Sterol Methyltransferase:  Functional Analysis of Highly Conserved Residues by Site-Directed Mutagenesis

Abstract

Sterol methyltransferase (SMT), the enzyme from Saccharomyces cerevisiae that catalyzes the conversion of sterol acceptor in the presence of AdoMet to C-24 methylated sterol and AdoHcy, was analyzed for amino acid residues that contribute to C-methylation activity. Site-directed mutagenesis of nine aspartate or glutamate residues and four histidine residues to leucine (amino acids highly conserved in 16 different species) and expression of the resulting mutant proteins in Escherichia coli revealed that residues at H90, Asp125, Asp152, Glu195, and Asp276 are essential for catalytic activity. Each of the catalytically impaired mutants bound sterol, AdoMet, and 25-azalanosterol, a high energy intermediate analogue inhibitor of C-methylation activity. Changes in equilibrium binding and kinetic properties of the mutant enzymes indicated that residues required for catalytic activity are also involved in inhibitor binding. Analysis of the pH dependence of log kcat/Km for the wild-type SMT indicated a pH optimum for activity between 6 and 9. These results and data showing that only the mutant H90L binds sterol, AdoMet, and inhibitor to similar levels as the wild-type enzyme suggest that H90 may act as an acceptor in the coupled methylation-deprotonation reaction. Circular dichroism spectra and chromatographic information of the wild-type and mutant enzymes confirmed retention of the overall conformation of the enzyme during the various experiments. Taken together, our studies suggest that the SMT active center is composed of a set of acidic amino acids at positions 125, 152, 195, and 276, which contribute to initial binding of sterol and AdoMet and that the H90 residue functions subsequently in the reaction progress to promote product formation.

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

“…At the current time, a single base, histidine90, is thought to function as the C-28 deprotonating agent and a second, as yet, unknown polar residue can function as the base in C-27 deprotonation. Glutamic acid276 is considered to hydrogen bond to the C-3 hydroxyl group of the sterol [20]. The systematic perturbation of the enzymatic mechanism involving isotopically branching experiments and testing substrate analogs are in agreement with the suggestion [6] that distinct polar amino acids in the proximal and distal regions of the active center exist to recognize the acceptor nucleophilicity and channel the reaction pathway to ∆ 24(28) or ∆ 25 (27) -olefin formation [19,20].…”

“…In the case of yeast, the cDNA encoding SMT is the ERG6 gene and genetic studies indicate the importance of ERG6 for normal membrane functions and resistance to antifungal drugs [10][11][12]. Functional analysis of highly conserved amino acids in the primary structure of ERG6 by site-directed mutagenesis have led to a secondary structure prediction of the enzyme as shown in Figure 3 [18][19][20]. Different SMT isoforms are now recognized to be synthesized in the cycloartenol-sitosterol pathway and lanosterol/cycloartenol-ergosterol pathway (Scheme 1) [6,14,21].…”

“…The physical properties based on the secondary structure prediction from Circular Dichroism measurements of yeast and soybean SMT1 indicate different populations of α-helices, β-sheets and random coils (Figure 3) but the common core that contains the active center appears to be conserved based on sequence alignment of 16 SMTs and homology building experiments [19,20]. At the current time, a single base, histidine90, is thought to function as the C-28 deprotonating agent and a second, as yet, unknown polar residue can function as the base in C-27 deprotonation.…”

“…The ammonium and sulfonium analogs were prepared with the expectation that the SMT in the ground state would recognize the "high energy intermediate" mimic, the assumption being that the active site binds the C-24 or C-25 heteroatom (N or S) substituted sterols to the same active center as the substrates [42] and the enzyme will undergo a conformational change during catalysis to recognize the inhibitory group of the substrate analog [6]. In work with the yeast SMT, 25-azalanosterol was discovered to bind to the active center with a K d value of 4 µM similar to either the K d values of zymosterol or AdoMet at 4 µM [20]. The expectation for tight binding was borne out by the results of the 24-and 25-heteroatom substituted sterols assayed with SMT1 and SMT2, the carbocation mimic was bound to the Michaelis complex many orders more tightly than the sterol (or AdoMet) substrate generating a K m /K i ratio of approximately 1000 [16,23,38,[40][41][42][43][44][45][46].…”

An Essential Gene for Fruiting Body Initiation in the Basidiomycete Coprinopsis cinerea Is Homologous to Bacterial Cyclopropane Fatty Acid Synthase Genes

“…Low homology is found over the whole protein length between ERG6 of S. cerevisiae and Cfs1 of C. cinerea, with the highest conservation in the SAM-binding motif (Figure 6). In ERG6, directly at the N-terminal end of the SAMdependent MTase fold is a sequence (DFYEYGWGSS FHFS; residues 77-92) referred to as region I that is highly specific to all D 24 -sterol C-MTases, has sterol binding activity, and forms an a-helix with a loop structure that targets into the substrate pocket (Nes et al 1998(Nes et al , 2004). This sequence is not present in the family of CFA synthases (Figure 6).…”