ROULETTE MUTAGENESIS OF THE FMN-BINDING SITE OF KLEBSIELLA-PNEUMONIAE FLAVODOXIN

A method of randomising specific regions of coding sequences has been devised which utilises the Lac phenotype to identify mutants. Intact genes can be mutagenised, ma ng it unnecessary to reclone the mutations before examining mutant phenotypes. The method has been applied to three residues around the N-terminus of the first alpha helix of the Klebsiella pneumoniae nitrogenase flavodoxin, which are predicted to form part of the phosphate-binding subsite, Surprisingly, most substitutions at Gly12, a highly conserved residue in the chain reversal preceding the alpha helix, appeared to be fairly stable in vivo and were found to retain some function. Substitutions at Lys13, a surface residue which contributes to a patch of positive charge characteristic of the nitrogenase flavodoxins, had no major effect on stability or function. However, most substitutions at Thr14, which is predicted to hydrogen bond to the phosphate of the prosthetic group FMN, were much more destabilising and grossly reduced function. The exceptions were Ala, Cys, Ser and Val, which suggests that the bulk of the residue at this position is critical.