Characterization of Multifunctional Enzyme Complexes from Comamonas testosteroni KF1 and Thermomonospora curvata DSM 43183 Involved in Steroid Side Chain Degradation

Abstract

The genes fadI and fadJ from Comamonas testosteroni KF1 are homologous to genes encoding the components of multifunctional enzyme complexes involved in fatty acid side chain β-oxidation. Both of these genes are located within the steroid degradation locus in the genome. FadJ had hydratase activity towards the 5-carbon side chain of cholic acid metabolite; however, gene knockout studies revealed no phenotypic changes in growth patterns. In the Actinobacterium Thermomonospora curvata DSM 43183, the genes Tcur3480, Tcur3482 and ltp2Tcur, located within a steroid side chain degrading operon, together encode a multifunctional steroid degrading enzyme complex. It was previously determined that the aldolase Ltp2Tcur associates with the hydratase Tcur3480-Tcur3482 through the DUF35 domain, although the catalytic mechanism and kinetic parameters were unknown. The crystal structure of Ltp2Tcur-DUF35 was solved and wild-type and mutant variant kinetic parameters were determined to support a proposed, novel retro-aldol catalytic mechanism involving two catalytic tyrosine residues.