ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin b17
ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin b17
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DOI：
10.1021/bi980996e
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发表时间：
1998-09-22
期刊：
影响因子：
2.9
通讯作者：
Walsh, CT
中科院分区：
文献类型：
--
作者：
Milne, JC;Eliot, AC;Walsh, CT
在大肠杆菌抗生素Microcin B17的成熟过程中，mcbA基因的产物被多聚体Microcin合成酶复合物（由McbB、C和D组成）后修饰，以将四个Cys和四个Ser残基分别环化为四个噻唑和四个恶唑。纯化的合成酶在肽底物杂环化中显示出对ATP或GTP的绝对需求，在初始速率研究中GTP的有效性为ATP的三分之一。合成酶复合物的ATP酶/GTP酶活性是有条件的，因为ADP或GDP的形成需要底物的存在; McbA的非环化版本与合成酶结合，但不诱导NTR酶活性。对于含有两个潜在修饰位点的底物，ATP水解和杂环形成的化学计量比为5：1。然而，在高底物浓度（> 50 K（m））下，杂环的形成被抑制，而ATP酶活性未减弱，这与高底物浓度下NTP水解和杂环形成的解偶联一致。序列同源性揭示McbD亚基具有使人想起ATP利用酶中的步行者B盒和小G蛋白GTP酶中发现的基序的基序。在这些基序（D132、D147和D199）中将三个谷氨酸突变为丙氨酸减少了Microcin B17在体内的产生和在体外的杂环形成，这表明45 kDa McbD在其N-末端区域具有调节的ATP酶/GTdR结构域，这是肽杂环化所必需的。
In the maturation of the Escherichia coli antibiotic Microcin B17, the product of the mcbA gene is modified posttranslationally by the multimeric Microcin synthetase complex (composed of McbB, C, and D) to cyclize four Cys and four Ser residues to four thiazoles and four oxazoles, respectively. The purified synthetase shows an absolute requirement for ATP or GTP in peptide substrate heterocyclization, with GTP one-third as effective as ATP in initial rate studies. The ATPase/GTPase activity of the synthetase complex is conditional in that ADP or GDP formation requires the presence of substrate; noncyclizable versions of McbA bind to synthetase, but do not induce the NTPase activity. The stoichiometry of ATP hydrolysis and heterocycle formation is 5:1 for a substrate that contains two potential sites of modification. However, at high substrate concentrations (>50K(m)) heterocycle formation is inhibited, while ATPase activity occurs undiminished, consistent with uncoupling of NTP hydrolysis and heterocycle formation at high substrate concentrations. Sequence homology reveals that the McbD subunit has motifs reminiscent of the Walker B box in ATP utilizing enzymes and of motifs found in small G protein GTPases. Mutagenesis of three aspartates to alanine in these motifs (D132, D147, and D199) reduced Microcin B17 production in vivo and heterocycle formation in vitro, suggesting that the 45 kDa McbD has a regulated ATPase/GTPase domain in its N-terminal region necessary for peptide heterocyclization.