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, regulates sodium and phosphate excretion, and displays cardioprotectant action through a mechanism that is barely understood to date. It has been proposed to act as a catecholamine-degrading enzyme, via either O 2-dependent 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 flavin adenine dinucleotide (FAD)-binding domain and a putative substrate-binding domain, the latter of which contains a five-stranded anti-parallel β-sheet. A large cavity (228 Å 3), facing the flavin ring, presumably represents the active site. Compared to monoamine oxidase or polyamine oxidase, the renalase active site is fully solvent exposed and lacks an ‘aromatic cage’ for binding the substrate amino group. Renalase has an extremely low diaphorase activity, displaying lower k cat but higher k cat/ K m for NADH compared to NADPH. Moreover, its FAD prosthetic group becomes slowly reduced when it is incubated with NADPH under anaerobiosis, and binds NAD + or NADP + with K d values of ca 2 mM. The absence of a recognizable NADP-binding site in the protein structure and its poor affinity for, and poor reactivity towards, NADH and NADPH suggest that these are not physiological ligands of renalase. Although our study does not answer the question on the catalytic activity of renalase, it provides a firm framework for testing hypotheses on the molecular mechanism of its action. [Display omitted] ► Renalase is a flavoprotein that regulates blood pressure and heart function in mammals. ► We solved its crystal structure at 2.5 Å resolution. ► It has a two-domain organization based on the p-hydroxybenzoate hydroxylase fold. ► FAD reactivity and active-site structure indicate that it is not an oxidase enzyme. ► NADH and NADPH are nonphysiological ligands.