Enzymes involved in the degradation of steroids

本研究项目涉及酶的结构-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Rhodoins jostti RHA1的胆固醇和胆汁酸的降解途径中发现的异构酶复合体。醛缩酶-脱氢酶复合体(HsaF-HsaG)、酰基辅酶A脱氢酶(Acads)和烯醇辅酶A水合酶(EcHds)是类固醇降解途径中的酶，是解释蛋白质相互作用如何调节酶的活性和底物专一性的理想范例。*HsaF参与类固醇A环的降解；醛缩酶HsaF催化4-羟基-2-氧己酸(HOHA)C3和C4之间的C-C键断裂为丙酮酸和丙醛。后者通过分子间隧道被引导到脱氢酶HsaG，在那里它被转化为丙酰辅酶A。纯化的HsaF本身不具有活性，但当与纯化的HsaG预先孵育时，由于形成了异构体复合体，该酶恢复了活性。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。通过使用定点突变、结构和生化工具，我们建议检验这一假说与脱氢酶，HsaG，导致关键残基中HsaF的构象变化，这些残基参与催化或调节底物对活性部位的访问。Acads和EcHds参与类固醇D环侧链的降解。它们催化类似于脂肪酸β-氧化酶的反应。经典的Acads和EcHds利用脂肪底物是同源低聚物，但最近发现参与类固醇降解的是异构体或包含重复结构域的融合蛋白，关键残基没有显著差异。我们假设这些酶是从同源寡聚酶进化而来的，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对具有不同环替代物/结构和侧链长度的底物具有特异性。我们建议确定编码在结核分枝杆菌和R.jostti RHA1类固醇降解基因簇中的其他Acads和EcHds的底物专一性，以便能够确定序列/结构的差异如何控制底物专一性。我们还计划结晶其中一些带有结合底物的酶，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降解途径的醛缩酶-脱氢酶复合体的广泛机制工作，这是表现底物通道的最具特性的酶系统之一。对Acads和EcHds的研究将为调节酶底物专一性的不寻常机制提供新的见解，涉及四级结构的变化。**

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.**