title: Eukaryotic molybdopterin synthase. Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants.
abstract: 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.
authors.affiliation: Department of Microbiology, Monash University, Clayton, Victoria 3168, Australia. shiela.unkles@med.monash.edu.au
authors.forename: S E
authors.initials: SE
authors.lastname: Unkles
authors.forename: I S
authors.initials: IS
authors.lastname: Heck
authors.forename: M V
authors.initials: MV
authors.lastname: Appleyard
authors.forename: J R
authors.initials: JR
authors.lastname: Kinghorn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001233:Aspergillus nidulans / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002704:Chlorates / Q000494:pharmacology; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D013466:Sulfurtransferases / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
chemical_list: D002704:Chlorates; D019271:Hypoxanthine; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D013466:Sulfurtransferases; C085585:molybdopterin synthase