Petranovic Dina, Grangeasse Christophe, Macek Boris, Abdillatef Mohammad, Gueguen-Chaignon Virginie, Nessler Sylvie, Deutscher Josef, Mijakovic Ivan
Technical University of Denmark, Biosys, Center for Microbial Biotechnology, Lyngby, Denmark.
J Mol Microbiol Biotechnol. 2009;17(2):83-9. doi: 10.1159/000206635. Epub 2009 Mar 4.
The phosphorylation-dependent activation of bacterial UDP-glucose dehydrogenases by BY-kinases has been previously described in several bacterial model organisms, but the identity of phosphorylated tyrosine(s) and the exact activation mechanism remained unknown. A recent site-specific phosphoproteomic study indicated that tyrosine 70 is phosphorylated in the Bacillus subtilis UDP-glucose dehydrogenase Ugd. In this study we confirm that this tyrosine 70 is indeed the main residue phosphorylated by the cognate BY-kinase PtkA. Homology-based modeling of the Ugd structure using structures from UDP-glucose/GDP-mannose dehydrogenases revealed that this residue is in close proximity to the NAD-binding site. We identified lysine 108 as the second important residue involved in Ugd activation. Enzymatic characterization of the Ugd proteins mutated in residues tyrosine 70 or lysine 108 suggested a phosphorylation-based regulatory mechanism. This study represents the first attempt to understand the activation of a bacterial enzyme by tyrosine phosphorylation at the molecular level.
此前在几种细菌模式生物中已描述过细菌UDP - 葡萄糖脱氢酶被BY激酶磷酸化依赖性激活的情况，但磷酸化酪氨酸的身份以及确切的激活机制仍不清楚。最近一项位点特异性磷酸化蛋白质组学研究表明，枯草芽孢杆菌UDP - 葡萄糖脱氢酶Ugd中的酪氨酸70被磷酸化。在本研究中，我们证实该酪氨酸70确实是同源BY激酶PtkA磷酸化的主要残基。使用UDP - 葡萄糖/ GDP - 甘露糖脱氢酶的结构对Ugd结构进行基于同源性的建模表明，该残基紧邻NAD结合位点。我们确定赖氨酸108是参与Ugd激活的第二个重要残基。对酪氨酸70或赖氨酸108残基发生突变的Ugd蛋白进行酶学表征，提示了一种基于磷酸化的调节机制。本研究是在分子水平上理解酪氨酸磷酸化激活细菌酶的首次尝试。
