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Protein Engineering, Vol. 14, No. 9, 675-681, September 2001
© 2001 Oxford University Press

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

Dapeng Sun1, Limei H. Jones1, F.Scott Mathews2 and Victor L. Davidson1,3

1 Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MI 39216-4505 and 2 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine,St. Louis, MO 63110, USA

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the ß subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH ß subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH ß subunit, protein folding and TTQ biosynthesis are discussed.


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