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Eukaryotic Molybdopterin Synthase

1999; Elsevier BV; Volume: 274; Issue: 27 Linguagem: Inglês

10.1074/jbc.274.27.19286

1083-351X

Shiela E. Unkles, Immanuel S. Heck, M. Virginia C. L. Appleyard, James R. Kinghorn,

Peptidase Inhibition and Analysis

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-sensitivecnxH 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-sensitivecnxH 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. 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-sensitivecnxH 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-sensitivecnxH 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. high performance liquid chromatography rapid amplification of cDNA ends kilobase pair(s) Molybdoenzymes play essential roles in carbon, sulfur, and nitrogen cycles in most organisms. For instance, in higher eukaryotes including humans, sulfite oxidase is required for the degradation of sulfur amino acids converting sulfite to sulfate (1Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1994; 269: 272-276Abstract Full Text PDF PubMed Google Scholar). In certain lower eukaryotes such as Aspergillus nidulans, another molybdoenzyme nitrate reductase is required for the important ecological process of nitrate assimilation to ammonium (2Cove D.J. Biol. Rev. 1969; 54: 291-303Crossref Google Scholar). Additionally, a few molybdoenzymes exist in both eukaryotic groups, including xanthine dehydrogenase, which is important in the breakdown process of purines to uric acid (3Ichida K. Amaya Y. Kamatani N. Nishino T. Hosoya T. Sakai O. J. Clin. Invest. 1997; 99: 2391-2397Crossref PubMed Scopus (110) Google Scholar, 4Glatigny A. Scazzocchio C. J. Biol. Chem. 1995; 270: 3534-3550Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).For catalytic activity, these enzymes require the molybdenum cofactor, a prosthetic group that consists of a novel pterin called molybdopterin linked by its 6-alkyl side chain to a dithiolene group, which coordinates molybdenum. Its chemical structure and likely biosynthetic pathway were proposed by Rajagopalan (5Rajagopalan K.V. Neidhart F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 674-679Google Scholar) and are shown in Fig. 1. The presence of molybdenum cofactor has been demonstrated indirectly in a variety of biological material, such as cow milk and rabbit or fowl liver, by its ability to assemble active holo-nitrate reductase in cell-free extracts of a nit-1 mutant from the related fungusNeurospora crassa (6Ketchum P.A. Cambier H.Y. Frazier W.A. Madanski C.H. Nason A. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 1016-1023Crossref PubMed Scopus (98) Google Scholar). The nit-1 mutant lacks the cofactor but contains inactive apo-nitrate reductase. Similarly, the presence of the molybdenum cofactor has also been demonstrated indirectly in the fungus, A. nidulans (7Garrett R.G. Cove D.J. Mol. Gen. Genet. 1976; 147: 179-186Crossref PubMed Scopus (43) Google Scholar).Pleiotropic loss of human molybdoenzymes, including sulfite oxidase and xanthine dehydrogenase, results in a severe clinical disease for which no known therapy exists (8Mize C. Johnson J.L. Rajagopalan K.V. J. Inher. Metab. Dis. 1995; 18: 283-290Crossref PubMed Scopus (27) Google Scholar). In contrast to this extreme phenotype, impaired molybdoenzymes in the lower eukaryotes such as A. nidulans result in differences in simple growth characteristics,vis à visthe inability to grow on nitrate or hypoxanthine as the sole nitrogen source. Such phenotypes are easily observable and permit convenient genetic analyses (9Cove D.J. Pateman J.A. Nature. 1963; 198: 262-264Crossref PubMed Scopus (50) Google Scholar, 10Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar). These features make the study of the molybdenum cofactor in A. nidulans attractive and indeed allowed Cove and Pateman (9Cove D.J. Pateman J.A. Nature. 1963; 198: 262-264Crossref PubMed Scopus (50) Google Scholar) and Pateman et al. (10Pateman J.A. Cove D.J. Rever B.M. Roberts D.B. Nature. 1964; 201: 58-60Crossref PubMed Scopus (165) Google Scholar) to isolate molybdenum cofactor-deficient mutants some 3 decades ago. Their genetic analyses of mutants, isolated on the basis of screening for the inability to grow on nitrate as the sole source of nitrogen, indicated the presence of five unlinked loci. The mutants were shown to lack nitrate reductase and xanthine dehydrogenase, and this suggested that these loci were involved in the synthesis of a cofactor common to both nitrate reductase and xanthine dehydrogenase, accordingly designated as cnx(common component for nitrate reductase andxanthine dehydrogenase) with the gene allocations,cnxABC, cnxE, cnxF, cnxG, and cnxH.In addition to screening for lack of growth on nitrate, improved methods were found to allow selection of cnx mutants. One procedure involved direct selection for nitrate non-utilization by the putrescine starvation technique (11Cove D.J. Heredity. 1976; 36: 191-203Crossref PubMed Scopus (173) Google Scholar, 12Herman C. Clutterbuck A.J. Aspergillus Newsletter. 1966; 7: 13-14Google Scholar). Here a putrescine auxotrophic mutant strain (puA2) grown on minimal medium containing nitrate as the nitrogen source and a severely limiting concentration of putrescine produced small compact colonies. From these colonies, rapidly growing, spidery sectors of growth were observed which, on isolation, were found to result from a secondary mutation affecting nitrate utilization. Although the underlying mechanism and physiology of the putrescine starvation method are unclear, fortuitously a proportion of these sectors was found to be cnx mutants. In this way, cnx mutants could be easily and directly selected on the basis of nitrate non-utilization. A second selection technique involved the isolation of mutants resistant to the toxic substance, chlorate (13Aberg B. K. Lantbrukshögsk. Ann. 1947; 15: 37-107Google Scholar, 14Cove D.J. Mol. Gen. Genet. 1976; 146: 147-159Crossref PubMed Scopus (110) Google Scholar). Although the mechanisms of chlorate toxicity are unclear, this approach afforded the rapid and convenient isolation of large numbers of cnx mutants. Among the cnxmutants isolated on the basis of nitrate non-utilization (either by screening or by putrescine starvation selection), some were found to be chlorate-resistant, but intriguingly others were found to be sensitive to chlorate, both classes of mutants occurring in all fivecnx loci (11Cove D.J. Heredity. 1976; 36: 191-203Crossref PubMed Scopus (173) Google Scholar, 14Cove D.J. Mol. Gen. Genet. 1976; 146: 147-159Crossref PubMed Scopus (110) Google Scholar).The study of the A. nidulans cnx mutants yielded limited biochemical information regarding the function of their gene products (2Cove D.J. Biol. Rev. 1969; 54: 291-303Crossref Google Scholar). Mutations in the cnxH gene were found that resulted in a temperature-sensitive phenotype on nitrate, but not hypoxanthine, as sole nitrogen source. Unexpectedly, these mutants were reported to possess thermolabile nitrate reductase, and this led MacDonald and Cove (15MacDonald D.W. Cove D.J. Eur. J. Biochem. 1974; 47: 107-110Crossref PubMed Scopus (26) Google Scholar) to propose that the cnxH gene produces a protein that is an integral part of the molybdoenzyme, nitrate reductase.In this article, we describe biochemical and molecular characteristics of cnxG and cnxH genes and their products as well as the characterization of chlorate-resistant, chlorate-sensitive, and temperature-sensitive mutants.DISCUSSIONThe data presented here show that the cnxG andcnxH gene products of the eukaryote, A. nidulans, are involved in the conversion of precursor Z to molybdopterin, the intermediate section of the molybdenum cofactor biosynthetic pathway. First, cnxG4 and cnxH3 mutant strains, most likely loss-of-function, have negligible molybdopterin compared with wild-type levels while having vastly increased levels of precursor Z. Second, amino acid sequence comparisons with prokaryotes indicate that the CnxG and CnxH proteins are the eukaryotic homologues of the small and large subunits, respectively, of molybdopterin synthase, which is required to synthesize molybdopterin from precursor Z (5Rajagopalan K.V. Neidhart F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 674-679Google Scholar, 24Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 110-116Crossref PubMed Google Scholar, 25Johnson M.E. Rajagopalan K.V. J. Bacteriol. 1987; 169: 117-125Crossref PubMed Google Scholar). Between the eukaryotic CnxG and E. coli MoaD (29Rivers S.L. McNairn E. Blasco F. Giordano G. Boxer D.H. Mol. Microbiol. 1993; 8: 1071-1081Crossref PubMed Scopus (91) Google Scholar), the small subunit, the overall similarity is low (23% identity) and concentrated mainly in the 13 C-terminal amino acid residues with 9 of 13 identical. Several amino acid substitutions in mutant strains (see below) locate either in this C-terminal conserved region or in the first 21 residues at the N terminus, in contrast a region of limited similarity. Although only a small number of mutants have been analyzed at the nucleotide level, such localization of mutations could possibly indicate functional domains of the protein. The human homologue, MOCO1-A (32Sloan J. Kinghorn J.R. Unkles S.E. Nucleic Acids Res. 1999; 27: 854-858Crossref PubMed Scopus (23) Google Scholar), is more similar to the A. nidulans CnxG protein with 39% identity, and there are residues in the central section, as well as the N and C termini, conserved in both eukaryotic proteins that might additionally be involved in function. In contrast to the molybdopterin synthase small subunit proteins, similarity of the large subunit proteins (around 29% identity between the three species) is distributed more or less evenly over their primary structure, the bacterial protein, MoaE (29Rivers S.L. McNairn E. Blasco F. Giordano G. Boxer D.H. Mol. Microbiol. 1993; 8: 1071-1081Crossref PubMed Scopus (91) Google Scholar), being somewhat smaller than its eukaryotic counterparts, CnxH and MOCO1-B (32Sloan J. Kinghorn J.R. Unkles S.E. Nucleic Acids Res. 1999; 27: 854-858Crossref PubMed Scopus (23) Google Scholar).We have taken advantage of the ease of cnx mutant isolation in A. nidulans to identify amino acid residues important for function of molybdopterin synthase small and large subunits. Six amino acid residues were found to be essential in CnxG, the small subunit. Of these residues, three are conserved in the bacterial, fungal, and human proteins. Ala-10 and Glu-21 are within the region of low similarity near the N termini of the proteins. The third, the C-terminal residue of the small subunit, Gly-91, has been implicated in the catalytic mechanism of the conversion of precursor Z to molybdopterin. In the mechanism proposed from studies in E. coli, molybdopterin synthase adds dithiolene sulfur to the pterin side chain of precursor Z to form molybdopterin (5Rajagopalan K.V. Neidhart F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 674-679Google Scholar). The available evidence suggests that molybdopterin formation requires molybdopterin synthase to be in a sulfur-charged state. This activation of the synthase is thought to occur when molybdopterin synthase sulfurylase, encoded by thecnxF gene in A. nidulans (22Appleyard M.V.C.L. Sloan J. Kana'n G.J.M. Heck I.S. Kinghorn J.R. Unkles S.E. J. Biol. Chem. 1998; 273: 14869-14876Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and moeBin E. coli (30Pitterle D.M. Rajagopalan K.V. J. Bacteriol. 1989; 171: 3373-3378Crossref PubMed Google Scholar, 31Pitterle D.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13499-13505Abstract Full Text PDF PubMed Google Scholar, 34Nohno T. Kasai Y. Saito T. J. Bacteriol. 1988; 170: 4097-4102Crossref PubMed Google Scholar), adds sulfur atoms to the molybdopterin synthase small subunit, i.e. CnxG in A. nidulans. The precise mechanisms are unclear but the proposed sulfur transfer might resemble the activation of ubiquitin by ubiquitin-activating enzyme E1 (35Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar). Central to this mechanism is the presence of a C-terminal Gly in CnxG, which forms a thioester with an internal Cys residue of CnxF and subsequently acts as the acceptor for reactive sulfur via a thiocarboxylate bond from an as yet unidentified sulfur donor (Fig. 7). The significance of the short region of identity between the three organisms, immediately upstream of this C-terminal Gly residue, is unknown but could be associated with this sulfur transfer process. It has been suggested (5Rajagopalan K.V. Neidhart F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 674-679Google Scholar) that Gly-Gly may be required at the C terminus, but, whereas MOCO1-A and MoaD both have C-terminal Gly-Gly residues, Ser is the penultimate residue in the A. nidulans CnxG protein. Of the remaining three substitution mutations, which occur in residues conserved in both eukaryotes but not in the prokaryotic protein, Ile-85 is located in this region of high similarity toward the C terminus. Near the N terminus, replacement of Tyr-8 with a non-aromatic residue (Cys) in the short sequence Tyr-Phe-Ala, conserved between fungus and human and Phe-Phe-Ala in E. coli, suggests that an aromatic residue at this position is necessary for protein function. Finally Ala-12, present in fungal and human proteins, is not conserved in the prokaryotic protein.Figure 7Model for the interaction of molybdopterin synthase and CnxF. Sulfur is transferred from an unknown donor (X-S) by CnxF to the carboxyl group of the small subunit (CnxG) C-terminal Gly forming a reactive thiocarboxylate (5Rajagopalan K.V. Neidhart F.C. Escherichia coli and Salmonella Cellular and Molecular Biology. ASM Press, Washington, D. C.1996: 674-679Google Scholar). The sulfur is passed from CnxG to precursor Z, forming molybdopterin and allowing the small subunit to be recharged with sulfur by CnxF (22Appleyard M.V.C.L. Sloan J. Kana'n G.J.M. Heck I.S. Kinghorn J.R. Unkles S.E. J. Biol. Chem. 1998; 273: 14869-14876Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).View Large Image Figure ViewerDownload (PPT)In organisms such as bacteria and higher plants, generation of mutants defective in molybdenum cofactor biosynthesis is usually achieved by selection for chlorate resistance. In A. nidulans, mutants can additionally be selected on the basis of nitrate non-utilization using the putrescine starvation technique, and a proportion of these mutants are sensitive to chlorate. Therefore, in A. nidulanstwo classes of cnx mutants with regard to chlorate toxicity can be obtained. Chlorate-resistant mutants cnxG4 andcnxH3 fail to produce molybdopterin and completely lack the ability to grow on nitrate even after prolonged incubation. In contrast, the chlorate-sensitive mutants cnxG2 andcnxH1 elaborate some molybdopterin and show slow but significant growth on nitrate after long incubation periods (7 days) probably reflecting low in vivo levels of nitrate reductase activity. Such results suggest that in order to become chlorate-resistant, a mutant requires the complete abolition of nitrate reductase activity, whereas the retention of some activity renders a mutant strain sensitive to chlorate. In this way, we can predict whichcnx mutants are leaky, producing low levels of molybdopterin, and which are loss-of-function, completely lacking molybdopterin, simply by testing for sensitivity or resistance to chlorate toxicity. Therefore, we would expect that low but detectable levels of molybdopterin are present in chlorate-sensitive strains,cnxG24 and cnxG20. On this basis, replacement of CnxG residues, Ala-10 or Ala-12, in these mutants leads to reduced molybdopterin synthase activity but not loss-of-function. By extension, in chlorate-resistant strains, cnxG141, cnxG100,cnxH89, and cnxH43, substitution of residues Tyr-7 or Glu-21 of CnxG, and Gly-74 or Glu-166 of CnxH, respectively, results in absence of molybdopterin due to inactivity of the molybdopterin synthase.Temperature-sensitive mutants cnxH255 and cnxH604affect the same location in the CnxH protein, cnxH255 having an addition of a Gly residue after Gly-148 and cnxH604 being an alteration in Gly-148 itself. This region of the protein does not appear to show similarity to the human or bacterial proteins. A likely interpretation is that the conformation of the CnxH protein in these mutant strains is altered substantially at the nonpermissive temperature leading to complete loss-of-function of the molybdopterin synthase. Because no molybdopterin is produced, no active nitrate reductase is formed, and hence the strains are chlorate-resistant. At the permissive temperature, the conformation of the CnxH polypeptide is such that a small but significant level of molybdopterin synthase activity is possible, molybdenum cofactor is synthesized permitting active nitrate reductase, and the strains are chlorate-sensitive. Strains cnxH35 and cnxH36 have a less extreme temperature-sensitive phenotype than cnxH255 andcnxH604. Replacement of Ala-108 therefore also effects reduced molybdopterin synthase efficiency due to alteration of protein conformation, although the structural perturbation is less severe than changes affecting Gly-148. In contrast to mutants cnxH255and cnxH604, at the nonpermissive temperature of 37 °C, strains cnxH35 and cnxH36 are chlorate-sensitive and thus probably produce a low level of molybdopterin. Finally, it is noteworthy that no temperature-sensitive mutations have been isolated in the cnxG gene despite fairly extensive searches previously (15MacDonald D.W. Cove D.J. Eur. J. Biochem. 1974; 47: 107-110Crossref PubMed Scopus (26) Google Scholar) and in this study. The significance of this is unclear, but the lack of temperature-sensitive mutations may reflect the fact that cnxG is a small gene.A further point emerges from the study of the chlorate-sensitive mutants with regard to the interaction of the molybdopterin synthase subunits and the cnxF gene product, molybdopterin synthase sulfurylase. Previous analysis of pairwise double mutants between chlorate-sensitive strains, cnxH1, cnxG2, andcnxF11, showed that a cnxH1cnxF11 double mutant remains chlorate-sensitive (therefore producing molybdopterin), whereascnxG2cnxH1 and cnxG2cnxF11 double mutants become resistant (unable to produce molybdopterin) (14Cove D.J. Mol. Gen. Genet. 1976; 146: 147-159Crossref PubMed Scopus (110) Google Scholar). Study of combinations of mutations leading to chlorate sensitivity could therefore yield information on the tolerance of conformational changes during protein-protein interactions.The demonstration that temperature-sensitive cnxH mutants possessed thermolabile nitrate reductase led to the hypothesis advanced by MacDonald and Cove (15MacDonald D.W. Cove D.J. Eur. J. Biochem. 1974; 47: 107-110Crossref PubMed Scopus (26) Google Scholar) that the CnxH protein is an integral part of the nitrate reductase molecule, a homodimer of around 180 kDa (36MacDonald D.W. Coddington A. Eur. J. Biochem. 1974; 46: 169-178Crossref PubMed Scopus (52) Google Scholar, 37Minagawa N. Yoshimoto A. J. Biochem. (Tokyo). 1982; 91: 761-774Crossref PubMed Scopus (22) Google Scholar) consisting of two identical 91-kDa polypeptide subunits (38Cooley R.N. Tomsett A.B. Biochim. Biophys. Acta. 1985; 831: 89-93Crossref Scopus (7) Google Scholar). Because the validity of this proposal has never been resolved and the originalcnxH mutants with thermolabile nitrate reductase properties are lost or not available to us, two new temperature-sensitive strains,cnxH255 and cnxH604, were isolated in this study. These mutants were found to have nitrate reductase with wild-type thermolability. The cnxH gene encodes the molybdopterin synthase large subunit, and there is no evidence of nitrate reductase containing polypeptides other than the two subunits in any organism in which the enzyme has been purified including A. nidulans(38Cooley R.N. Tomsett A.B. Biochim. Biophys. Acta. 1985; 831: 89-93Crossref Scopus (7) Google Scholar). These observations, coupled with the wild-type thermal stability of nitrate reductase in our mutants, makes the proposition that CnxH is part of the nitrate reductase molecule appear less attractive.Chlorate-sensitive mutants, such as cnxG2 andcnxH1, grow better on nitrate than hypoxanthine after extended growth periods. This is also true for the temperature-sensitive mutant cnxH255 at the permissive temperature. Such mutants have been shown to produce low but measurable levels of molybdopterin. The reasons for the growth differences on nitrate and hypoxanthine are unclear, but it has been suggested that xanthine dehydrogenase has a more stringent requirement for the integrity of molybdenum cofactor than nitrate reductase (15MacDonald D.W. Cove D.J. Eur. J. Biochem. 1974; 47: 107-110Crossref PubMed Scopus (26) Google Scholar) or that nitrate reductase has a greater affinity for the cofactor than xanthine dehydrogenase (39Arst H.N. Microbiology. 1997; 143: 1437Google Scholar).The study of the A. nidulans molybdenum cofactor biosynthetic genes may aid the identification of essential residues in other eukaryotic homologues. These are clearly observed in EST data bases including worm, rice, fruit fly, mouse, and human.2Until now, human molybdenum cofactor deficiency has been recognized as a rare disease that leads to fatality at a very young age (1Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1994; 269: 272-276Abstract Full Text PDF PubMed Google Scholar, 3Ichida K. Amaya Y. Kamatani N. Nishino T. Hosoya T. Sakai O. J. Clin. Invest. 1997; 99: 2391-2397Crossref PubMed Scopus (110) Google Scholar), but recently, late-onset symptoms have been described in two individuals who appear to have less severe forms of molybdenum cofactor deficiency (40Hughes E.F. Fairbanks L. Simmonds H.A. Robinson R.O. Dev. Med. Child Neurol. 1998; 40: 57-61Crossref PubMed Scopus (33) Google Scholar). By extrapolation from the studies of A. nidulanschlorate-sensitive cnx mutants, these milder symptoms in humans could be the result of alteration of certain residues leading to low molybdopterin production with concomitant decrease in the elaboration of molybdoenzymes. The position of the A. nidulans CnxG residues which result in a leaky phenotype is conserved in the human protein. Reduced levels of molybdopterin due to residue changes may be less easily diagnosed in humans than severe deficiency and escape detection. Therefore, milder forms of human molybdenum cofactor deficiency could be more common in the population than originally thought.It is perhaps surprising that both cnxG and cnxHgenes are absent from the yeast Saccharomyces cerevisiae,2 an organism of immense technological interest as well as being a model eukaryote. Their absence however supports our inability to demonstrate the presence of precursor Z and molybdopterin in yeast. 3I. S. Heck, unpublished data. From the genome sequence, it is clear that S. cerevisiae does not possess nitrate reductase and xanthine dehydrogenase (or probably any molybdoenzyme) which reflects much earlier observations that the yeast cannot grow on nitrate or hypoxanthine as sources of nitrogen. Because molybdoenzymes and the molybdenum cofactor biosynthetic pathway are found in most organisms from bacteria to man, it seems likely that at some point during evolution S. cerevisiae has lost one of the cofactor biosynthetic genes, making redundant the genes encoding the remainder of the pathway and those specifying molybdoenzymes.Note Added in ProofThe half-life of nitrate reductase in temperature-sensitive mutants cnxH255 and cnxH604was reported to be approximately wild-type. However, the nitrate reductase assay used was that normally used for N. crassa(26Heck I.S. Ninnemann H. Phytochem. Photobiol. 1995; 61: 54-60Crossref PubMed Scopus (11) Google Scholar). To circumvent the unlikely possibility that this was the reason our results differed from MacDonald and Cove (15MacDonald D.W. Cove D.J. Eur. J. Biochem. 1974; 47: 107-110Crossref PubMed Scopus (26) Google Scholar), we repeated the analysis of cnxH604 and wild-type with the A. nidulans nitrate reductase assay procedure used by MacDonald and Cove (15MacDonald D.W. Cove D.J. Eur. J. Biochem. 1974; 47: 107-110Crossref PubMed Scopus (26) Google Scholar). The results are presented in the Fig. 8. Nitrate reductase activity in the wild-type was maximal after 2.5 min. In contrast, the maximum activity of cnxH604 was not reached until 10 min, after an initial increase in activity of 27%. The higher standard deviation values in the mutant result from variation in this initial increase (which was not observed in two of three experiments). The average half-life calculated for cnxH604 was 22.2 min compared with 19.25 min for wild-type. Even although formation of active nitrate reductase may not have a significant effect on the apparent rate of inactivation in the later stages of the experiment, there is a clear and significant superimposition of nitrate reductase formation and inactivation at the start of the incubation period. Consistent with the low specific activity of nitrate reductase from the mutant, these findings might be expolained by a kinetic limitation of nitrate reductase formation in vivo, resulting in accumulation of precursor form(s) that can form nitrate reductasein vitro. As discussed in the text, however, we can see no evidence that the half-life of nitrate reductase is significantly lower in our temperature-sensitive cnxH mutants than in the wild