Probing the function of a ligand-modulated dynamic tunnel in bifunctional proline utilization A (PutA).
Probing the function of a ligand-modulated dynamic tunnel in bifunctional proline utilization A (PutA).
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DOI：
10.1016/j.abb.2021.109025
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发表时间：
2021-11-15
影响因子：
3.9
通讯作者：
Tanner JJ
中科院分区：
文献类型：
--
作者：
Korasick DA;Christgen SL;Qureshi IA;Becker DF;Tanner JJ
在许多细菌中，Pro分解代谢的反应是由被称为Pro利用A(PUTA)的双功能酶催化的。PUTA通过不同的脯氨酸脱氢酶(PROH)和L-谷氨酸-γ-半醛脱氢酶(GSALDH)活性中心催化L-脯氨酸两步氧化成L-谷氨酸，这两个活性中心由一个复杂的隧道系统连接。该隧道系统由一个主隧道和六个功能未知的辅助隧道组成，主隧道连接底物通道中的两个活性部位和功能。在这里，我们使用隧道阻断突变来探索动态辅助隧道(隧道2a)的作用，该隧道的形状受与脯氨酸脱氢酶活性位点结合的配体的调节。硫还原地杆菌变异体A206W的1.90？分辨率晶体结构验证了Trp206的侧链在不干扰周围结构的情况下干净地阻断了隧道2a。稳态动力学测量表明，突变削弱了PRODH活性，而不影响GSALDH活性。单周转实验证实，A206W的PROSH活性严重受损，黄素含量比野生型减少了近600倍。底物沟道也受到显著影响，因为A206W在耦合的PROH-GSALDH活性测试中显示出3000倍的催化效率降低，这是衡量NADH形成与脯氨酸的函数。这一结构表明，Trp206通过阻止保守的谷氨酸-精氨酸离子对的形成和PROSH活性部位的关闭来抑制底物L-Pro的结合。我们的数据与隧道2a是一致的，因为它是一个开放的空间，离子对的谷氨酸在活性中心的打开和关闭过程中通过它来响应与L-脯氨酸的结合。这些结果证实了保守的离子对在结合L-脯氨酸中的重要性，并支持了离子对作为门的作用，控制着PROSH活性中心的访问。
In many bacteria, the reactions of proline catabolism are catalyzed by the bifunctional enzyme known as proline utilization A (PutA). PutA catalyzes the two-step oxidation of L-proline to L-glutamate using distinct proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites, which are separated by over 40 Å and connected by a complex tunnel system. The tunnel system consists of a main tunnel that connects the two active sites and functions in substrate channeling, plus six ancillary tunnels whose functions are unknown. Here we used tunnel-blocking mutagenesis to probe the role of a dynamic ancillary tunnel (tunnel 2a) whose shape is modulated by ligand binding to the proline dehydrogenase active site. The 1.90 Å resolution crystal structure of Geobacter sulfurreducens PutA variant A206W verified that the side chain of Trp206 cleanly blocks tunnel 2a without perturbing the surrounding structure. Steady-state kinetic measurements indicate the mutation impaired PRODH activity without affecting the GSALDH activity. Single-turnover experiments corroborated a severe impairment of PRODH activity with flavin reduction decreased by nearly 600-fold in A206W relative to wild-type. Substrate channeling is also significantly impacted as A206W exhibited a 3000-fold lower catalytic efficiency in coupled PRODH-GSALDH activity assays, which measure NADH formation as a function of proline. The structure suggests that Trp206 inhibits binding of the substrate L-proline by preventing the formation of a conserved glutamate-arginine ion pair and closure of the PRODH active site. Our data are consistent with tunnel 2a serving as an open space through which the glutamate of the ion pair travels during the opening and closing of the active site in response to binding L-proline. These results confirm the essentiality of the conserved ion pair in binding L-proline and support the hypothesis that the ion pair functions as a gate that controls access to the PRODH active site.
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DOI：
10.1016/j.str.2017.10.009
发表时间：
2017-12-05
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Gore S;Sanz García E;Hendrickx PMS;Gutmanas A;Westbrook JD;Yang H;Feng Z;Baskaran K;Berrisford JM;Hudson BP;Ikegawa Y;Kobayashi N;Lawson CL;Mading S;Mak L;Mukhopadhyay A;Oldfield TJ;Patwardhan A;Peisach E;Sahni G;Sekharan MR;Sen S;Shao C;Smart OS;Ulrich EL;Yamashita R;Quesada M;Young JY;Nakamura H;Markley JL;Berman HM;Burley SK;Velankar S;Kleywegt GJ
通讯作者：
Kleywegt GJ
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：
Liu LK;Becker DF;Tanner JJ
通讯作者：
Tanner JJ
影响因子：
2.9
作者：
Arentson, Benjamin W.;Luo, Min;Pemberton, Travis A.;Tanner, John J.;Becker, Donald F.
通讯作者：
Becker, Donald F.
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K