The sterol C-14 reductase encoded by the Neurospora crassa erg-3 gene: essential charged and polar residues identified by site-specific mutagenesis.
Sterol C-14 reductase catalyses the reduction of the Delta(14,15) bond in intermediates in the sterol biosynthesis pathway using NADPH as a cofactor. We have undertaken a systematic site-directed mutational analysis of all the conserved charged and potentially proton-donating residues of the sterol C-14 reductase from Neurospora crassa. The effect of each mutation was determined using an in vivo assay based on the complementation of the corresponding N. crassa mutant ( erg-3). The non-complementing mutations were also tested in the erg24 mutant of Saccharomyces cervisiae. The results are discussed with reference to the predicted topology of the enzyme and to its proposed catalytic mechanism, which involves addition of a proton from an appropriately positioned charged or polar residue to the substrate double bond, followed by addition of hydride ion from NADPH.
Prakash
A
A
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Kasbekar
D P
DP
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004563:Electrochemistry; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics*; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D010088:Oxidoreductases; C022672:delta(14)-sterol reductase