THE ASPERGILLUS-NIDULANS CNXF GENE AND ITS INVOLVEMENT IN MOLYBDOPTERIN BIOSYNTHESIS - MOLECULAR CHARACTERIZATION AND ANALYSIS OF IN-VIVO GENERATED MUTANS

The product of the Aspergillus nidulans cnxF gene was found by biochem ical analysis of cnxF mutants to be involved in the conversion of prec ursor Z to molybdopterin, Mutants cnxF1242 and cnxF8 accumulate precur sor Z, while the level of molybdopterin is undetectable. The DNA seque nce of the cnxF gene was determined, and the inferred protein of 560 a mino acids was found to contain a central region (residues around 157 to 396) similar in sequence to the prokaryotic proteins MoeB, which is thought to encode molybdopterin synthase sulfurylase, ThiF, required for thiamine biosynthesis, and HesA, involved in heterocyst formation, as well as eukaryotic ubiquitin-activating protein E1. Based on these similarities, a possible mechanism of action is discussed. Sequence c omparisons indicate the presence of one and possibly two nucleotide bi nding motifs, Gly-X-Gly-X-X-Gly, as well as two metal binding Cys-X-X- Cys motifs in this central region of the CnxF protein, Seven in vivo g enerated A. nidulans cnxF mutants were found to have amino acid substi tutions of conserved residues within this central region of similarity to molybdopterin synthase sulfurylase, indicating that these seven am ino acids are essential and that this domain is crucial for function. Of these seven, the cnxF1285 mutation results in the replacement of Gl y-178, the last glycine residue of the N-proximal Gly-X-Gly-X-X-Gly mo tif, indicating that this motif is essential. Mutation of the conserve d Arg-208, also probably involved in nucleotide binding, leads to a lo ss-of-function phenotype in cnxF200, Alteration of Cys-263, the only c onserved Cys residue (apart from the metal binding motifs), in cnxF472 suggests this residue as a candidate for thioester formation between molybdopterin synthase and the sulfurylase, Substitution of Gly-160 in two independently isolated mutants, cnxF21 and cnxF24, results in tem perature-sensitive phenotypes and indicates that this residue is impor tant in protein conformation. The C-terminal CnxF stretch (residues 39 7-560) shows substantial sequence conservation to a yeast hypothetical protein, Yhr1, such conservation between species suggesting that this region has function. Not inconsistent with this proposition is the ob servation that mutant cnxF8 results from loss of the 34 C-terminal res idues of CnxF, There is no obvious similarity of the CnxF C-terminal r egion with other proteins of known function. Two cnxF transcripts are found in low abundance and similar levels were observed in nitrate- or ammonium-grown cells.