Deletion of the cnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans.

The Aspergillus nidulans cnxE gene, required for molybdenum cofactor biosynthesis, was isolated by functional complementation of an Escherichia coli mogA mutant strain. The deduced CnxE polypeptide consists of two domains which display similarity to the E. coli proteins MoeA and MogA, respectively, separated by a putative hinge region of around 58 amino acid residues which is notably histidine rich. A deletion mutant lacking the entire cnxE gene, including both MoeA-like and MogA-like domains, was identified. Compared to the wild type, a small increase in the intermediate precursor Z was observed in the deletion strain but was significant only under conditions in which the molybdoenzyme nitrate reductase was induced. Elevated levels of the pathway intermediate molybdopterin were found both under nitrate reductase-inducing and non-inducing conditions in the deletion mutant compared to the wild type. This increase is in contrast to previous results for cnxABC, cnxF, cnxG, and cnxH mutants, in which the levels of molybdopterin were substantially reduced, and therefore supports previously published classical genetic and biochemical studies that indicated that the CnxE protein is likely to be involved in the final stages of molybdenum cofactor biosynthesis. We have found no evidence during our chemical analysis for any involvement of this protein in the intermediate section of the molybdenum cofactor biosynthetic pathway (i.e. in the synthesis of molybdopterin from precursor Z), as has been suggested previously for E. coli MoeA. The 2.5-kb cnxE transcript is not abundant and appears to be expressed constitutively.

X-ray crystal structure of the trimeric N-terminal domain of gephyrin.

Gephyrin is a ubiquitously expressed protein that, in the central nervous system, forms a submembraneous scaffold for anchoring inhibitory neurotransmitter receptors in the postsynaptic membrane. The N- and C-terminal domains of gephyrin are homologous to the Escherichia coli enzymes MogA and MoeA, respectively, both of which are involved in molybdenum cofactor biosynthesis. This enzymatic pathway is highly conserved from bacteria to mammals, as underlined by the ability of gephyrin to rescue molybdenum cofactor deficiencies in different organisms. Here we report the x-ray crystal structure of the N-terminal domain (amino acids 2-188) of rat gephyrin at 1.9-A resolution. Gephyrin-(2-188) forms trimers in solution, and a sequence motif thought to be involved in molybdopterin binding is highly conserved between gephyrin and the E. coli protein. The atomic structure of gephyrin-(2-188) resembles MogA, albeit with two major differences. The path of the C-terminal ends of gephyrin-(2-188) indicates that the central and C-terminal domains, absent in this structure, should follow a similar 3-fold arrangement as the N-terminal region. In addition, a central beta-hairpin loop found in MogA is lacking in gephyrin-(2-188). Despite these differences, both structures show a high degree of surface charge conservation, which is consistent with their common catalytic function.

Eukaryotic molybdopterin synthase. Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants.

We describe the primary structure of eukaryotic molybdopterin synthase small and large subunits and compare the sequences of the lower eukaryote, Aspergillus nidulans, and a higher eukaryote, Homo sapiens. Mutants in the A. nidulans cnxG (encoding small subunit) and cnxH (large subunit) genes have been analyzed at the biochemical and molecular level. Chlorate-sensitive mutants, all the result of amino acid substitutions, were shown to produce low levels of molybdopterin, and growth tests suggest that they have low levels of molybdoenzymes. In contrast, chlorate-resistant cnx strains have undetectable levels of molybdopterin, lack the ability to utilize nitrate or hypoxanthine as sole nitrogen sources, and are probably null mutations. Thus on the basis of chlorate toxicity, it is possible to distinguish between amino acid substitutions that permit a low level of molybdopterin production and those mutations that completely abolish molybdopterin synthesis, most likely reflecting molybdopterin synthase activity per se. Residues have been identified that are essential for function including the C-terminal Gly of the small subunit (CnxG), which is thought to be crucial for the sulfur transfer process during the formation of molybdopterin. Two independent alterations at residue Gly-148 in the large subunit, CnxH, result in temperature sensitivity suggesting that this residue resides in a region important for correct folding of the fungal protein. Many years ago it was proposed, from data showing that temperature-sensitive cnxH mutants had thermolabile nitrate reductase, that CnxH is an integral part of the molybdoenzyme nitrate reductase (MacDonald, D. W., and Cove, D. J. (1974) Eur. J. Biochem. 47, 107-110). Studies of temperature-sensitive cnxH mutants isolated in the course of this study do not support this hypothesis. Homologues of both molybdopterin synthase subunits are evident in diverse eukaryotic sources such as worm, rat, mouse, rice, and fruit fly as well as humans as discussed in this article. In contrast, molybdopterin synthase homologues are absent in the yeast Saccharomyces cerevisiae. Precursor Z and molybdopterin are undetectable in this organism nor do there appear to be homologues of molybdoenzymes.

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.

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.

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.

Molybdenum cofactor biosynthesis in Neurospora crassa: biochemical characterization of pleiotropic molybdoenzyme mutants nit-7, nit-8, nit-9A, B and C.

Available mutants of molybdenum cofactor (MoCo) biosynthesis of Neurospora crassa were studied for converting factor activity and for in vitro molybdate repair of nitrate reductase (NR) activity. Mutant nit-7 was found to contain an activity that fits the functional definition of converting factor activity in Escherichia coli. Its high molecular weight fraction converts a low molecular weight compound from nit-1 and nit-8 into biologically active molybdopterin (MPT). Like nit-1, mutant nit-8 is devoid of this activity. Mutants nit-9 A, B and C contain a protein-bound precursor form of MoCo, which is presumed to be MPT bound to apo-NR. It is converted into active MoCo as part of NR in the presence of reduced glutathione and high exogenous molybdate concentrations. The NR apoenzyme of nit-1 is needed to detect the total amount of MoCo after molybdate repair, because mutants nit-9 A, B and C build no detectable content of functional NR apoenzyme. Evidence is presented for the transfer of MPT from demolybdo-NR to free NR apoenzyme.