Isolation and characterisation of molybdenum cofactor biosynthesis genes for 'Aspergillus nidulans'

Abstract

The genes cnxG and cnxE from Aspergillus nidulans have been cloned and characterised. cnxG was isolated by functional complementation of the A. nidulans cnxG4 mutant strain. Within the cnxG4 mutant a single amino acid change from valine 250 to phenylalanine has been identified suggesting that this residue is critical for CnxG protein functionality. cnxG exhibits similarity at the amino acid level to several enzymes involved in catalysing transsulphuration reactions during the biosynthesis of cysteine and methionine. HPLC analysis of several cnxG mutant strains has demonstrated that precursor Z is accumulated to 17.2 -25.3 times that observed in the wild-type and that molybdopterin is not detectable. The evidence suggests that CnxG may be involved in the initial donation of sulphur to the MoCo biosynthesis pathway. cnxE was isolated by functional complementation of an Escherichia coli mogA mutant strain. cnxE exhibits similarity at the amino acid level to the E. coli MoeA, MogA and MoaB proteins and to the eukaryotic proteins Gephyrin from rat, Cnx1 from Arabidopsis thaliana and Cinnamon from Drosophila melanogaster. Analysis of the deduced amino acid sequences of several cnxE mutants has allowed the identification of residues which are critical for CnxE to function. HPLC analysis of the cnxE14 mutant indicated a modest accumulation of precursor Z and molybdopterin levels in comparison to the wild-type however the increase was too slight to conclude a significant effect. It would appear that CnxE is involved in the processing and/or incorporation of molybdenum into molybdopterin. Northern analysis has indicated that the transcription of both cnxG and cnxE is not subject to regulation by nitrate or ammonium. Mutations within cnxABC were shown to result in a significant reduction of precursor Z levels and abolition of molybdopterin levels in comparison to the wild-type, suggesting a function for CnxABC in the synthesis of precursor Z. In the wild-type, precursor Z levels were found to be 3.5 times higher in cells grown on nitrate than in those grown on ammonium as sole nitrogen source, perhaps indicating a degree of nitrate induction exerted on the early part of the pathway.