TY - JOUR
T1 - Sterol C24-methyltransferase
T2 - Physio- and stereo-chemical features of the sterol C3 group required for catalytic competence
AU - Howard, Alicia L.
AU - Liu, Jialin
AU - Elmegeed, Gamal A.
AU - Collins, Emily K.
AU - Ganatra, Kalgi S.
AU - Nwogwugwu, Chizaram A.
AU - Nes, W. David
N1 - Copyright:
Copyright 2012 Elsevier B.V., All rights reserved.
PY - 2012/5
Y1 - 2012/5
N2 - Sterol C24-methyltransferases (24-SMTs) catalyze the electrophilic alkylation of Δ24-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 Vmax/Km 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-NH2 (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.
AB - Sterol C24-methyltransferases (24-SMTs) catalyze the electrophilic alkylation of Δ24-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 Vmax/Km 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-NH2 (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.
KW - Fluorinated sterol
KW - Hydrogen bonding
KW - Lanosterol
KW - Paracoccidioides brasiliensis
KW - Sterol C24-methyltransferase
KW - Sterol catalysis
UR - http://www.scopus.com/inward/record.url?scp=84860460208&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2012.03.002
DO - 10.1016/j.abb.2012.03.002
M3 - Article
C2 - 22446159
AN - SCOPUS:84860460208
VL - 521
SP - 43
EP - 50
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 1-2
ER -