In Vitro Determined Kinetic Properties of Mutant Phosphoglucomutases and Their Effects on Sugar Catabolism in Escherichia coli
In Vitro Determined Kinetic Properties of Mutant Phosphoglucomutases and Their Effects on Sugar Catabolism in Escherichia coli
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DOI：
10.1006/mben.1999.0145
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发表时间：
2000-04-01
影响因子：
8.4
通讯作者：
Valla, Svein
中科院分区：
文献类型：
--
作者：
Brautaset, Trygve;Petersen, Steffen B.;Valla, Svein
通过与其他磷酸糖糖转换酶的初级氨基酸序列比较，木醋杆菌（Acetobacter xylinum）磷酸糖糖转换酶（CelB）中的12个保守残基被位点定向诱变取代，导致突变酶的K-cat值[葡萄糖-1-磷酸（G-1-P）至葡萄糖-6-磷酸]相对于野生型酶的K-cat值为0至46%。这些蛋白与一组多用途的质粒表达载体结合，用于大肠杆菌糖分解代谢的代谢工程研究。缺乏磷酸葡萄糖糖化酶的大肠杆菌突变体在半乳糖上生长时，由于G-1-P的积累而合成细胞内直链淀粉。野生型细胞b可以补足这种病变，我们在这里表明，突变酶的补足能力对它们各自体外测定的K-cat和K-m （G-1-p）值的变化很敏感。降低的催化效率可以通过增加CelB表达水平来弥补，这样，一个催化效率降低7600倍的突变蛋白（将Thr-45替换为Ala）可以用来消除直链淀粉的积累。与同源磷酸葡萄糖糖化酶的互补实验表明，明显低于CelB的K-m （G-1-p）值对底物的体内转化并不重要。(C) 2000年学术出版社
Based on primary amino acid sequence comparisons with other phosphoglucomutases, 12 conserved residues in the Acetobacter xylinum phosphoglucomutase (CelB) were substituted by site-directed mutagenesis, resulting in mutant enzymes with K-cat values [glucose-1-phosphate (G-1-P) to glucose-6-phosphate] ranging from 0 to 46% relative to that of the wild-type enzyme. In combination with a versatile set of plasmid expression vectors these proteins were used in a metabolic engineering study on sugar catabolism in Escherichia coli. Mutants of E. coli deficient in phosphoglucomutase synthesize intracellular amylose when grown on galactose, due to accumulation of G-1-P. Wild-type celB can complement this lesion, and we show here that the ability of the mutant enzymes to complement is sensitive to variations in their respective in vitro determined K-cat and K-m (G-1-p) values. Reduced catalytic efficiencies could be compensated by increasing the CelB expression level, and in this way a mutant protein (substitution of Thr-45 to Ala) displaying a 7600-fold reduced catalytic efficiency could be used to eliminate the amylose accumulation. Complementation experiments with the homologous phosphoglucomutase indicated that a K-m (G-1-p) value significantly below that of CelB is not critical for the in vivo conversion of the substrate. (C) 2000 Academic Press