Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MufF synthetases as established by chemical rescue experiments
Evidence of a functional requirement for a carbamoylated lysine residue in MurD, MurE and MufF synthetases as established by chemical rescue experiments
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DOI：
10.1046/j.0014-2956.2001.02524.x
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发表时间：
2001-11-01
期刊：
影响因子：
--
通讯作者：
Blanot, D
中科院分区：
文献类型：
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作者：
Dementin, S;Bouhss, A;Blanot, D
MURD、MURE、MURF、叶基多谷氨酸合成酶和蓝藻素合成酶属于MUR合成酶超家族，含有一个不变的赖氨酸残基(在大肠杆菌MURD编号中为K198)。最近对MURD和MURE的晶体分析表明，该残基以氨基甲酸酯类化合物的形式存在，这一修饰可能是镁离子结合和酰基磷酸形成所必需的。在本工作中，通过定点诱变和化学救援实验，研究了氨甲酰化残基在MURD、MURE和MURF中的重要性。突变蛋白Murd K198A/F、Mure K224A和Murf K202a的酶活性较低，但与短链羧酸孵育后可被挽救，但不能与胺作用。MURD K198A为醋酸盐，K198F为甲酸盐，MURE K224A和MURF K202A为丙酸盐。在最后一次实验中，野生型的活动水平得到了恢复。注意到取代赖氨酸的残基的体积和酸的碳链长度之间的互补性。这些观察结果支持氨基甲酸酯在三种Mur合成酶中的功能作用。还进行了旨在通过引入酸性残基来取代不变赖氨酸残基来恢复活性酶的实验。突变蛋白Murd K198E活性较弱，可被甲酸盐解救，表明其酸性功能需要正确定位于肽骨架。共价挽救突变蛋白Murd K198C的尝试失败了，因为它对卤酸缺乏反应性。
Enzymes MurD, MurE, MurF, folylpolyglutamate synthetase and cyanophycin synthetase, which belong to the Mur synthetase superfamily, possess an invariant lysine residue (K198 in the Escherichia coli MurD numbering). Crystallographic analysis of MurD and MurE has recently shown that this residue is present as a carbamate derivative, a modification presumably essential for Mg2+ binding and acyl phosphate formation. In the present work, the importance of the carbamoylated residue was investigated in MurD, MurE and MurF by site-directed mutagenesis and chemical rescue experiments. Mutant proteins MurD K198A/F, MurE K224A and MurF K202A, which displayed low enzymatic activity, were rescued by incubation with short-chain carboxylic acids, but not amines. The best rescuing agent was acetate for MurD K198A, formate for K198F, and propionate for MurE K224A and MurF K202A. In the last of these, wild-type levels of activity were recovered. A complementarity between the volume of the residue replacing lysine and the length of the carbon chain of the acid was noted. These observations support a functional role for the carbamate in the three Mur synthetases. Experiments aimed at recovering an active enzyme by introducing an acidic residue in place of the invariant lysine residue were also undertaken. Mutant protein MurD K198E was weakly active and was rescued by formate, indicating the necessity of correct positioning of the acidic function with respect to the peptide backbone. Attempts at covalent rescue of mutant protein MurD K198C failed because of its lack of reactivity towards haloacids.