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首页> 外文期刊>Biochemistry >Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease
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Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease

机译：突变和计算证据，UreD中的镍转移隧道被用于激活产气克雷伯氏菌脲酶

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Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD/UreF/UreG complex, which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous, structurally characterized UreH/UreF/UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 angstrom predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-disrupting variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-down studies of cell-free extracts from tunnel-disrupting mutant cultures showed no loss of UreD interactions with urease or UreF/UreG. The nickel contents of urease samples enriched from activity-deficient cultures were decreased, while zinc and iron incorporation increased. Molecular dynamics simulations revealed size restrictions in the internal channels of the UreD variants. These findings support the role of a molecular tunnel in UreD as a direct facilitator of nickel transfer into urease, illustrating a new paradigm in active site metallo center assembly.
机译：产气克雷伯菌中的含镍脲酶需要四个辅助蛋白才能使活性位点金属化。金属伴侣蛋白UreE向UreG传递镍，UreG是一种形成UreD / UreF / UreG复合物的GTP酶，该复合物通过UreD与脲酶载脂蛋白结合。先前对来自幽门螺杆菌的同源，结构特征化的UreH / UreF / UreG复合物进行了计算机分析，结果确定了一条水通道，该水通道起源于UreG中可能的镍结合基序，穿过UreF，然后离开UreH，表明该通道在提供金属以脲酶载脂蛋白的活化;但是，没有关于该隧道的意义的实验性支持报道。在这里，设计了特定的变体，以破坏可比的34.6埃预测的内部通道，替代通道和UreD的表面位点。产生一组破坏UreD的破坏隧道的变体的细胞表现出大大降低的脲酶比活性，而其他突变体对活性没有明显影响。对破坏隧道的突变体培养物中无细胞提取物的亲和力下拉研究表明，与脲酶或UreF / UreG的UreD相互作用没有损失。从缺乏活性的培养物中富集的脲酶样品的镍含量降低，而锌和铁的掺入增加。分子动力学模拟揭示了UreD变体内部通道的尺寸限制。这些发现支持了UreD中分子隧道作为镍直接转化为脲酶的直接促进者的作用，说明了活性位点金属中心组装的新范例。

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