THE FLAVINYLATION REACTION OF TRIMETHYLAMINE DEHYDROGENASE - ANALYSISBY DIRECTED MUTAGENESIS AND ELECTROSPRAY MASS-SPECTROMETRY

The flavinylation reaction products of wild-type and mutant forms of t rimethylamine dehydrogenases purified from Methylophilus methylotrophu s (bacterium W(3)A(1)) and Escherichia coli were studied by electrospr ay mass spectrometry (ESMS). The ESMS analyses demonstrated for the fi rst time that wild-type enzyme expressed in M. methylotrophus is predo minantly in the holoenzyme form, although a small proportion is presen t as the deflavo enzyme. ESMS demonstrated that the deflavo forms of t he recombinant wild-type and mutant enzymes are not post-translational ly modified and therefore prevented from assembling with flavin mononu cleotide (FMN) because of previously unrecognized modifications. The d ata suggest that the higher proportion of deflavo enzyme observed for the recombinant wild-type enzyme is a consequence of the higher expres sion levels in E. coli. Mutagenesis of the putative flavinylation base (His-29 to Gln-29) did not prevent flavinylation, but the relative pr oportion of flavinylated product was substantially less than that seen for the recombinant mild-type enzyme. No flavinylation products were observed for a double mutant (His-29 to Cys-29; Cys-30 to His-30), in which the positions of the putative flavinylation base and cysteine nu cleophile were exchanged. Taken together, the data indicate that the a ssembly of trimethylamine dehydrogenase with FMN occurs during the fol ding of the enzyme, and in the fully folded form, deflavo enzyme is un able to recognize FMN. Results of site-directed mutagenesis experiment s in the FMN-binding site suggest that following mutation the affinity for FMN during the folding process is reduced. Consequently, in the f olded mutant enzymes, less flavin is trapped in the active site, and r educed levels of flavinylated product are obtained.