The Aspergillus nidulans cnxF gene and its involvement in molybdopterin biosynthesis. Molecular characterization and analysis of in vivo generated mutants.
The product of the Aspergillus nidulans cnxF gene was found by biochemical analysis of cnxF mutants to be involved in the conversion of precursor Z to molybdopterin. Mutants cnxF1242 and cnxF8 accumulate precursor Z, while the level of molybdopterin is undetectable. The DNA sequence of the cnxF gene was determined, and the inferred protein of 560 amino 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 comparisons indicate the presence of one and possibly two nucleotide binding 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 generated A. nidulans cnxF mutants were found to have amino acid substitutions of conserved residues within this central region of similarity to molybdopterin synthase sulfurylase, indicating that these seven amino acids are essential and that this domain is crucial for function. Of these seven, the cnxF1285 mutation results in the replacement of Gly-178, the last glycine residue of the N-proximal Gly-X-Gly-X-X-Gly motif, indicating that this motif is essential. Mutation of the conserved Arg-208, also probably involved in nucleotide binding, leads to a loss-of-function phenotype in cnxF200. Alteration of Cys-263, the only conserved 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 temperature-sensitive phenotypes and indicates that this residue is important in protein conformation. The C-terminal CnxF stretch (residues 397-560) 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 observation that mutant cnxF8 results from loss of the 34 C-terminal residues of CnxF. There is no obvious similarity of the CnxF C-terminal region 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.
Appleyard
M V
MV
School of Environmental and Evolutionary Biology, University of St. Andrews, St. Andrews, Fife KY16 9TH, United Kingdom.
Sloan
J
J
Kana'n
G J
GJ
Heck
I S
IS
Kinghorn
J R
JR
Unkles
S E
SE
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D003001:Cloning, Molecular; D003067:Coenzymes; D005656:Fungal Proteins / Q000737:chemistry*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D000090006:Molybdenum Cofactors; D009154:Mutation / Q000235:genetics; D011621:Pteridines / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013466:Sulfurtransferases / Q000378:metabolism
D003067:Coenzymes; D005656:Fungal Proteins; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; D012333:RNA, Messenger; D013466:Sulfurtransferases; C112988:CNXF protein, Emericella nidulans; C022261:molybdopterin; C085585:molybdopterin synthase