Abstract
Sterol methyltransferase (SMT) from Saccharomyces cerevisiae was purified from Escherichia coli BL21(DE3) and labeled with the mechanism-based irreversible inhibitor [3-3H]26,27-dehydrozymosterol (26,27-DHZ). A 5-kDa tryptic digest peptide fragment containing six acidic residues at positions Glu-64, Asp-65, Glu-68, Asp-79, Glu-82, and Glu-98 was determined to contain the substrate analog covalently attached to Glu-68 by Edman sequencing and radioanalysis using C18 reverse phase high performance liquid chromatography. Site-directed mutagenesis of the six acidic residues to leucine followed by activity assay of the purified mutants confirmed Glu-68 as the only residue to participate in affinity labeling. Equilibration studies indicated that zymosterol and 26,27-DHZ were bound to native and the E68L mutant with similar affinity, whereas S-adenosylmethionine was bound only to the native SMT, K(d) of about 2 microm. Analysis of the incubation products of the wild-type and six leucine mutants by GC-MS demonstrated that zymosterol was converted to fecosterol, 26,27-DHZ was converted to 26-homo-cholesta-8(9),23(24)E,26(26')-trienol as well as 26-homocholesta-8(9),26(26')-3beta,24beta-dienol, and in the case of D79L and E82L mutants, zymosterol was also converted to a new product, 24-methylzymosta-8,25(27)-dienol. The structures of the methylenecyclopropane ring-opened olefins were determined unambiguously by a combination of (1)H and (13)C NMR techniques. A K(m) of 15 microm, K(cat) of 8 x 10(-4) s(-1), and partition ratio of 0.03 was established for 26,27-DHZ, suggesting that the methylenecyclopropane can serve as a lead structure for a new class of antifungal agents. Taken together, partitioning that leads to loss of enzyme function is the result of 26,27-DHZ catalysis forming a highly reactive cationic intermediate that interacts with the enzyme in a region normally not occupied by the zymosterol high energy intermediate as a consequence of less than perfect control. Alternatively, the gain in enzyme function resulting from the production of a delta(25(27))-olefin originates with the leucine substitution directing substrate channeling along different reaction channels in a similar region at the active site.
Highlights
The sterol methyltransferases (SMTs)1 are appealing targets in the design of enzyme inhibitors, because they lie on a pathway with no counterpart in human physiology (1, 2)
Recent studies with Sterol methyltransferase (SMT) from fungi and plants employing a range of sterol acceptor molecules and isotopically branching sensitive experiments evoke a common set of enzymatic intermediates involved with the stereochemically related events, electrophilic alkylations, 1 The abbreviations used are: SMT, sterol methyltransferases; 26,27DHZ, [3-3H]26,27-dehydrozymosterol; AdoMet, S-adenosylmethionine; HPLC, high performance liquid chromatography; methoxy-2-phenyl-2-(trifluoromethyl)acetic acid (MTPA), 2-methoxy-2phenyl-2-(trifluoromethyl)acetic acid; ⌬25(27)-olefin, 24-methylzymosta25(27)-dienol; ⌬24(28)-olefin, fecosterol
The mechanism of inhibition and covalent attachment of 26,27-DHZ responsible for trapping an active site nucleophile suggested nucleophilic attack of the ⌬24-bond of the S-methyl group of AdoMet leading to a reactive ring-opened intermediate, ⌬25(27)olefin 14, shown in Scheme 2, in an analogous manner to the mechanism proposed for the catalysis of 6-cyclopropylidene3E-methyl-hex-2-en-1-yl-diphosphate for monoterpene cyclase (21)
Summary
The sterol methyltransferases (SMTs) are appealing targets in the design of enzyme inhibitors, because they lie on a pathway with no counterpart in human physiology (1, 2). Comparison of protein sequences deduced from the cDNA for fungal SMTs (Fig. 1) revealed a highly conserved region rich in aromatic amino acids, referred to as Region I, containing a signature motif Y81EYGWG86 not present in other AdoMet-dependent methyltransferases As each of these enzymes uses zymosterol 1A as the sterol acceptor molecule, the aromaticrich domain of Region I has been proposed to be involved in substrate binding, and possibly product formation, by stabilizing intermediate carbenium ions generated during sterol Cmethylation (11). Many of the mechanistic and stereochemical details of the C-24 alkylation pathways have been verified (18, 19), little is known about the active site of any SMT or the manner in which a SMT enzyme imposes a particular conformation on its acceptor molecule, precisely controls the coupled-methylation deprotonation reaction, and establishes kinetic and substrate specificity for C1and the successive C1-methyltransfers giving rise to the side chains of fungal ergosterol and plant sitosterol, respectively. The mechanism of inhibition and covalent attachment of 26,27-DHZ responsible for trapping an active site nucleophile suggested nucleophilic attack of the ⌬24-bond of the S-methyl group of AdoMet leading to a reactive ring-opened intermediate, ⌬25(27)olefin 14, shown in Scheme 2, in an analogous manner to the mechanism proposed for the catalysis of 6-cyclopropylidene3E-methyl-hex-2-en-1-yl-diphosphate for monoterpene cyclase (21)
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