FAD-Binding Site and NADP Reactivity in Human Renalase: A New Enzyme Involved in Blood Pressure Regulation

Renalase is a recently discovered flavoprotein that regulates blood pressure, sodium and phosphate excretion, and has a cardioprotectant action through a mechanism that is scarcely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, either via O2- or NADH-dependent mechanisms. Here we report the renalase crystal structure at 2.5 Å resolution together with new data on its interaction with nicotinamide dinucleotides. Renalase adopts the p-hydroxybenzoate hydroxylase fold topology, comprising a Rossmann fold based FAD-binding domain, and a putative substrate-binding domain, containing a five-stranded antiparallel -sheet. A large cavity (228 Å3), facing the flavin ring, presumably represents the active site. Compared to mono- or poly-amine oxidases, the renalase active-site is fully solvent exposed and lacks the 'aromatic cage' for binding the substrate amino-group. Renalase has extremely low diaphorase activity, displaying lower kcat but higher kcat/Km for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NAD(P)H under anaerobiosis, and binds NAD+ or NADP+ with Kd values of ca. 2 mM. The absence of a recognizable NAD(P)-binding site in the protein structure and its poor affinity/reactivity towards NAD(H) and NADP(H) suggest that these are not physiological ligands of renalase. Although our study does not answer the question of what the catalytic activity of renalase is, it provides a firm framework for testing hypotheses on the molecular mechanism of its action.