Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase.
Candida albicans LYS1-encoded saccharopine dehydrogenase (CaLys1p, SDH) catalyzes the final biosynthetic step (saccharopine to lysine + alpha-ketoglutarate) of the novel alpha-aminoadipate pathway for lysine synthesis in fungi. The reverse reaction catalyzed by lysine-alpha-ketoglutarate reductase (LKR) is used exclusively in animals and plants for the catabolism of excess lysine. The 1,146 bp C. albicans LYS1 ORF encodes a 382 amino acid SDH. In the present investigation, we have used E. coli-expressed recombinant C. albicans Lys1p for the determination of both forward and reverse SDH activities in vitro, compared the sequence identity of C. albicans Lys1p with other known SDHs and LKRs, performed extensive site-directed mutational analyses of conserved amino acid residues and analyzed the phylogenetic relationship of C. albicans Lys1p to other known SDHs and LKRs. We have identified 14 of the 68 amino acid substitutions as essential for C. albicans Lys1p SDH activity, including two highly conserved functional motifs, H93XXF96XH98 and G138XXXG142XXG145. These results provided new insight into the functional and phylogenetic characteristics of the distinct biosynthetic SDH in fungi and catabolic LKR in higher eukaryotes.
Guo
Shujuan
S
Department of Microbiology, Miami University, Pearson Hall, Room 46, Oxford, OH 45056, USA.
Garrad
Richard C
RC
Bhattacharjee
J K
JK
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017354:Point Mutation; D012443:Saccharopine Dehydrogenases / Q000737:chemistry* / Q000235:genetics
D005656:Fungal Proteins; D007656:Ketoglutaric Acids; D012443:Saccharopine Dehydrogenases; C029496:saccharopine dehydrogenase (NADP+, lysine-forming); D008239:Lysine