The Aspergillus nidulans cnxF Gene and Its Involvement in Molybdopterin Biosynthesis

Abstract

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-XGly, 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.

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans

“…Eukaryotic MoeB homologues have been identified in fungi (Aspergillus nidulans, CnxF) (19), plants (Arabidopsis thaliana, Cnx5) (3), humans (MOCS3) (3), and other organisms showing C-and N-terminal extensions in comparison to E. coli MoeB. Although the Ϸ50 aa or longer N-terminal extensions are not conserved, the Ϸ158 aa C-terminal extensions show homologies to RLDs with a conserved cysteine residue proposed to be involved in sulfur transfer by formation of a cysteine persulfide (Fig.…”

The Biosynthesis of Methylated Amino Acids in the Active Site Region of Methyl-coenzyme M Reductase

“…The closest analogies observed in nature are thiopeptide antibiotics like promoinducine (23) or thiostreptone (24) that are produced by Streptomyces species. Also, C-terminal amino acid sulfurylation is known to occur during the biosynthesis of thiamine (25) and molybdopterin (26).…”

Eukaryotic Molybdopterin Synthase

“…Transcriptional start points were determined by sequencing 5ЈRACE products obtained using a 5ЈRACE System (Life Technologies, Inc.) according to manufacturer's instructions and primers GGSP1 (5Ј-GGCAACCTCATCGCCGC, nucleotide position 249) and GGSP2 (5Ј-TCGAGCGTATCGAAGAGCGAGG, position 110) for cnxG and primers HF (5Ј-CCATCCTGTCAAGTCTTG, position 618) and HF4 (5Ј-GCGACGGGACGGTCATC, position 257) for cnxH. Automated sequencing and analysis of DNA from mutant strains was carried out as described before (21,22) following polymerase chain reaction amplification of genomic DNA with primers GF (5Ј-GACT-TACGTCGCGTACCGG, position Ϫ216) and GR (5Ј-CTGCGCCTGAT-GGTATCAG, position 435) for cnxG and primers HR (5Ј-GGCAGTG-CAGTTCAAGC, position Ϫ87) and HF for cnxH. A. nidulans transformation and self-cloning procedures on the basis of complementation of mutants using the autonomous replicating plasmid, pHELP (23), were as described before for the characterization of the A. nidulans cnxF gene (22).…”

“…Automated sequencing and analysis of DNA from mutant strains was carried out as described before (21,22) following polymerase chain reaction amplification of genomic DNA with primers GF (5Ј-GACT-TACGTCGCGTACCGG, position Ϫ216) and GR (5Ј-CTGCGCCTGAT-GGTATCAG, position 435) for cnxG and primers HR (5Ј-GGCAGTG-CAGTTCAAGC, position Ϫ87) and HF for cnxH. A. nidulans transformation and self-cloning procedures on the basis of complementation of mutants using the autonomous replicating plasmid, pHELP (23), were as described before for the characterization of the A. nidulans cnxF gene (22).…”

“…Cloning and Structure of the cnxG and cnxH Genes-Both genes were isolated by self-cloning techniques assisted by the autonomous plasmid, pHELP (23), as used for the isolation of the A. nidulans cnxF gene (22). Clones obtained were used as probes to isolate genomic copies from an independently generated A. nidulans genomic library to circumvent potential problems of sequence rearrangement during the original isolation procedure.…”