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.
Highlights
Introduction of Covalent FAD in Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuORh)A394C—In a previous study we constructed a model of PuORh that is based on the structure of the sequence-related monoamine oxidase B (MAO-B) [11]
The determined mass spectrum of unfolded PuORh confirms that PuORh contains noncovalently bound FAD: besides for a species that matches the primary sequence of PuORh, a species is observed with a mass of 785 Da, which corresponds to one FAD molecule
By ESI-MS experiments we observed that PuORh is present in three different dimeric species which contain: 1) two FAD molecules, 2) one FAD and one adenosine 5Ј-diphosphate (ADP) molecule, or 3) two ADP molecules per dimer
Summary
Based on a structural model of PuORh that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuORh 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. Like PuOMr, PuORh is a homodimer of about 100 kDa and, interestingly, contains only one mol of noncovalently bound FAD per mol of dimeric protein Such a low flavin cofactor occupancy is remarkable as inspection of the PuO sequences suggests that each monomer contains a Rossmanfold domain capable of FAD binding.
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