Enzymes involved in the degradation of steroids

该研究计划涉及酶的结构-功能关系的研究。重点是烯酰辅酶A水合酶（EcHds），醛缩酶和3-羟酰辅酶A水解酶（3-HDH）负责降解的甾醇和胆汁酸侧链放线菌和变形菌。这些酶中的一些与脂肪族脂肪酸β-氧化酶相关，但在结构上适于大体积类固醇底物的利用。这些独特的酶的基础研究将阐明酶的底物特异性如何通过其寡聚结构进行调节，这是酶学领域尚未详细探索的概念。这项工作可能会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环侧链降解之前降解，而胆固醇降解途径中发生相反的顺序。我们推测，特异性的EcHds决定了这些途径的顺序。我们将通过表征缺乏EcHd基因的突变体和通过用含有完整ABCD环或降解AB环的类固醇烯酰CoA衍生物测试纯化的EcHds的特异性来测试该假设。我们将与结合配体的EcHds结晶，以确定在这些酶的底物识别的分子基础。 我们以前表明，Ltp 2催化的C-C键醛解裂解的胆固醇和胆汁酸的异丙基侧链。β-谷甾醇侧链的醛解裂解需要两种Ltp 2同系物Ltp 3和Ltp 4的参与，而变形菌中胆酸盐侧链的降解需要Ltp 2同系物Sal。Sal、Ltp 3和Ltp 4似乎与含有DUF 35结构域（功能未知的结构域35）的蛋白质形成复合物，类似于Ltp 2。然而，Ltp 3、Ltp 4和Sal仅共享Ltp 2中鉴定的催化酸和碱的部分保守性。我们建议表达，纯化和表征这些进化上不同的醛缩酶，以确定其结构，催化机制和底物特异性。来自细菌类固醇降解途径的3-HDH与氧化皮质醇并将胆固醇侧链转化为胆汁酸的酶具有同源性。然而，他们缺乏的N-和C-末端延伸重要的四级结构，有助于类固醇底物结合在这些同系物。我们建议从类固醇降解途径表征推定的3-HDHs，并将其底物特异性与X射线晶体学确定的结构相关联。 这里描述的侧链转化酶的表征和环与侧链降解的顺序的决定因素的鉴定可以促进使用工程化类固醇降解细菌合成具有新型侧链的新一代类固醇药物。

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.