Enzymes involved in the degradation of steroids

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。******HsaF-HsaG参与类固醇A环降解；醛缩酶HsaF催化4-羟基-2-氧己酸（HOHA）的C3和C4之间的C-C键裂解生成丙酮酸和丙醛。后者通过分子间通道进入脱氢酶HsaG，在那里转化为丙酰辅酶a。纯化的HsaF本身没有活性，但当与纯化的HsaG预孵卵时，酶由于异质复合物的形成而恢复活性。据推测，在没有脱氢酶的情况下，这可以防止醛缩酶形成有毒的醛。利用位点特异性诱变、结构和生化工具，我们提出了一种假说，即与脱氢酶（HsaG）的关联会导致HsaF在催化或调节底物进入活性位点的关键残基上的构象变化。******ACADs和EcHds参与类固醇d环侧链降解。它们催化类似于脂肪酸β -氧化酶的反应。利用脂肪底物的经典acad和echd是同源低聚物，但最近发现参与类固醇降解的acad和echd是异质的，或者是包含关键残基有显著差异的重复结构域的融合蛋白。我们假设这些酶是从同聚寡聚酶进化而来的，以适应体积庞大的类固醇底物。我们发现不同的ACADs对具有不同环取代基/结构和侧链长度的底物具有特异性，这证实了这一点。我们建议确定在结核分枝杆菌和约氏杆菌RHA1的类固醇降解基因簇中编码的其他ACADs和EcHds的底物特异性，以便能够确定序列/结构的差异如何控制底物特异性。我们还计划用结合底物结晶这些酶，以确定它们的三维结构。******对haf - hsag的研究将增加我们对芳香降解途径的醛缩酶-脱氢酶复合物的广泛机制研究，醛缩酶-脱氢酶复合物是表现底物通道的最佳表征酶系统之一。对ACADs和echd的研究将为研究涉及四级结构变化的调节酶底物特异性的不寻常机制提供新的见解

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.**