OleB from Bacterial Hydrocarbon Biosynthesis Is a β-Lactone Decarboxylase That Shares Key Features with Haloalkane Dehalogenases,Biochemistry

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OleB from Bacterial Hydrocarbon Biosynthesis Is a β-Lactone Decarboxylase That Shares Key Features with Haloalkane Dehalogenases
Biochemistry ( IF 2.9 ) Pub Date : 2017-09-19 00:00:00 , DOI: 10.1021/acs.biochem.7b00667
James K. Christenson _{1,

2} , Serina L. Robinson _{2,

3} , Tiffany A. Engel ₂ , Jack E. Richman _{1,

2} , An N. Kim ₂ , Larry P. Wackett _{1,

2,

4}

Affiliation

OleB is an α/β-hydrolase found in bacteria that biosynthesize long-chain olefinic hydrocarbons, but its function has remained obscure. We report that OleB from the Gram-negative bacterium Xanthomonas campestris performs an unprecedented β-lactone decarboxylation reaction, to complete cis-olefin biosynthesis. OleB reactions monitored by ¹H nuclear magnetic resonance spectroscopy revealed a selectivity for decarboxylating cis-β-lactones and no discernible activity with trans-β-lactones, consistent with the known configuration of pathway intermediates. Protein sequence analyses showed OleB proteins were most related to haloalkane dehalogenases (HLDs) and retained the canonical Asp-His-Asp catalytic triad of HLDs. Unexpectedly, it was determined that an understudied subfamily, denoted as HLD-III, is comprised mostly of OleB proteins encoded within oleABCD gene clusters, suggesting a misannotation. OleB from X. campestris showed very low dehalogenase activity only against haloalkane substrates with long alkyl chains. A haloalkane substrate mimic alkylated wild-type X. campestris OleB but not OleB_D114A, implicating this residue as the active site nucleophile as in HLDs. A sequence-divergent OleB, found as part of a natural OleBC fusion and classified as an HLD-III, from the Gram-positive bacterium Micrococcus luteus was demonstrated to have the same activity, stereochemical preference, and dependence on the proposed Asp nucleophile. H₂¹⁸O studies with M. luteus OleBC suggested that the canonical alkyl–enzyme intermediate of HLDs is hydrolyzed differently by OleB enzymes, as ¹⁸O is not incorporated into the nucleophilic aspartic acid. This work defines a previously unrecognized reaction in nature, functionally identifies some HLD-III enzymes as β-lactone decarboxylases, and posits an enzymatic mechanism of β-lactone decarboxylation.

中文翻译：

来自细菌碳氢化合物生物合成的OleB是与卤代烷脱卤酶具有关键特征的β-内酯脱羧酶

OleB是生物合成长链烯烃的细菌中发现的一种α/β水解酶，但其功能仍然不清楚。我们报道从革兰氏阴性细菌油菜黄单胞菌（Xanthomonas campestris）的OleB进行了前所未有的β-内酯脱羧反应，以完成顺式-烯烃的生物合成。通过¹ H核磁共振波谱监测的OleB反应显示了对顺式-β-内酯脱羧的选择性，而反式没有可识别的活性-β-内酯，与途径中间体的已知构型一致。蛋白质序列分析显示OleB蛋白质与卤代烷脱卤酶（HLDs）最为相关，并保留了HLDs的经典Asp-His-Asp催化三联体。出乎意料的是，已确定一个未充分研究的亚家族，称为HLD-III，主要由oleABCD基因簇内编码的OleB蛋白组成，提示存在误解。来自野油菜X. Campestris的OleB仅对具有长烷基链的卤代烷底物显示出非常低的脱卤酶活性。卤代烷底物模拟烷基化的野生型喜树油菜OleB，但不模拟_{OleB D114A}，暗示此残基是HLD中的活性位点亲核试剂。从天然革兰氏阳性细菌微球菌（Micrococcus luteus）中发现的序列差异型OleB，是天然OleBC融合的一部分，分类为HLD-III，具有相同的活性，立体化学偏好和对拟议的Asp亲核试剂的依赖性。ħ _2个¹⁸带O研究藤黄微球菌OleBC建议规范烷基-酶中间体的HLDS通过OleB酶不同水解，如¹⁸O未结合到亲核天冬氨酸中。这项工作定义了自然界以前无法识别的反应，在功能上将某些HLD-III酶鉴定为β-内酯脱羧酶，并提出了β-内酯脱羧酶机制。

更新日期：2017-09-20

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