Exploring triterpene cyclization mechanism : binding sites mapping and mutagenesis

Update Item Information
Publication Type dissertation
School or College College of Pharmacy
Department Medicinal Chemistry
Author Dang, Tongyun
Title Exploring triterpene cyclization mechanism : binding sites mapping and mutagenesis
Date 2000-08
Description Squalene: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.
Type Text
Publisher University of Utah
Subject Antimicrobial Agents; Catalytic
Subject MESH Terpenes; Cholestanol; Mutagenesis
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Exploring triterpene cyclization mechanism: binding sites mapping and mutagenesis.". Spencer S. Eccles Health Sciences Library. Print version of "Exploring triterpene cyclization mechanism: binding sites mapping and mutagenesis.". available at J. Willard Marriott Library Special Collection. QP6.5 2000 .D35
Rights Management © Tongyun Dang.
Format Medium application/pdf
Format Extent 2,890,530 bytes
Identifier undthes,4590
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Master File Extent 2,890,565 bytes
ARK ark:/87278/s6bv7jfb
Setname ir_etd
ID 191115
Reference URL https://collections.lib.utah.edu/ark:/87278/s6bv7jfb