The flavinylation reaction of trimethylamine dehydrogenase. Analysis by directed mutagenesis and electrospray mass spectrometry.
The flavinylation reaction products of wild-type and mutant forms of trimethylamine dehydrogenases purified from Methylophilus methylotrophus (bacterium W3A1) and Escherichia coli were studied by electrospray mass spectrometry (ESMS). The ESMS analyses demonstrated for the first time that wild-type enzyme expressed in M. methylotrophus is predominantly in the holoenzyme form, although a small proportion is present as the deflavo enzyme. ESMS demonstrated that the deflavo forms of the recombinant wild-type and mutant enzymes are not post-translationally modified and therefore prevented from assembling with flavin mononucleotide (FMN) because of previously unrecognized modifications. The data suggest that the higher proportion of deflavo enzyme observed for the recombinant wild-type enzyme is a consequence of the higher expression levels in E. coli. Mutagenesis of the putative flavinylation base (His-29 to Gln-29) did not prevent flavinylation, but the relative proportion of flavinylated product was substantially less than that seen for the recombinant wild-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 nucleophile were exchanged. Taken together, the data indicate that the assembly of trimethylamine dehydrogenase with FMN occurs during the folding of the enzyme, and in the fully folded form, deflavo enzyme is unable to recognize FMN. Results of site-directed mutagenesis experiments in the FMN-binding site suggest that following mutation the affinity for FMN during the folding process is reduced. Consequently, in the folded mutant enzymes, less flavin is trapped in the active site, and reduced levels of flavinylated product are obtained.
Packman
L C
LC
Department of Biochemistry, University of Cambridge, United Kingdom.
Mewies
M
M
Scrutton
N S
NS
D001483:Base Sequence; D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005415:Flavins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase