dissertationSqualene:hopene cyclases (SHCs) are bacterial enzymes that convert squalene into hopanoids, a function analogous to the action of oxidosqualene cyclases (OSCs) in eukaryotic steroid and triterpenoid biosynthesis. Selective inhibitors for these two enzymes would uniquely suppress the committed step in triterpene biosynthesis, thus acting as cholesterol lowering drugs as well as antifungal or antimicrobial agents demonstrating minimal side effects. Ro48-8071, a benzophenone-containing hypocholesteremic drug, is a selective, potent, photoactivatable inhibitor of SHC. Identification of the binding site of Ro48-8071 for SHC from Alicyclobacillus acidocaldarius is described herein, Edman degradation of a peptide fragment of covalently modified SHC confirmed that Ala44 was specifically modified. Molecular modeling, using X-ray derived protein, coordinates and a single point constraint for the inhibitor, was then employed to define the enzyme inhibitor complex. Subsequent mutagenesis studies provide evidence that the nucleophilicity and positioning of Glu45 is crucial for its stabilization of the carbocation intermediates. Asp or Ala substitution resulted in significant lower cyclization activity, in addition to altered products ratios. Replacement of the conserved "DDTAV V" motif is SHC with the DCTAEA" motif from OSC changes the substrate specificity. This mutant cyclizes, 2,3-oxidosqualene but cannon process squalene. Mono- and pent acyclic 3-hydrosy triterpenes were isolated and characterized from the cyclization mixture for this mutant. (3S)-29-Methylidene-2,3-oxidosqualene (29-MOS) was a mechanism-based irreversible inhibitor of both OSC and SHC. A peptide fragment of SHC was identified to bind 29-MOS, and the specific residues were further defined. A dammarene derivative was isolated from the incubation mixture as a major cyclization product of 29-MOS. Sequence comparison led to the identification of a 501-residue protein from M. tuberculosis that was homologous to SHC and OSC. This gene was cloned and express in E. coli, but no cyclization activity for squalene and OS could be detected. However, this protein was found to be specifically labeled by [3H]Ro18-8071, and it was also shown to cyclize 2,3:22,23-dioxidosqualence (DOS) into a novel product. Catalytic function of this enzyme was further discussed
