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Updated: Jul 3, 2026

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
Published on: June 24, 2016
ADP competes with FAD binding in putrescine oxidase
Erik W van Hellemond1, Hortense Mazon, Albert J Heck
1Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Abstract:
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.
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