Active-site residues are critical for the folding and stability of methylamine dehydrogenase.
Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta 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 beta 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 beta subunit, protein folding and TTQ biosynthesis are discussed.
Sun
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D
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MI 39216-4505, USA.
Jones
L H
LH
Mathews
F S
FS
Davidson
V L
VL
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003461:Crystallography; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D015964:Gene Expression Regulation, Bacterial; D006860:Hydrogen Bonding; D045563:Indolequinones; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011809:Quinones / Q000737:chemistry*; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
D004220:Disulfides; D045563:Indolequinones; D011809:Quinones; C070569:tryptophan tryptophylquinone; D014364:Tryptophan; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors