Functional analysis of the Escherichia coli molybdopterin cofactor biosynthesis protein MoeA by site-directed mutagenesis.
Five moeA mutants were generated by replacing some conserved amino acids of MoeA by site-directed mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Mo-molybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterin-derived dephospho FormA as compared to bacteria transformed with wild-type moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.
Sandu
Cristinel
C
Institut für Biochemie und Molekularbiologie, Universität Freiburg, Freiburg i. Br., Germany.
Brandsch
Roderich
R
D015153:Blotting, Western; D003067:Coenzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008667:Metalloproteins / Q000096:biosynthesis*; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D011621:Pteridines; D013466:Sulfurtransferases / Q000235:genetics*
D003067:Coenzymes; D029968:Escherichia coli Proteins; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D011621:Pteridines; D013466:Sulfurtransferases; C022261:molybdopterin; C111302:MoeA protein, E coli