THE ASPERGILLUS-NIDULANS CNXABC LOCUS IS A SINGLE-GENE ENCODING 2 CATALYTIC DOMAINS REQUIRED FOR SYNTHESIS OF PRECURSOR-Z, AN INTERMEDIATE IN MOLYBDENUM COFACTOR BIOSYNTHESIS

The Aspergillus nidulans complex locus, cnxABC, has been shown to be r equired for the synthesis of precursor Z, an intermediate in the molyb dopterin cofactor pathway. The locus was isolated by chromosome walkin g a physical distance of 65-kilobase pairs from the brlA gene and defi nes a single transcript that encodes, most likely, a difunctional prot ein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affect ing the CNXA domain, mutants (cnxC) in the CNXC domain, and frameshift (cnxB) mutants disrupting both domains have greatly reduced levels of precursor Z compared with the wild type. The CNXA domain is similar a t the amino acid level to the Escherichia coli moaA gene product, whil e CNXC is similar to the E. coli moaC product, with both E. coli produ cts encoded by different cistrons. In the wild type, precursor Z level s are 3-4 times higher in nitrate-grown cells than in those grown on a mmonium, and there is an approximately parallel increase in the 2.4-ki lobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduc ed amino acid sequence of several mutants has identified residues crit ical for the function of the protein. In the CNXA section of the prote in, insertion of three amino acid residues into a domain thought to bi nd an iron-sulfur cofactor leads to a null phenotype as judged by comp lete loss of activity of the molybdoenzyme, nitrate reductase. More sp ecifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in b inding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids b etween residues valine 139 and serine 140, leads to a temperature-sens itive phenotype, suggesting a conformational change affecting the cata lytic activity of the CNXA region only. A single base pair deletion le ading to an in frame stop codon in the CNXC region, which causes a nul l phenotype, effectively deletes the last 20 amino acid residues of th e protein, indicating that these residues are necessary for catalytic function.