Molecular analysis of the LYS2 gene of Candida albicans : homology to peptide antibiotic synthetases and the regulation of the ⋅-aminoadipate reductase

Abstract

The unique alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi and involves eight enzyme steps. alpha-Aminoadipate semialdehyde dehydrogenase, commonly called alpha-aminoadipate reductase (AAR), catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic semialdehyde by a novel mechanism. Two genes, LYS2 and LYS5, encode the heterodimeric enzyme in Saccharomyces cerevisiae. The LYS2 gene of Candida albicans was shown to be contained in the 4.8-kb insert of the plasmid pCaLYS2. This plasmid complemented lys2 mutants of both S. cerevisiae and C. albicans. The S. cerevisiae and C. albicans Lys2(+) transformants exhibited 138% and 160% of wild-type AAR activity, respectively. The DNA-sequence analysis of the 4.8-kb region in plasmid pCaLYS2 and a PCR product from genomic DNA which overlapped with the 4.8-kb insert revealed a continuous ORF of 4173 nucleotides encoding 1391 amino-acid residues. The C. albicans LYS2 ORF exhibited 63.0% identity at the nucleotide level and 56.2% identity at the amino-acid level to the LYS2 gene of S. cerevisiae. The ORF is preceded by consensus sequences for the TATA-, CAAT- and GCN4-box elements. An S. cerevesiae-type transcription termination signal is seen in the 3' flanking region. The deduced amino-acid sequence revealed a motif for an AMP-binding site and also the highly conserved core sequences common to peptide antibiotic synthetases. The LYS2 mRNA and alpha-aminoadipate reductase activity were repressed to a higher level in YEPD-grown cells than in cells grown in the presence of lysine or minimal medium. Additionally, AAR was shown to be feedback-inhibited by lysine and the lysine analog, thialysine. The results of the present report reveal the molecular characteristics of the LYS2 gene of C. albicans, its homology to peptide antibiotic synthetases, its divergence from the LYS2 gene of S. cerevisiae, and the regulation of AAR in C. albicans.

Novel Posttranslational Activation of the LYS2- Encoded α-Aminoadipate Reductase for Biosynthesis of Lysine and Site-Directed Mutational Analysis of Conserved Amino Acid Residues in the Activation Domain of Candida albicans

“…The activation of Lys2p and its AAR activity were measured using a previously described assay (16,34). The AAR reaction mixture contained 12.5 mM DL-␣-aminoadipate, 15 mM ATP, 10 mM MgCl 2 , 1 mM reduced glutathione, 0.625 mM ␤-NADPH, and 250 mM Tris, pH 8.0.…”

“…These genes and the encoded approximately 150-kDa proteins exhibit more than 60% identity at the nucleotide level and 55% identity at the amino acid level. Additionally, there exist several highly conserved core sequences and functional domains in the Lys2p which correspond to those in the nonribosomal peptide synthetases such as ␣-aminoadipyl-L-cysteinyl-D-valine synthetase for penicillin biosynthesis (8,11,18,34). Computer analysis also revealed the presence of a highly conserved phosphopantetheinylation domain (LGGHSI, amino acid residues 880 to 885) in Lys2p (34).…”

“…Additionally, there exist several highly conserved core sequences and functional domains in the Lys2p which correspond to those in the nonribosomal peptide synthetases such as ␣-aminoadipyl-L-cysteinyl-D-valine synthetase for penicillin biosynthesis (8,11,18,34). Computer analysis also revealed the presence of a highly conserved phosphopantetheinylation domain (LGGHSI, amino acid residues 880 to 885) in Lys2p (34). The LYS5 gene (816 nt encoding 272 amino acid residues) of S. cerevisiae does not contain any of the functional domains for the catalytic activity of AAR (23).…”

“…The open reading frame of the LYS2 gene consists of 4,176 nucleotides (nt) encoding 1,392 amino acid residues in S. cerevisiae (1,25), 4,173 nt encoding 1,391 amino acid residues in C. albicans (21,34), 4,330 nt encoding 1,409 amino acid residues in Penicillium chrysogenum (11), and 4,245 nt encoding 1,415 amino acid residues in Schizosaccharomyces pombe (8). These genes and the encoded approximately 150-kDa proteins exhibit more than 60% identity at the nucleotide level and 55% identity at the amino acid level.…”

LYS2 gene and its mutation in Kluyveromyces lactis

“…The sequence of this enzyme appears to be highly conservative in the fungal kingdom, since the α-AA reductase of K. lactis was also similar to that of Pichia sorbitophila (identity of 57.2%; Accession No. AJ288950; Bleykasten-Grosshans et al, 2001), Candida albicans (55.7%; Accession No.U58 133; Suvarna et al, 1998) Bhattacherjee and Bhattacherjee, 1998), Penicillium chrysogenum (48.7%;Accession No.Y13 967;Casqueiro et al, 1998), Acremonium chrysogenum (46.8%; Accession No. AJ261064; Hijarrubia et al, 2001) and Neurospora crassa (48.9%; Accession No.AL389890; Schulte et al, unpublished).…”