Enzymes involved in the degradation of steroids

该研究计划涉及酶的结构-功能关系和酶进化的研究。 重点是在结核分枝杆菌和土壤细菌Rhodococcus jostti RHA 1的胆固醇和胆汁酸降解途径中发现的异聚酶复合物。醛缩酶-脱氢酶复合物（HsaF-HsaG）、酰基辅酶A水解酶（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-氧代己酸（HOHA）的C3和C4之间的C-C键裂解为丙酮酸和丙醛。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预孵育时，由于异聚复合物的形成，酶恢复活性。据推测，这防止了在没有脱氢酶的情况下由醛缩酶形成有毒醛。 使用位点特异性诱变，结构和生化工具，我们建议测试的假设，与脱氢酶，HsaG，导致构象变化的HsaF中的关键残基参与催化或调节底物进入活性位点。 **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。利用脂肪族底物的经典ACADs和EcHds是同源寡聚体，但最近发现参与类固醇降解的ACADs和EcHds是异聚体或融合蛋白，其含有在关键残基中具有显著差异的重复结构域。 我们假设这些酶从同源寡聚酶进化而来，以适应庞大的类固醇底物。我们的发现证实了这一点，即不同的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。 我们建议确定其他ACADs和EcHds内的类固醇降解基因簇的M编码的底物特异性。结核和R. jostti RHA 1，以便能够确定序列/结构的差异如何支配底物特异性。我们还计划将其中一些酶与结合底物一起结晶，以确定它们的3D结构。** 对HsaF-HsaG的拟议研究将增加我们对芳香族降解途径的醛缩酶-脱氢酶复合物的广泛机械工作，这是表现出底物通道的最佳表征酶系统之一。 对ACADs和EcHds的研究将为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.**