Faulkner Nicholas W, Joyce John B, Smith Christy, Swartz Paul, Rose Robert B, Miller Eric S, Hyman Michael R
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, USA.
Appl Environ Microbiol. 2025 Sep 17;91(9):e0039325. doi: 10.1128/aem.00393-25. Epub 2025 Aug 26.
Isobutylene (IB) is produced on a large scale by the petrochemical industry and is metabolized by the aerobic alkene-metabolizing bacterium sp. ELW1. The initial metabolite of IB catabolism by this bacterium is proposed to be 2-methyl-1,2-epoxypropane (isobutylene oxide [IBO]). The epoxide is then thought to be rapidly converted into 2-methyl-1,2-propanediol (MPD) by an epoxide hydrolase. A gene () encoding a hydrolase is in a putative IB catabolism gene cluster on a ~222-kbp megaplasmid. This gene was cloned, heterologously expressed, and purified by Ni-NTA affinity chromatography. The purified protein rapidly and stoichiometrically hydrolyzed IBO to MPD with a specific activity of 29 µmoles min mg protein. Additional epoxides were also hydrolyzed by IbcK, including 1,2-epoxypropane, 1,2-epoxybutane, 1,2-epoxypentane, epichlorohydrin, and cyclohexane oxide, albeit at lower rates than IBO. IbcK also slowly hydrolyzed both - and -2,3-epoxybutane, which are the only other epoxides other than IBO known to support the growth of sp. ELW1. Furthermore, IbcK also appears to be enantioselective towards chiral 2,3-epoxybutane. The crystal structure of IbcK was determined at 2.29 Å resolution, revealing a two-domain structure with an α/β hydrolase fold topology at its core. IbcK has high similarity to the epoxide hydrolase EchA from AD1, including the key active site residues Asp 117, Asp 256, and His 284. IbcK was observed to be in monomer-dimer equilibrium, which we propose occurs through interactions between the "cap" domains.IMPORTANCEThe initial metabolites generated during catabolism of volatile alkenes by aerobic alkene-oxidizing bacteria are consistently epoxides. These bacteria employ several different mechanisms to protect DNA, lipids, and proteins from damage by these reactive metabolites. The most common mechanisms are conjugation with coenzyme M or glutathione. In contrast, the role for hydrolases in the bacterial metabolism of volatile alkenes and their epoxides has not been frequently observed. The enzymatic, functional, and structural characterization of an epoxide hydrolase (IbcK) from the IB-utilizing bacterium sp. ELW1 described here advances our understanding of these enzymes and suggests their potential application as an enantioselective catalyst. This study advances our understanding of how microorganisms utilize aliphatic alkenes, such as carbon and energy sources, including the role of epoxide hydrolases in these catabolic pathways.
异丁烯（IB）由石油化学工业大规模生产，并由需氧烯烃代谢细菌sp. ELW1进行代谢。该细菌对IB分解代谢的初始代谢产物被认为是2-甲基-1,2-环氧丙烷（异丁烯氧化物[IBO]）。然后，这种环氧化物被认为会通过一种环氧化物水解酶迅速转化为2-甲基-1,2-丙二醇（MPD）。一个编码水解酶的基因（）位于一个约222 kbp的大质粒上的假定IB分解代谢基因簇中。该基因被克隆、异源表达，并通过镍-氮三乙酸亲和层析进行纯化。纯化后的蛋白质能快速且化学计量地将IBO水解为MPD，比活性为29微摩尔每分钟毫克蛋白质。IbcK还能水解其他环氧化物，包括1,2-环氧丙烷、1,2-环氧丁烷、1,2-环氧戊烷、环氧氯丙烷和环己烷氧化物，尽管水解速率比IBO低。IbcK也能缓慢水解α-和β-2,3-环氧丁烷，这是已知的除IBO外仅有的能支持sp. ELW1生长的其他环氧化物。此外，IbcK似乎也对手性2,3-环氧丁烷具有对映选择性。IbcK的晶体结构在2.29 Å分辨率下测定，揭示了一种双结构域结构，其核心具有α/β水解酶折叠拓扑结构。IbcK与来自AD1的环氧化物水解酶EchA具有高度相似性，包括关键活性位点残基天冬氨酸117、天冬氨酸256和组氨酸284。观察到IbcK处于单体-二聚体平衡状态，我们认为这是通过“帽”结构域之间的相互作用发生的。
需氧烯烃氧化细菌在挥发性烯烃分解代谢过程中产生的初始代谢产物一直是环氧化物。这些细菌采用几种不同机制来保护DNA、脂质和蛋白质免受这些反应性代谢产物的损害。最常见的机制是与辅酶M或谷胱甘肽结合。相比之下，水解酶在挥发性烯烃及其环氧化物细菌代谢中的作用并不常见。本文描述的来自利用IB的细菌sp. ELW1的环氧化物水解酶（IbcK）的酶学、功能和结构特征，增进了我们对这些酶的理解，并表明它们作为对映选择性催化剂的潜在应用。这项研究增进了我们对微生物如何利用脂肪族烯烃作为碳源和能源以及环氧化物水解酶在这些分解代谢途径中的作用的理解。
