Mutational analysis of the gephyrin-related molybdenum cofactor biosynthetic gene cnxE from the lower eukaryote Aspergillus nidulans.
We report the identification of a number of mutations that result in amino acid replacements (and their phenotypic characterization) in either the MogA-like domain or domains 2 and 3 of the MoeA-like region of the Aspergillus nidulans cnxE gene. These domains are functionally required since mutations that result in amino acid substitutions in any one domain lead to the loss or to a substantial reduction in all three identified molybdoenzyme activities (i.e., nitrate reductase, xanthine dehydrogenase, and nicotinate hydroxylase). Certain cnxE mutants that show partial growth with nitrate as the nitrogen source in contrast do not grow on hypoxanthine or nicotinate. Complementation between mutants carrying lesions in the MogA-like domain or the MoeA-like region, respectively, most likely occurs at the protein level. A homology model of CnxE based on the dimeric structure of E. coli MoeA is presented and the position of inactivating mutations (due to amino acid replacements) in the MoeA-like functional region of the CnxE protein is mapped to this model. Finally, the activity of nicotinate hydroxylase, unlike that of nitrate reductase and xanthine dehydrogenase, is not restored in cnxE mutants grown in the presence of excess molybdate.
Heck
Immanuel S
IS
Institute for Plant Biochemistry, D-72076 Tübingen, Germany.
Schrag
Joseph D
JD
Sloan
Joan
J
Millar
Lindsey J
LJ
Kanan
Ghassan
G
Kinghorn
James R
JR
Unkles
Shiela E
SE
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001233:Aspergillus nidulans / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008667:Metalloproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism; D011621:Pteridines / Q000378:metabolism; D016415:Sequence Alignment; D014968:Xanthine Dehydrogenase / Q000378:metabolism
D002352:Carrier Proteins; D003067:Coenzymes; D005656:Fungal Proteins; D008565:Membrane Proteins; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011621:Pteridines; D014968:Xanthine Dehydrogenase; C073441:gephyrin; C022261:molybdopterin; C021862:nicotinate dehydrogenase