Structure and Mutational Analysis of the PhoN Protein of Salmonella typhimurium Provide Insight into Mechanistic Details
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- 作者：Ravindra D. Makde ; Suresh K. Mahajan ; Vinay Kumar
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：February 27, 2007
- 年：2007
- 卷：46
- 期：8
- 页码：2079 - 2090
- 全文大小：703K
- 年卷期：v.46,no.8(February 27, 2007)
- ISSN：1520-4995
文摘
The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs tothe phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures ofphosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functionalcharacterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123,Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishesphosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the activesite resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hithertounknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 andHis-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201Nmutation completely abolished phosphatase activity. On the basis of this observation and in silico analysiswe suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on thephosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, locatedwithin hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions thatAsp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzymeintermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyprolinemotif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that thevarying quaternary structure of PhoN orthologues may have functional significance.