Characterization of Candida albicans dihydrofolate reductase.
Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.
Baccanari
D P
DP
Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Tansik
R L
RL
Joyner
S S
SS
Fling
M E
ME
Smith
P L
PL
Freisheim
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology; D005796:Genes; D005800:Genes, Fungal; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D007202:Indicators and Reagents; D013762:Tetrahydrofolate Dehydrogenase