The alpha-aminoadipate reductase, a novel enzyme in the alpha-aminoadipic acid pathway for the biosynthesis of lysine in fungi, catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde in the presence of ATP, NADPH and MgCl(2). This reaction requires two distinct gene products, Lys2p and Lys5p. In the presence of CoA, Lys5p posttranslationally activates Lys2p for the alpha-aminoadipate reductase activity. Sequence alignments indicate the presence of all functional domains required for the activation, adenylation, dehydrogenation and alpha-aminoadipic acid binding in the Lys2p. In this report we present the results of site-directed mutational analysis of the conserved amino acid residues in the catalytic domains of Lys2p from the pathogenic yeast Candida albicans. Mutants were generated in the LYS2 sequence of pCaLYS2SEI by PCR mutagenesis and expressed in E. coli BL21 cells. Recombinant mutants and the wild-type Lys2p were analyzed for their alpha-aminoadipate reductase activity. Substitution of threonine 416, glycine 418, serine 419, and lysine 424 of the adenylation domain (TXGSXXXXK, residues 416-424) resulted in a significant reduction in alpha-aminoadipate reductase activity compared to the unmutagenized Lys2p control. Similarly replacement of glycine 978, threonine 980, glycine 981, phenylalanine 982, leucine 983 and glycine 984 of the NADPH binding domain (GXTGFLG, residues 978-984) caused a drastic decrease in alpha-aminoadipate reductase activity. Finally, substitution of histidine 460, aspartic acid 461, proline 462, isoleucine 463, glutamine 464, arginine 465, and aspartic acid 466 of the putative alpha-aminoadipic acid binding domain (HDPIQRD, residues 460-466) resulted in a highly reduced alpha-aminoadipate reductase activity. These results confirm the hypothesis that specific amino acid residues in highly conserved catalytic domains of Lys2p are essential for the alpha-aminoadipate reductase activity.