The ARO4 gene of Candida albicans encodes a tyrosine-sensitive DAHP synthase: evolution, functional conservation and phenotype of Aro3p-, Aro4p-deficient mutants The GenBank accession number for the sequence reported in this paper is U53216.

Abstract

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.

DhARO4, an amino acid biosynthetic gene, is stimulated by high salinity in Debaryomyces hansenii

“…Similar values have been reported for the expression of ScARO4 mRNA under 10 m M 3AT treatment (Grundmann et al , 2001). The results indicated that DhARO4 is induced by amino acid limitation, like ScARO4 and CaARO4 (Sousa et al , 2002), and is probably also regulated by the transcription factor Gcn4p (Natarajan et al , 2001; Grundmann et al , 2001). Since the induction of DhARO4 upon amino acid limitation was similar to that occurring under high salt treatment (Figure 4, lane 3), as was found previously (Figures 1B, 3), we decided to combine the effects of amino acid limitation with high salinity and measure DhARO4 ‐mRNA levels.…”

“…Since our results supported the identity of the isolated gene as that coding for the DAHP Tyr enzyme, the regulatory sequences were analysed, taking into consideration that D. hansenii is poorly studied in this respect. ARO4 from S. cerevisiae and C. albicans predominantly responds to amino acid starvation through the well‐characterized Gcn4p, which activates its transcription by means of Gcn4p‐responsive element (GCRE), defined by the consensus sequence ATGACTCAA (Braus, 1991; Künzler et al , 1992; Grundmann et al , 2001; Sousa et al , 2002; Natarajan et al , 2001). After analysing the regulatory region of DhARO4 with the RSAT programme, it became apparent that the upstream region contains two putative GCREs sites, at positions −135 (ATGACTCAT, GCRE1) and −143 (ATGATTCAA, GCRE2), differing from the one GCRE sequence of ScARO4 by one substitution in both cases (Table 3).…”

“…To experimentally support the finding that the promoter of DhARO4 may be regulated by the Gcn4p factor, we examined the effects of the histidine analogue 3‐aminotriazole (3AT) that is routinely used to induce amino acid starvation in S. cerevisiae (reviewed by Sousa et al , 2002). When D. hansenii cells were grown in the presence of 10 m M 3AT, DhARO4 ‐mRNA levels increased 2.5 relative units over the control (Figure 4).…”

“…DAHP synthase in S. cerevisiae and Candida albicans is encoded by two genes, ARO3 and ARO4 . The two isoenzymes can be distinguished by a selective inhibition growth assay; the ARO3 ‐encoded enzyme is feedback‐inhibited by phenylalanine (DAHP Phe ), while the other (DAHP Tyr ) is inhibited by tyrosine (Braus, 1991; Hinnebusch, 1992; Sousa et al , 2002; Hartmann et al , 2003; Helmstaedt et al , 2005).…”

“…The lack of an amino acid increases the translation of Gcn4p that binds to specific promoter sequences known as Gcn4‐protein‐Responsive‐Elements (GCREs), stimulating transcription of its target genes (Braus, 1991; Natarajan et al , 2001). ARO4 is a gene that responds to amino acid limitation in S. cerevisiae and C. albicans, being activated by Gcn4p (Künzler et al , 1992; Sousa et al , 2002).…”