Active-site residues are critical for the folding and stability of methylamine dehydrogenase

Site-directed mutagenesis was used to alter active-site residues of methyla mine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of t he beta subunit of MADH which are in close proximity to the tryptophan tryp tophylquinone (TTQ) prosthetic group were modified. The crystal structure o f MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively con servative mutations which removed the potentially reactive oxygens on the s ide chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly redu ced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts w ith antisera specific for the MADH beta subunit. No activity or cross-react ive protein was detected in extracts of cells expressing D76N, T122A and T1 22C MADH mutants. Very low levels of active MADH were produced by cells exp ressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutan ts were purified from cell extracts and found to be significantly less stab le than wild-type MADH. Only the T122S MADH mutant was produced at near wil d-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and T TQ biosynthesis are discussed.