Ma N, Ikeda S, Guo S, Fieno A, Park I, Grimme S, Ikeda T, Ives D H
Department of Biochemistry, Ohio State University, Columbus 43210-1292, USA.
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14385-90. doi: 10.1073/pnas.93.25.14385.
Three of the four deoxynucleoside kinases required for growth of Lactobacillus acidophilus R-26 exist as heterodimeric pairs specific for deoxyadenosine (dAK) and deoxycytidine (dCK) or dAK and deoxyguanosine (dGK). However, only two tandem genes, dak/dgk, are found, and are expressed only as dAK/dGK in transformed Escherichia coli. Sequencing peptides spanning 63% of the native dCK subunit revealed a sequence identical to that deduced from dgk (beginning MTVIVL...), except that dCK lacks residues 2 and 3 (dCK is M..IVL; dGK is .TVIVL). Also, mass spectrometry indicates that native dCK and dGK subunits are identical in mass adjusted for the first three residues. Furthermore, the native enzymes have identical isoelectric pH values, indicating an equal number of charged residues. To enable E. coli to express peptide having the native dCK sequence, codons 2 and 3 were deleted from the dgk portion of the tandem genes, resulting in expression of protein having the specificities and regulatory properties of native dAK/dCK, including heterotropic stimulation of dAK activity by deoxycytidine or dCTP (not deoxyguanosine or dGTP) and end-product inhibition of the respective activities by dATP and dCTP. Subcloning normal and mutant dgk yielded homodimeric dGK and dCK, respectively. The dCK homodimer strongly resembles human dCK, with a low K(m) for deoxycytidine, the ability to phosphorylate deoxyadenosine and deoxyguanosine at much higher K(m) values, and end-product inhibition by dCTP. Thus two distinct and specific enzymes evidently are derived from a single Lactobacillus gene. The mechanism by which this occurs in vivo has yet to be elucidated.
嗜酸乳杆菌R - 26生长所需的四种脱氧核苷激酶中的三种以脱氧腺苷（dAK）和脱氧胞苷（dCK）或dAK和脱氧鸟苷（dGK）特异性的异二聚体对形式存在。然而，仅发现两个串联基因，即dak/dgk，并且在转化的大肠杆菌中仅表达为dAK/dGK。对跨越天然dCK亚基63%的肽段进行测序，结果显示其序列与从dgk推导的序列相同（起始为MTVIVL...），只是dCK缺少第2和第3位残基（dCK为M..IVL；dGK为.TVIVL）。此外，质谱分析表明，经前三个残基质量校正后，天然dCK和dGK亚基的质量相同。而且，天然酶具有相同的等电pH值，表明带电残基数量相等。为使大肠杆菌表达具有天然dCK序列的肽段，从串联基因的dgk部分删除了密码子2和3，从而表达出具有天然dAK/dCK特异性和调节特性的蛋白质，包括脱氧胞苷或dCTP（而非脱氧鸟苷或dGTP）对dAK活性的异促刺激以及dATP和dCTP对各自活性的终产物抑制。亚克隆正常和突变的dgk分别产生了同二聚体dGK和dCK。dCK同二聚体与人类dCK非常相似，对脱氧胞苷的K(m)值较低，能够在高得多的K(m)值下磷酸化脱氧腺苷和脱氧鸟苷，并且受到dCTP的终产物抑制。因此，两种不同且特异的酶显然源自单个乳酸杆菌基因。其在体内发生的机制尚待阐明。
