An unusual diphosphatase from the PhnP family cleaves reactive FAD photoproducts

Abstract

Flavins are notoriously photolabile, but while the photoproducts derived from the iso-alloxazine ring are well known the other photoproducts are not. In the case of FAD, typically the main cellular flavin, the other photoproducts are predicted to include four- and five-carbon sugars linked to ADP. These FAD photoproducts were shown to be potent glycating agents, more so than ADP-ribose. Such toxic compounds would require disposal via an ADP-sugar diphosphatase or other route. Comparative analysis of bacterial genomes uncovered a candidate disposal gene that is chromosomally clustered with genes for FAD synthesis or transport and is predicted to encode a protein of the PhnP cyclic phosphodiesterase family. The representative PhnP family enzyme from Koribacter versatilis (here named Fpd, FAD photoproduct diphosphatase) was found to have high, Mn2+-dependent diphosphatase activity against FAD photoproducts, FAD, and ADP-ribose, but almost no phosphodiesterase activity against riboflavin 4′,5′-cyclic phosphate, a chemical breakdown product of FAD. To provide a structural basis of the unique Fpd activity, the crystal structure of K. versatilis Fpd was determined. The results place Fpd in the broad metallo-β-lactamase-like family of hydrolases, a diverse family commonly using two metals for hydrolytic catalysis. The active site of Fpd contains two Mn2+ ions and a bound phosphate, consistent with a diphosphatase mechanism. Our results characterize the first PhnP family member that is a diphosphatase rather than a cyclic phosphodiesterase and suggest its involvement in a cellular damage-control system that efficiently hydrolyzes the reactive, ADP-ribose-like products of FAD photodegradation.