The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins.
Filamentous phage assembly in vivo shows an absolute requirement for thioredoxin and a partial requirement for thioredoxin reductase. Mutants in which one or both of the active site cysteine residues of thioredoxin were changed to alanine or serine were constructed and shown to support filamentous phage assembly. Some of the mutants were almost as effective as wild-type thioredoxin, while others supported phage assembly only when high levels of the mutant protein were present in the infected cell. The mutant proteins were all inactive in an assay which couples oxidation of NADPH to reduction of 5,5'-dithiobis-2-nitrobenzoic acid) via thioredoxin reductase and thioredoxin. These active site mutants make phage assembly completely independent of thioredoxin reductase, which suggests that the phage needs, and the active site mutants provide, the proteins in the reduced conformation. Other mutants were isolated on the basis of their failure to support filamentous phage growth. These specified mutant thioredoxin proteins with varying levels of redox activity in vivo and in vitro. The locations of these mutations suggest that the surface of thioredoxin thought to interact with thioredoxin reductase also interacts with the filamentous phage assembly machinery. An in vivo assay for thioredoxin redox function, based on the ability of cells to utilize methionine sulfoxide, was developed. Met- cells containing mutant thioredoxins that are inactive in vitro do not form colonies on plates containing methionine sulfoxide as the sole methionine source.
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003090:Coliphages / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005838:Genotype; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D013045:Species Specificity; D013879:Thioredoxins / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D013879:Thioredoxins