title: Characterization of Chlamydomonas 102 and 104 mutants reveals intermolecular complementation in the molybdenum cofactor protein CNX1E.
abstract: Chlamydomonas reinhardtii is a eukaryotic, unicellular, biflagellate green alga. In Chlamydomonas nitrate assimilation depends on the activity of the enzyme nitrate reductase that requires the molybdenum cofactor (Moco). We have characterized the Chlamydomonas strains 102 (nit5nit6), 104 (nit4nit5) and 106 (nit6). These mutations lead to the exchanges V171A (nit4) and G183D (nit6) in the CNX1E protein of Moco biosynthesis that result in Moco deficiency and hence inability to grow on nitrate. CNX1E inserts molybdenum into molybdopterin (MPT) to yield Moco through the intermediate MPT-AMP. The CNX1E variant G183D is unable to bind MPT-AMP. However, the CNX1E variant V171A is able to bind MPT-AMP but is not able to hydrolyze it and insert the molybdenum to obtain Moco. We show that intermolecular complementation between the CNX1E variants V171A and G183D both in vivo and in vitro restored its activity. The molecular characterization of strain 106 led us to discover the presence of wild type and nit6 CNX1E alleles in this strain (nit6/+). Then, we found out that 106 is indeed a diploid strain. It is proposed that 102, the strain from which 106 derived, may contain a genetic alteration that produces an aberrant meiotic division of the mature zygotes.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA3, Campus de Rabanales, Edificio Severo Ochoa, Córdoba 14071, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Chamizo-Ampudia
authors.forename: Aurora
authors.initials: A
authors.lastname: Galvan
authors.forename: Emilio
authors.initials: E
authors.lastname: Fernandez
authors.forename: Angel
authors.initials: A
authors.lastname: Llamas
mesh_terms: D020418:Algal Proteins / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D005816:Genetic Complementation Test; D006868:Hydrolysis; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009566:Nitrates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011621:Pteridines / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D020418:Algal Proteins; D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050505:Mutant Proteins; D009566:Nitrates; D011621:Pteridines; C022261:molybdopterin