Aromatic amino-acid biosynthesis inCandida albicans: identification of theARO4 gene encoding a second DAHP synthase

Abstract

The primary step in the aromatic amino-acid biosynthetic pathway inSaccharomyces cerevisiae is catalyzed by two redundant isozymes of 3-deoxy-d-arabino-heptulosonate-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 theARO3 gene) is feedback-inhibited by phenylalanine, whereas the activity of the other isozyme (encoded by theARO4 gene) is feedback-inhibited by tyrosine. Transcription of both genes is controlled by GCN4. We previously cloned theARO3 gene from the opportunistic pathogenCandida albicans and found that: (1) it can complement anaro3 aro4 double mutation inS. 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 inC. albicans, we have constructed a homozygousaro3-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 homozygousaro3-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 fromS. cerevisiae andC. albicans, but even greater homology (78% identity) toS. cerevisiae Aro4p. We conclude that cells ofC. albicans contain a second Aro4p-related DAHP synthase.