Enzymes involved in the degradation of steroids

点击翻译按钮获取中文摘要

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.