title: Sterol C24-methyltransferase: Physio- and stereo-chemical features of the sterol C3 group required for catalytic competence.
abstract: Sterol C24-methyltransferases (24-SMTs) catalyze the electrophilic alkylation of Δ(24)-sterols to a variety of sterol side chain constructions, and the C3- moiety is the primary determinant for substrate binding by these enzymes. To determine what specific structural features of the C3-polar group ensure sterol catalysis, a series of structurally related C3-analogs of lanosterol that differed in stereochemistry, bulk and electronic properties were examined against the fungal 24-SMT from Paracoccidioides brasiliensis (Pb) which recognize lanosterol as the natural substrate. Analysis of the magnitude of sterol C24-methylation activity (based on the kinetic constants of V(max)/K(m) and product distributions determined by GC-MS) resulting from changes at the C3-position in which the 3β-OH was replaced by 3α-OH, 3β-acetyl, 3-oxo, 3-OMe, 3β-F, 3β-NH(2) (protonated species) or 3H group revealed that lanosterol and five substrate analogs were catalyzed and yielded identical side chain products whereas neither the 3H- or 3α-OH lanosterol derivatives were productively bound. Taken together, our results demonstrate a chemical complementarity involving hydrogen bonding formation of specific active site contacts to the nucleophilic C3-group of sterol is required for proper orientation of the substrate C-methyl intermediate in the activated complex.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA.
authors.forename: Alicia L
authors.initials: AL
authors.lastname: Howard
authors.forename: Jialin
authors.initials: J
authors.lastname: Liu
authors.forename: Gamal A
authors.initials: GA
authors.lastname: Elmegeed
authors.forename: Emily K
authors.initials: EK
authors.lastname: Collins
authors.forename: Kalgi S
authors.initials: KS
authors.lastname: Ganatra
authors.forename: Chizaram A
authors.initials: CA
authors.lastname: Nwogwugwu
authors.forename: W David
authors.initials: WD
authors.lastname: Nes
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D007810:Lanosterol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D008958:Models, Molecular; D010228:Paracoccidioides / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013261:Sterols / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D013261:Sterols; D007810:Lanosterol; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase