Enzymes involved in the degradation of steroids

参与类固醇降解的酶

• 批准号: RGPIN-2015-05366
• 负责人: Seah, Stephen
• 金额: $ 3.28万
• 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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613507
• 关键词: Enzymes; involved; degradation; steroids

This research program involves the study of structure-function relationships of enzymes and enzyme evolution.  The focus is on heteromeric enzymes complexes found in the cholesterol and bile acids degradation pathways of Mycobacterium tuberculosis and the soil bacterium Rhodococcus jostti RHA1. The aldolase-dehydrogenase complex (HsaF-HsaG), acyl CoA dehydrogenases (ACADs) and enoyl CoA hydratases (EcHds) are enzymes within the steroid degradation pathway that are ideal paradigms to decipher how protein-protein interactions can modulate the activity and substrate specificity of enzymes. HsaF-HsaG are involved in steroid A ring degradation; the aldolase, HsaF, catalyzes the C-C bond cleavage between C3 and C4 of 4-hydroxy-2-oxohexanoate (HOHA) to pyruvate and propionaldehyde. The latter is channeled via an intermolecular tunnel to the dehydrogenase HsaG where it is converted to propionyl CoA. Purified HsaF is not active by itself, but when preincubated with purified HsaG, the enzyme recovers activity due to heteromeric complex formation. Presumably this prevents the formation of toxic aldehydes by the aldolase in the absence of the dehydrogenase.  Using site-specific mutagenesis, structural and biochemical tools we propose to test the hypothesis that association with the dehydrogenase, HsaG, causes conformational changes in HsaF in key residues either involved in catalysis or regulating substrate access to the active site.  ACADs and EcHds are involved in steroid D-ring side chain degradation.  They catalyze reactions analogous to fatty acid beta-oxidation enzymes. Classical ACADs and EcHds that utilize aliphatic substrate are homo-oligomers but those involved in steroid degradation have recently been found to be heteromeric or are fusion proteins containing duplicated domains with significant differences in key residues.  We hypothesize that these enzymes evolved from homo-oligomeric enzymes to accommodate the bulky steroid substrates. This is substantiated by our finding that different ACADs have specificity for substrates with different ring subsitutents/structure and side chain length.  We propose to determine the substrate specificities of other ACADs and EcHds encoded within the steroid degradation gene clusters of M. tuberculosis and R. jostti RHA1 in order to be able to determine how differences in sequences/structures govern substrate specificity. We also plan to crystallize some of these enzymes, with bound substrates, to determine their 3D structures.  Proposed studies on HsaF-HsaG will add to our extensive mechanistic work on the aldolase-dehydrogenase complex of aromatic degradation pathways, one of the best characterized enzyme systems that exhibit substrate channeling.  Studies on ACADs and EcHds will provide novel insight on an unusual mechanism for modulating enzyme substrate specificity involving changes in quaternary structure.

本专业主要研究酶的结构-功能关系和酶的进化。重点是在结核分枝杆菌和土壤细菌约氏红球菌RHA1的胆固醇和胆汁酸降解途径中发现的异质酶复合物。醛缩酶-脱氢酶复合物（HsaF-HsaG）、酰基辅酶a脱氢酶（ACADs）和烯酰辅酶a水合酶（EcHds）是类固醇降解途径中的酶，是解释蛋白质-蛋白质相互作用如何调节酶的活性和底物特异性的理想范例。