Enzymes involved in the degradation of steroids

这个研究项目涉及酶的结构-功能关系的研究。重点是在放线菌和变形菌中负责降解甾酸和胆汁酸侧链的烯酰辅酶a水合酶（EcHds），醛缩酶和3-羟基辅酶a脱氢酶（3-HDHs）。其中一些酶与脂肪脂肪酸β -氧化酶有关，但在结构上适合于大体积类固醇底物的利用。对这些独特酶的基础研究将揭示酶的底物特异性如何通过它们的寡聚结构来调节，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环侧链降解之前被降解，而在胆固醇降解途径中则发生相反的顺序。我们假设echd的特异性决定了这些通路的顺序。我们将通过表征缺乏EcHd基因的突变体，并通过含有完整ABCD环或降解AB环的类固醇烯酰辅酶a衍生物测试纯化的EcHd的特异性来验证这一假设。我们将用结合的配体结晶EcHds，以确定这些酶识别底物的分子基础。我们之前已经证明Ltp2催化了胆固醇和胆汁酸的异丙基侧链的C-C键醛解裂解。ß-谷甾醇侧链的醛水解裂解需要两个Ltp2同源物Ltp3和Ltp4的参与，而变形杆菌中胆酸侧链的降解需要Ltp2同源物Sal的参与。Sal， Ltp3和Ltp4似乎与含有DUF35结构域（未知功能结构域35）的蛋白质形成复合物，类似于Ltp2。然而，Ltp3、Ltp4和Sal在Ltp2中只保留了部分催化酸和碱。我们打算表达、纯化和表征这些进化上不同的醛缩酶，以确定它们的结构、催化机制和底物特异性。细菌类固醇降解途径中的3-HDHs与氧化皮质醇和将胆固醇侧链转化为胆汁酸的酶具有同源性。然而，它们缺乏对维持这些同源物中有助于类固醇底物结合的四级结构至关重要的N端和c端延伸。我们建议从类固醇降解途径表征假定的3-HDHs，并将其底物特异性与x射线晶体学确定的结构联系起来。本文描述的侧链转化酶的表征和环的顺序与侧链降解的决定因素的鉴定可能有助于利用工程类固醇降解细菌合成具有新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.