Guo Shujuan, Garrad Richard C, Bhattacharjee J K
Department of Microbiology, Miami University, Pearson Hall, Room 46, Oxford, OH 45056, USA.
Mol Genet Genomics. 2006 Jan;275(1):74-80. doi: 10.1007/s00438-005-0062-z. Epub 2005 Nov 15.
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.
白色念珠菌LYS1编码的酵母氨酸脱氢酶（CaLys1p，SDH）催化真菌中赖氨酸合成的新α-氨基己二酸途径的最后生物合成步骤（酵母氨酸转化为赖氨酸+α-酮戊二酸）。赖氨酸-α-酮戊二酸还原酶（LKR）催化的逆反应仅用于动植物中过量赖氨酸的分解代谢。1146 bp的白色念珠菌LYS1开放阅读框编码一个382个氨基酸的SDH。在本研究中，我们使用大肠杆菌表达的重组白色念珠菌Lys1p在体外测定正向和反向SDH活性，比较白色念珠菌Lys1p与其他已知SDH和LKR的序列同一性，对保守氨基酸残基进行广泛的定点突变分析，并分析白色念珠菌Lys1p与其他已知SDH和LKR的系统发育关系。我们已经确定68个氨基酸取代中的14个对于白色念珠菌Lys1p SDH活性至关重要，包括两个高度保守的功能基序，H93XXF96XH98和G138XXXG142XXG145。这些结果为真菌中独特的生物合成SDH和高等真核生物中分解代谢LKR的功能和系统发育特征提供了新的见解。
