Cloning and Prokaryotic Expression of the Gene Encoding Phosphite Dehydrogenase from the Soil Bacterium Alcaligenes faecalis
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摘要: 亚磷酸盐脱氢酶(PTDH/PtxD)在基于亚磷酸盐的磷利用和辅酶再生等方面有着巨大的应用潜力。本研究直接从土壤宏基因组中扩增得到粪产碱菌亚磷酸盐脱氢酶全长基因AfPtxD，由其推导出的Af PtxD蛋白含有辅酶NAD+的结合位点和关键的酶催化残基，并经保守结构域预测属于PTDH家族成员。在同家族的系统进化树上，Af PtxD蛋白与脓杆菌PtxD几乎完全相同，而与已具晶体结构分析的罗尔斯顿菌PtxD和施氏假单胞菌PtxD的同源性并不很高，亲缘关系相对较远；但同源建模结果表明Af PtxD蛋白在高级结构与罗尔斯顿菌PtxD几乎完全相同，与施氏假单胞菌PtxD也很相似，且都以二聚体形式存在。另外，AfPtxD基因经IPTG诱导能在大肠杆菌中获得高效表达，但可溶性不高。进一步用His标签亲合层析对重组蛋白Af PtxD纯化，经测定，其以亚磷酸钠盐为底物的酶比活性为4.285 U/mg。该AfPtxD功能基因的获得为其后续应用研究打下了必要的基础。Abstract: Phosphite dehydrogenase（PTDH/PtxD） has large application potentials in the aspects of phosphite-based phosphorus utilization, coenzyme regeneration etc. Herein, the full-length gene of Alcaligenes faecalis PtxD（Af PtxD） was directly amplified from the soil metagenome. Its deduced Af PtxD protein contains the binding site for coenzyme NAD+and key catalytic residues, and also belongs to a member of PTDH family via conserved domain predictions. Meanwhile, phylogenetic tree analysis showed that in PTDH family the Af PtxD protein was almost identical to Pusillimonas PtxD, but clearly different from Ralstonia PtxD and Pseudomonas stutzeri PtxD already with crystal structural analysis. However, the results of homology modelling demonstrated that the Af PtxD protein was almost the same as Ralstonia PtxD in high-level structure and also very similar to P. stutzeri PtxD, existing in dimer form. We also found that the AfPtxD gene could be efficiently expressed in Escherichia coli by IPTG induction, despite poor solubility. The recombinant Af PtxD protein was further purified by His-tag affinity chromatography, and detected with a specific enzymatic activity of 4.285 U/mg towards its substrate, sodium phosphite. Prospectively, this cloned AfPtxD gene with verified activity should be of fundamental significance for its future application studies.
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