The enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyses the first step in aromatic amino acid biosynthesis in prokaryotes, plants and fungi. Cells of Saccharomyces cerevisiae contain two catalytically redundant DAHP synthases, encoded by the genes ARO3 and ARO4, whose activities are feedback-inhibited by phenylalanine and tyrosine, respectively. ARO3/4 gene transcription is controlled by GCN4. The authors previously cloned an ARO3 gene orthologue from Candida albicans and found that : (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine, and (2) a homozygous aro3-deletion mutant of C. albicans is phenotypically Aro M , suggesting the existence of another isozyme(s). They now report the identification and functional characterization of the C. albicans orthologue of S. cerevisiae Aro4p. The two Aro4p enzymes share 68 % amino acid identity. Phylogenetic analysis places the fungal DAHP synthases in a cluster separate from prokaryotic orthologues and suggests that ARO3 and ARO4 arose from a single gene via a gene duplication event early in fungal evolution. C. albicans ARO4 mRNA is elevated upon amino acid starvation, consistent with the presence of three putative Gcn4p-responsive elements (GCREs) in the gene promoter sequence. C. albicans ARO4 complements an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess tyrosine. The authors engineered ∆aro3/∆aro3 ∆aro4/MET3p ::ARO4 cells of C. albicans (with one wild-type copy of ARO4 placed under control of the repressible MET3 promoter) and found that they fail to grow in the absence of aromatic amino acids when ARO4 expression is repressed, and that this growth defect can be partially rescued by aromatic amino acids and certain aromatic amino acid pathway intermediates. It is concluded that, like S. cerevisiae, C. albicans contains two DAHP synthases required for the first step in the aromatic amino acid biosynthetic pathway.

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The control of amino acid and purine biosynthesis in the yeast Saccharomyces cerevisiae is mediated by the transcriptional activator GCN4. We previously identified the presence of two putative GCN4 responsive elements (GCREs) in the promoter sequence of the Candida albicans ARO3 gene, which encodes an enzyme in the aromatic amino acid pathway. We now show that amino acid deprivation results in a dramatic rise in the steady-state level of ARO3-specific mRNA, indicative of a GCN-like pathway in C. albicans.

The primary step in the aromatic amino-acid biosynthetic pathway in Saccharomyces cerevisiae is catalyzed by two redundant isozymes of 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase, either of which alone is sufficient to permit growth on synthetic complete media lacking aromatic acids (SC-Aro). The activity of one isozyme (encoded by the ARO3 gene) is feedback-inhibited by phenylalanine, whereas the activity of the other isozyme (encoded by the ARO4 gene) is feedback-inhibited by tyrosine. Transcription of both genes is controlled by GCN4. We previously cloned the ARO3 gene from the opportunistic pathogen Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine; and (2) its expression is induced in response to amino-acid deprivation, consistent with the presence of two putative GCN4-responsive promoter elements (Pereira and Livi 1993, 1995). To determine whether other DAHP synthases exist in C. albicans, we have constructed a homozygous aro3-deletion mutant strain. Such a mutant was found to be phenotypically Aro+, i. e., capable of normal growth on SC-Aro media, suggesting the presence of at least one additional isozyme. To confirm this result, a 222-bp DNA fragment was amplified by the polymerase chain reaction (PCR) from genomic DNA prepared from the homozygous aro3-deletion mutant, using a degenerate primer based on a conserved N-terminal region of Aro3p plus a degenerate comeback primer encoding a conserved region of the protein that lies within the deleted portion of the gene. The nucleotide sequence of this PCR fragment predicts a 74-amino acid DAHP synthase-related protein which shows strong homology to Aro3p from S. cerevisiae and C. albicans, but even greater homology (78% identity) to S. cerevisiae Aro4p. We conclude that cells of C. albicans contain a second Aro4p-related DAHP synthase.