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

Site-directed mutagenesis was used to alter active-site residuesof methylamine dehydrogenase (MADH) from Paracoccus denitrificans.Four residues of the ß subunit of MADH which are inclose proximity to the tryptophan tryptophylquinone (TTQ) prostheticgroup were modified. The crystal structure of MADH reveals thateach of these residues participates in hydrogen bonding interactionswith other active-site residues, TTQ or water. Relatively conservativemutations which removed the potentially reactive oxygens onthe side chains of Thr122, Tyr119, Asp76 and Asp32 each resultedin greatly reduced or undetectable levels of MADH production.The reduction of MADH levels was determined by assays of activityand Western blots of crude extracts with antisera specific forthe MADH ß subunit. No activity or cross-reactiveprotein was detected in extracts of cells expressing D76N, T122Aand T122C MADH mutants. Very low levels of active MADH wereproduced by cells expressing D32N, Y119F, Y119E and Y119K MADHmutants. The Y119F and D32N mutants were purified from cellextracts and found to be significantly less stable than wild-typeMADH. Only the T122S MADH mutant was produced at near wild-typelevels. Possible roles for these amino acid residues in stabilizingunusual structural features of the MADH ß subunit,protein folding and TTQ biosynthesis are discussed.