesophilic properties, in contrast with monomer- and dimer-type IDHs of C. maris as cold adapted and mesophilic enzymes, respectively. The substitution of Glu55 residue in the P. psychrophila monomeric IDH for Lys, which is the corresponding residue conserved between the cold-adapted monomeric IDHs from C. maris and C. psychrerythraea, by site-directed mutagenesis resulted in the decreased thermostability and the lowered optimum temperature of activity, suggesting that this residue is involved in the mesophilic properties of the P. psychrophila monomeric IDH.
从嗜冷菌假单胞菌（Pseudomonas psychrophila）中克隆并测序了编码单体型和二聚体型异柠檬酸脱氢酶（IDH）同工酶的基因。这些基因的开放阅读框分别长 2226 和 1257bp，分别对应由 741 和 418 个氨基酸组成的多肽。推断的氨基酸序列与嗜冷细菌科尔韦尔氏菌（Colwellia maris）和科尔韦尔氏菌（Colwellia psychrerythraea）具有很高的序列同一性（约 70%的同一性），以及假单胞菌属中其他细菌的各自类型的推定 IDH 基因（超过 80%的同一性）。这两个基因位于染色体 DNA 上的二聚体型和单体型 IDH 基因的相反方向，间隔 463 个碱基，但 northern 印迹和 mRNA 5'末端区域的分析表明它们是独立转录的。在 C. maris 中，单体型和二聚体型 IDH 基因的表达分别受低温和乙酸诱导，而在 P. psychrophila 单体型和二聚体型 IDH 基因的表达中仅观察到低温和/或乙酸的轻微诱导。这两种 IDH 同工酶在大肠杆菌中过度表达表现出中温特性，与 C. maris 的单体型和二聚体型 IDH 分别作为适应低温和中温的酶相反。通过定点突变将 P. psychrophila 单体型 IDH 中的 Glu55 残基替换为 Lys，这是科尔韦尔氏菌和科尔韦尔氏菌冷适应单体型 IDH 之间保守的相应残基，导致热稳定性降低和最适温度降低，表明该残基参与了 P. psychrophila 单体型 IDH 的中温特性。
