Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.
Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.
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DOI：
10.1021/bi5007404
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发表时间：
2014-08-12
期刊：
影响因子：
2.9
通讯作者：
Becker, Donald F.
中科院分区：
文献类型：
--
作者：
Arentson, Benjamin W.;Luo, Min;Pemberton, Travis A.;Tanner, John J.;Becker, Donald F.
大豆慢生根瘤菌脯氨酸利用A（Proline utilization A from Bradyrhizobium japonicum，BjPutA）是一种双功能黄素酶，其利用融合的脯氨酸脱氢酶（PRODH）和Δ1-吡咯啉-5-羧酸脱氢酶（P5 CDH）结构域催化脯氨酸氧化为谷氨酸。最近的晶体结构和动力学数据表明，分子内通道连接两个活性位点，促进中间体Δ1-吡咯啉-5-羧酸/谷氨酸-γ-半醛（P5 C/GSA）的底物通道。在这项工作中，通过在BjPutA中通道的四个位置沿着插入大的侧链残基来探索通道的结构。不同突变体的动力学分析显示，用Tyr（D 779 Y）或Trp（D 779 W）替换D 779显著降低了PRODH-P5 CDH通道反应的总体速率。D 779 Y和D 779 W的X射线晶体结构显示，大的侧链在通道的中心部分引起收缩，因此可能阻碍P5 C/GSA在通道中的行进。D 779 Y和D 779 W突变体具有与野生型BjPutA相似的PRODH活性，但表现出显著较低的P5 CDH活性，表明外源P5 C/GSA进入Asp 779上游的通道。用Tyr（D 778 Y）替换附近的Asp 778不影响BjPutA通道活性。与动力学结果一致，D 778 Y的X射线晶体结构显示主通道路径不受影响;然而，离腔路径与通道关闭。这些发现提供的证据表明，离腔途径是不是必不可少的基板通道BjPutA。
Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ1-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH–P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA.
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DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
多功能脯氨酸利用A蛋白质中脯氨酸脱氢酶的快速反应动力学。
影响因子：
2.9
作者：
Moxley, Michael A.;Becker, Donald F.
通讯作者：
Becker, Donald F.
影响因子：
3.9
作者：
Moxley MA;Tanner JJ;Becker DF
通讯作者：
Becker DF
影响因子：
2.9
作者：
Krishnan, N;Becker, DF
通讯作者：
Becker, DF
影响因子：
11.6
作者：
Cobessi, David;Dumas, Renaud;Alban, Claude
通讯作者：
Alban, Claude