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 - Funding Information:
This research was supported in part by grants from the National Science Foundation ( MCB-0929212 ). Partial support from the National Science Foundation (REU program) and Howard Hughes Medical Institute (award to Texas Tech University) to the undergraduates E.K.C., K.S.G. and C.A.N. is greatly appreciated.
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
SN - 0003-9861
VL - 521
SP - 43
EP - 50
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1-2
ER -