Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans.
Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K(m) 50 μM and k(cat) of 0.01 s⁻¹. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K(i)=54 nM and 24 (R,S),25-epiminolanosterol, K(i)=11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K(i) 9 μM) and k(inact)=0.03 min⁻¹) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K(i) values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC(50) values that ranged from 3 to 20 μM.
Ganapathy
Kulothungan
K
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, 79409, USA.
Kanagasabai
Ragu
R
Nguyen
Thi Thuy Minh
TT
Nes
W David
WD
D055162:Biocatalysis / Q000187:drug effects; D002176:Candida albicans / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004875:Ergosterol / Q000096:biosynthesis; D007700:Kinetics; D008745:Methylation / Q000187:drug effects; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D013261:Sterols / Q000737:chemistry / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013261:Sterols; D008780:Methyltransferases; D004875:Ergosterol