Enzymes involved in the degradation of steroids

参与类固醇降解的酶

• 批准号: RGPIN-2020-04099
• 负责人: Seah, Stephen
• 金额: $ 2.62万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739663
• 关键词: Enzymes; involved; degradation; steroids

This research program involves the study of structure-function relationships of enzymes. The focus is on enoyl CoA hydratases (EcHds), aldolases and 3-hydroxyacyl CoA dehydrogenases (3-HDHs) responsible for the degradation of sterol and bile acids side chains within Actinobacteria and Proteobacteria. Some of these enzymes are related to aliphatic fatty acids beta-oxidation enzymes but are structurally adapted for bulky steroid substrates utilization. The fundamental studies of these unique enzymes will shed light on how substrate specificities of enzymes can be modulated by their oligomeric structures, a concept that is not yet explored in detail in the enzymology field. This work may lead to new ways to engineer enzymes that involve the modifications of their quaternary structures. We hypothesize that in the bile acid degradation pathway, the AB steroid rings are degraded prior to D-ring side chain degradation while the opposite sequence occurs in the cholesterol degradation pathway. We hypothesize that the specificity of the EcHds determines the order of these pathways. We will test the hypothesis by characterizing mutants lacking EcHd genes and by testing the specificity of purified EcHds with steroid enoyl CoA derivatives containing intact ABCD rings or degraded AB rings. We will crystallize the EcHds with bound ligands to determine the molecular basis of substrate recognition in these enzymes. We showed previously that Ltp2 catalyzed the C-C bond aldolytic cleavage of the isopropyl side chains of cholesterol and bile acids. Aldolytic cleavage of ß-sitosterol side chain requires the participation of two Ltp2 homologues, Ltp3 and Ltp4 while the degradation of the cholate side chain in Proteobacteria requires the Ltp2 homolog, Sal. Sal, Ltp3 and Ltp4 appear to form complexes with proteins containing DUF35 domains (Domains of unknown function 35), similar to Ltp2. Ltp3, Ltp4 and Sal, however, share only partial conservation of the catalytic acid and base identified in Ltp2. We propose to express, purify and characterize these evolutionarily divergent aldolases to determine their structures, catalytic mechanisms and substrate specificities. The 3-HDHs from bacterial steroid degradation pathways share homology with enzymes that oxidize cortisol and transform cholesterol side chain to bile acids. However, they lack the N- and C-terminal extensions important for maintaining the quaternary structures that contribute to steroid substrate binding in these homologs. We propose to characterize putative 3-HDHs from steroid degradation pathways and to correlate their substrate specificity with structures determined by X-ray crystallography. Characterization of the side chain transformation enzymes and identification of the determinants of the order of rings versus side chain degradation described here may facilitate the synthesis of a new generation of steroid drugs with novel side chains using engineered steroid degrading bacteria.

这项研究计划涉及酶的结构-功能关系的研究。重点是烯醇辅酶A水合酶(EcHds)、醛缩酶和3-羟基酰辅酶A脱氢酶(3-HDHS)，它们负责降解放线菌和变形杆菌中的甾醇和胆汁酸侧链。这些酶中的一些与脂肪脂肪酸β-氧化酶有关，但在结构上适合于使用笨重的类固醇底物。对这些独特酶的基础研究将揭示酶的底物特异性如何受到其寡聚体结构的调节，这一概念在酶学领域尚未得到详细探索。这项工作可能导致新的方法来工程酶，涉及其四级结构的修改。我们假设，在胆汁酸降解途径中，AB类固醇环在D-环侧链降解之前被降解，而在胆固醇降解途径中则出现相反的序列。我们假设ECHDS的特异性决定了这些途径的顺序。我们将通过鉴定缺乏EcHd基因的突变体，以及通过测试含有完整ABCD环或降解AB环的类固醇Enoyl CoA衍生物纯化的EcHd的特异性来检验这一假设。我们将用结合配体结晶ECHDS，以确定这些酶中底物识别的分子基础。我们以前已经证明，Ltp2催化胆固醇和胆汁酸的异丙基侧链的C-C键醇解裂解。β-谷甾醇侧链的醇解裂解需要两个Ltp2同系物的参与，而变形杆菌中胆酸侧链的降解则需要Ltp2同系物Sal。SAL、LUP3和LUP4似乎与含有DUF35结构域(功能未知的区域)的蛋白质形成复合体，类似于LUP2。然而，Ltp3、Ltp4和Sal只部分保守了Ltp2中确定的催化酸和碱。我们建议表达、纯化和鉴定这些进化分化的醛缩酶，以确定它们的结构、催化机制和底物特异性。来自细菌类固醇降解途径的3-HDHS与氧化皮质醇并将胆固醇侧链转化为胆汁酸的酶有同源性。然而，它们缺乏对维持四级结构至关重要的N-末端和C-末端延伸，这些四元结构有助于这些同系物中的类固醇底物结合。我们建议表征类固醇降解途径中可能的3-HDHS，并将它们的底物特异性与X射线结晶学确定的结构相关联。本文描述的侧链转化酶的表征和环序与侧链降解的决定因素的鉴定，可能有助于利用工程类固醇降解菌合成具有新侧链的新一代类固醇药物。