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首页> 外文期刊>Journal of Molecular Biology >Tracking the evolution of porphobilinogen synthase metal dependence in vitro.
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Tracking the evolution of porphobilinogen synthase metal dependence in vitro.

机译：在体外跟踪胆色素原合酶金属依赖性的演变。

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Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+). Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+). The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination. Structurally and functionally it mimicsmycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges.
机译：金属离子是通过多种酶进行化学催化必不可少的辅助因子。根据其对Zn（2+）的依赖性，可归因于催化四吡咯生物合成的第二步的胆色素原合酶（PBGS）。使用定点诱变，我们着手将独立于Zn（2+）的铜绿假单胞菌PBGS转换为依赖Zn（2+）的酶。通过将三个半胱氨酸残基和另外两个残基置换引入酶的活性位点来产生九个PBGS变体，以匹配来自大肠杆菌的同源含Zn（2+）的PBGS。解决了七个酶变体的晶体结构，以阐明高分辨率的Zn（2+）配位的性质。始终发现三个单半胱氨酸变体在酶上无活性，并且仅发现一个（D139C）结合可检测量的Zn（2+）。双重突变体A129C / D139C具有酶活性，并以四面体配位结合Zn（2+）。在结构上和功能上，它都模拟了分枝杆菌PBGS，其具有等效的Zn（2+）配位位点。剩下的两个双突变体，没有已知的自然等效物，显示强烈扭曲的四面体Zn（2+）结合位点。变体A129C / D131C具有较弱的PBGS活性，而D131C / D139C不活跃。最后，三重突变体A129C / D131C / D139C显示了几乎理想的四面体Zn（2+）结合几何形状和重要的Zn（2+）依赖酶活性。两次额外的氨基酸交换进一步优化了针对大肠杆菌酶的活性位点结构，并增加了活性。我们的研究勾画出无Zn（2+）和依赖Zn（2+）的PBGS酶之间的潜在进化路径，表明PBGS酶的刚性骨架是通过有限数量的氨基酸创建或消除金属依赖性的理想框架。交流。

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