title: The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis.
abstract: The Aspergillus nidulans complex locus, cnxABC, has been shown to be required for the synthesis of precursor Z, an intermediate in the molybdopterin cofactor pathway. The locus was isolated by chromosome walking a physical distance of 65-kilobase pairs from the brlA gene and defines a single transcript that encodes, most likely, a difunctional protein with two catalytic domains, CNXA and CNXC. Mutations (cnxA) affecting 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 at the amino acid level to the Escherichia coli moaA gene product, while CNXC is similar to the E. coli moaC product, with both E. coli products encoded by different cistrons. In the wild type, precursor Z levels are 3-4 times higher in nitrate-grown cells than in those grown on ammonium, and there is an approximately parallel increase in the 2.4-kilobase pair transcript following growth on nitrate, suggesting nitrate induction of this early section of the pathway. Analysis of the deduced amino acid sequence of several mutants has identified residues critical for the function of the protein. In the CNXA section of the protein, insertion of three amino acid residues into a domain thought to bind an iron-sulfur cofactor leads to a null phenotype as judged by complete loss of activity of the molybdoenzyme, nitrate reductase. More specifically, a mutant has been characterized in which tyrosine replaces cysteine 345, one of several cysteine residues probably involved in binding the cofactor. This supports the proposition that these residues play an essential catalytic role. An insertion of seven amino acids between residues valine 139 and serine 140, leads to a temperature-sensitive phenotype, suggesting a conformational change affecting the catalytic activity of the CNXA region only. A single base pair deletion leading to an in frame stop codon in the CNXC region, which causes a null phenotype, effectively deletes the last 20 amino acid residues of the protein, indicating that these residues are necessary for catalytic function.
authors.affiliation: School of Environmental and Evolutionary Biology, University of St. Andrews, St. Andrews, Fife KY16 9TH, United Kingdom. shiela.unkles@med.monash.edu.au
authors.forename: S E
authors.initials: SE
authors.lastname: Unkles
authors.forename: J
authors.initials: J
authors.lastname: Smith
authors.forename: G J
authors.initials: GJ
authors.lastname: Kanan
authors.forename: L J
authors.initials: LJ
authors.lastname: Millar
authors.forename: I S
authors.initials: IS
authors.lastname: Heck
authors.forename: D H
authors.initials: DH
authors.lastname: Boxer
authors.forename: J R
authors.initials: JR
authors.lastname: Kinghorn
mesh_terms: D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003067:Coenzymes; D003360:Cosmids; D004271:DNA, Fungal / Q000737:chemistry; D004792:Enzyme Precursors / Q000096:biosynthesis*; D005656:Fungal Proteins / Q000235:genetics*; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D009097:Multienzyme Complexes / Q000235:genetics*; D011621:Pteridines / Q000378:metabolism*; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
chemical_list: D003067:Coenzymes; D004271:DNA, Fungal; D004792:Enzyme Precursors; D005656:Fungal Proteins; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009097:Multienzyme Complexes; D011621:Pteridines; C109155:CnxABC protein, Emericella nidulans; C022261:molybdopterin