Functional Analysis of the Escherichia coli Molybdopterin Cofactor Biosynthesis Protein MoeA by Site-Directed Mutagenesis
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C. Sandu
Abstract
Five moeA mutants were generated by replacing some conserved amino acids of MoeA by sitedirected 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 Momolybdopterin 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 molybdopterinderived dephospho FormA as compared to bacteria transformed with wildtype moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.
Copyright © 2002 by Walter de Gruyter GmbH & Co. KG
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- To Our Authors, Readers and Subscribers
- Editors Note
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- TCF: Lady Justice Casting the Final Verdict on the Outcome of Wnt Signalling
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