ADP competes with FAD binding in putrescine oxidase.
Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuO(Rh)) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuO(Rh) uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuO(Rh) that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuO(Rh) A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.
van Hellemond
Erik W
EW
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Mazon
Hortense
H
Heck
Albert J
AJ
van den Heuvel
Robert H H
RH
Heuts
Dominic P H M
DP
Janssen
Dick B
DB
Fraaije
Marco W
MW
D000244:Adenosine Diphosphate / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008995:Monoamine Oxidase / Q000737:chemistry; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D012240:Rhodococcus / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D000244:Adenosine Diphosphate; C022488:putrescine oxidase; D008995:Monoamine Oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010100:Oxygen