Inactivation of C30A trimethylamine dehydrogenase by N-cyclopropyl-alpha-methylbenzylamine, 1-phenylcyclopropylamine, and phenylhydrazine

Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methy lotrophus (sp. W(3)A(1)) and its C30A mutant were inactivated with three kn own inactivators of monoamine oxidase, namely, phenylhydrazine, N-cycloprop yl-alpha -methylbenzylamine, and 1-phenylcyclopropylamine. All three compou nds irreversibly inactivated both the wild-type and C30A mutant enzymes, al though phenylhydrazine was 10 times more potent than N-cyclopropyl-alpha -m ethylbenzylamine, which was much more potent than 1-phenylcyclopropylamine. The change in the UV-visible absorption spectra upon modification indicate d that the flavin was modified. In the case of the C30A mutant, the absence of a covalent attachment of the flavin to the polypeptide has permitted LC -electrospray mass spectrometry of the reaction product to be undertaken, d emonstrating new mass peaks corresponding to various chemically modified fo rms of the flavin cofactor. In the case of N-cyclopropyl-alpha -methylbenzy lamine, masses corresponding to hydroxy-FMN and hydroxyriboflavin were dete cted. 1-Phenylcyclopropylamine inactivation of the C30A mutant produced thr ee modified flavins, as evidenced by the electrospray mass spectrum: hydrox y-FMN, FMN plus C6H5COCH2CH2, and hydroxy-FMN plus C6H5COCH2CH2. Phenylhydr azine inactivation of the C30A mutant gave at least seven different modifie d flavins: hydroxyriboflavin, hydroxy-FMN, two apparently isomeric compound s corresponding to hydroxy-FMN plus one phenyl group, two apparently isomer ic compounds corresponding to FMN plus one phenyl group, and FMN plus two p henyl groups. Covalent flavin adduct formation appears to be the only modif ication because dialysis of the inactive enzyme followed by reconstitution with FMN restores the enzyme activity to that of a noninactivated control.