The crystal structure reveals the molecular mechanism of bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (Rv1415) from Mycobacterium tuberculosis
The crystal structure reveals the molecular mechanism of bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (Rv1415) from Mycobacterium tuberculosis
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DOI：
10.1107/s0907444913011402
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发表时间：
2013-09-01
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影响因子：
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通讯作者：
Karthikeyan, Subramanian
中科院分区：
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作者：
Singh, Mirage;Kumar, Pankaj;Karthikeyan, Subramanian
酶3，4-二羟基-2-丁酮4-磷酸合酶（DHBPS）和GTP环化水解酶II（GCHII）催化细菌核黄素生物合成途径的两个分支的初始步骤。DHBPS和GCHII作为单独的多肽的结构和分子机制是已知的;然而，它们作为双功能酶的组织和分子机制迄今为止是未知的。在这里，晶体结构的基本双功能DHBPS/GCHII酶从结核分枝杆菌（Mtb-ribA 2）报告在3.0埃分辨率。晶体结构揭示了在不对称单元中的两个构象不同的Mtb-ribA 2分子，其通过其GCHII结构域形成二聚体。有趣的是，晶体包装的分析揭示了一个长的“螺旋状低聚物”形成的DHBPS和GCHII功能同源二聚体，从而产生一个“开放式”的晶胞晶格。然而，尺寸排阻色谱研究表明，Mtb-ribA 2作为二聚体存在于溶液中。为了理解在溶液中观察到的寡聚化与在晶体结构中观察到的寡聚化之间的差异，Mtb-ribA 2的DHBPS（Mtb-DHBPS）和GCHII（Mtb-GCHII）结构域已被克隆、表达并纯化为His标记的蛋白质。尺寸排阻色谱研究表明，Mtb-GCHII是一个二聚体，而Mtb-DHBPS作为单体存在于溶液中。此外，动力学研究表明，Mtb-ribA 2和Mtb-GCHII的GCHII活性相似，而Mtb-ribA 2的DHBPS活性远高于单独的Mtb-DHBPS。综上所述，结果强烈表明Mtb-ribA 2作为通过其GCHII结构域形成的二聚体存在，并且需要全长Mtb-ribA 2以获得最佳DHBPS活性。
The enzymes 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS) and GTP cyclohydrolase II (GCHII) catalyze the initial steps of both branches of the bacterial riboflavin-biosynthesis pathway. The structures and molecular mechanisms of DHBPS and GCHII as separate polypeptides are known; however, their organization and molecular mechanism as a bifunctional enzyme are unknown to date. Here, the crystal structure of an essential bifunctional DHBPS/GCHII enzyme from Mycobacterium tuberculosis (Mtb-ribA2) is reported at 3.0 angstrom resolution. The crystal structure revealed two conformationally different molecules of Mtb-ribA2 in the asymmetric unit that form a dimer via their GCHII domains. Interestingly, analysis of the crystal packing revealed a long 'helical-like oligomer' formed by DHBPS and GCHII functional homodimers, thus generating an 'open-ended' unit-cell lattice. However, size-exclusion chromatography studies suggest that Mtb-ribA2 exists as a dimer in solution. To understand the discrepancy between the oligomerization observed in solution and in the crystal structure, the DHBPS (Mtb-DHBPS) and GCHII (Mtb-GCHII) domains of Mtb-ribA2 have been cloned, expressed and purified as His-tagged proteins. Size-exclusion chromatography studies indicated that Mtb-GCHII is a dimer while Mtb-DHBPS exists as a monomer in solution. Moreover, kinetic studies revealed that the GCHII activities of Mtb-ribA2 and Mtb-GCHII are similar, while the DHBPS activity of Mtb-ribA2 is much higher than that of Mtb-DHBPS alone. Taken together, the results strongly suggest that Mtb-ribA2 exists as a dimer formed through its GCHII domains and requires full-length Mtb-ribA2 for optimal DHBPS activity.