Molecular basis for enantioselectivity in the (R)- and (S)-hydroxypropylthioethanesulfonate dehydrogenases, a unique pair of stereoselective short-chain dehydrogenases/reductases involved in aliphatic epoxide carboxylation.
Molecular basis for enantioselectivity in the (R)- and (S)-hydroxypropylthioethanesulfonate dehydrogenases, a unique pair of stereoselective short-chain dehydrogenases/reductases involved in aliphatic epoxide carboxylation.
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DOI：
10.1021/bi100294m
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发表时间：
2010-04-27
期刊：
影响因子：
2.9
通讯作者：
Ensign, Scott A.
中科院分区：
文献类型：
--
作者：
Sliwa, Dariusz A.;Krishnakumar, Arathi M.;Peters, John W.;Ensign, Scott A.
(R)-和(S)-2-羟丙基-COM(R-HPC和S-HPC)分别是由辅酶M与(R)-环氧丙烷和(S)-环氧丙烷亲核加成而生成的。短链脱氢酶/还原酶家族中的两个高对映选择性脱氢酶(R-HPCDH和S-HPCDH)催化R-HPC和S-HPC转化为2-酮丙基-COM(2-KPC)，后者经过还原裂解和羧化生成乙酰乙酸酯。在本研究中，在自养黄杆菌PY2中线性巨质粒上存在的三个S-HPCDH酶中的一个已经被克隆和高效表达，这使得R-HPCDH酶和S-HPCDH酶的首次详细的并列表征成为可能。S-HPCDH型催化三联体由Y156、K160和S143组成。经鉴定，R211和K214是S高性能蛋白中与玉米赤霉素磺酸盐配位的氨基酸残基。R211A和K214A突变体在R-HPC的氧化和2-KPC的还原过程中受到严重损伤，而在脂肪醇和脂肪酮的氧化和还原过程中基本不受影响。以(R)-和(S)-HPC为底物的动力学分析表明，R-HPCDH944的对映体选择性主要由kCAT的差异决定，而S-HPCDH658的对映体选择性主要由Km的变化决定。S对2-丁酮合成(S)-2-丁醇具有很高的对映体选择性，不受与磺酸盐结合部位相互作用的调节剂的影响。叔醇2-甲基-2-羟丙基-COM(M-HPC)是R-HPCDH催化的R-HPC氧化的竞争性抑制剂，其Km与R-HPC的Km相似，但不是S-HPCDH的抑制剂。以伯醇2-羟乙基-COM为底物，R-羟基异丙基苯基脱氢酶和S-羟基异丙基苯基脱氢酶的Km值相同。动力学参数的pH依赖性表明，羟基对S-羟基磷灰石与S-羟基磷灰石结合的贡献大于对R-羟基磷灰石与R-羟基磷灰石结合的贡献。有人认为，S-HPCDH中的活性中心限制由于与C2碳上不正确排列的甲基发生空间碰撞而阻止了R-HPC和M-HPC的正确结合，导致了与R-HPCDH不同的底物专一性和对映选择性的控制机制。
(R)- and (S)-2-hydroxypropyl-CoM (R-HPC and S-HPC) are produced as intermediates in bacterial propylene metabolism from the nucleophilic addition of coenzyme M to (R)- and (S)-epoxypropane, respectively. Two highly enantioselective dehydrogenases (R-HPCDH and S-HPCDH) belonging to the short-chain dehydrogenase/reductase family catalyze the conversion of R-HPC and S-HPC to 2-ketopropyl-CoM (2-KPC), which undergoes reductive cleavage and carboxylation to produce acetoacetate. In the present study, one of three copies of S-HPCDH enzymes present on a linear megaplasmid in Xanthobacter autotrophicus strain Py2 has been cloned and overexpressed, allowing the first detailed side by side characterization of the R-HPCDH and S-HPCDH enzymes. The catalytic triad of S-HPCDH was found to consist of Y156, K160, and S143. R211 and K214 were identified as the amino acid residues coordinating the sulfonate of CoM in S-HPC. R211A and K214A mutants were severely impaired in the oxidation of R-HPC or reduction of 2-KPC but were largely unaffected in the oxidation and reduction of aliphatic alcohols and ketones. Kinetic analyses using (R)- and (S)-HPC as substrates revealed that enantioselectivity in R-HPCDH (value, 944) was dictated largely by differences in kcat while enantioselectivity for S-HPCDH (value, 658) was dictated largely by changes in Km. S-HPCDH had an inherent high enantioselectivity for producing (S)-2-butanol from 2-butanone that was unaffected by modulators that interact with the sulfonate binding site. The tertiary alcohol 2-methyl-2-hydroxypropyl-CoM (M-HPC) was a competitive inhibitor of R-HPCDH-catalyzed R-HPC oxidation, with a Kis similar to the Km for R-HPC, but was not an inhibitor of S-HPCDH. The primary alcohol 2-hydroxyethyl-CoM was a substrate for both R-HPCDH and S-HPCDH with identical Km values. The pH dependence of kinetic parameters suggests that the hydroxyl group is a larger contributor to S-HPC binding to S-HPCDH than for R-HPC binding to R-HPCDH. It is proposed that active site constraints within the S-HPCDH prevent proper binding of R-HPC and M-HPC due to steric clashes with the improperly aligned methyl group on the C2 carbon, resulting in a different mechanism for controlling substrate specificity and enantioselectivity than present in the R-HPCDH.
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影响因子：
5.1
作者：
Persson, Bengt;Kallberg, Yvonne;Bray, James E.;Bruford, Elspeth;Dellaporta, Stephen L.;Favia, Angelo D.;Gonzalez Duarte, Roser;Jornvall, Hans;Kavanagh, Kathryn L.;Kedishvili, Natalia;Kisiela, Michael;Maserk, Edmund;Mindnich, Rebekka;Orchard, Sandra;Penning, Trevor M.;Thornton, Janet M.;Adamski, Jerzy;Oppermann, Udo
通讯作者：
Oppermann, Udo
影响因子：
2.9
作者：
Ensign, SA
通讯作者：
Ensign, SA
影响因子：
3.2
作者：
Krum, JG;Ensign, SA
通讯作者：
Ensign, SA
影响因子：
2.9
作者：
GRIMSHAW, CE;COOK, PF;CLELAND, WW
通讯作者：
CLELAND, WW
影响因子：
2.9
作者：
JORNVALL, H;PERSSON, B;GHOSH, D
通讯作者：
GHOSH, D