Active site aspartate residues are critical for tryptophan tryptophylquinone biogenesis in methylamine dehydrogenase.
The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue betaTrp57 and covalent cross-linking of betaTrp57 to betaTrp108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues betaAsp76 and betaAsp32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative betaD76N and betaD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. betaD76N MADH possessed the six disulfides but had no oxygen incorporated into betaTrp57 and was completely inactive. The betaD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into betaTrp57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the betaD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the Km value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.
Jones
Limei H
LH
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Pearson
Arwen R
AR
Tang
Yu
Y
Wilmot
Carrie M
CM
Davidson
Victor L
VL
D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D045563:Indolequinones / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008744:Methylamines / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D015233:Models, Statistical; D009154:Mutation; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D010231:Paracoccus denitrificans / Q000378:metabolism*; D011487:Protein Conformation; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
D004220:Disulfides; D045563:Indolequinones; D008744:Methylamines; C070569:tryptophan tryptophylquinone; D001224:Aspartic Acid; D014364:Tryptophan; C027451:methylamine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D003545:Cysteine; D010100:Oxygen