Interaction of nicotinamide dinucleotides with human renalase, a novel multifaceted flavoprotein involved in the modulation of cardiovascular and renal functions

Renalase is a newly identified protein shown to regulate blood pressure, sodium and phosphate excretion, and to exert cardioprotective effects (1,2) through a molecular mechanism that is still obscure (3). Renalase has been proposed to be a catecholamine-degrading enzyme, either through an oxidative or a NADH-dependent mechanism. We have previously shown that renalase is a flavoprotein containing non-covalently bound FAD as prosthetic group (4). With the aim to shed light on the mechanism of its action, here we report on the interaction of the human protein with NAD and NADP, both in oxidized and reduced form, as potential cosubstrates of its enzymatic activity. Human renalase displays very low but significant NADH- and NADPH-diaphorase activities, with a marked preference for tetrazolium salts in comparison to other conventional dehydrogenase substrates as electron acceptors. The catalysis of these reactions involve the enzyme-bound FAD, as shown by pre-steady state studies in which we observed hydride-transfer from NAD(P)H to the prosthetic group. Renalase displays a higher catalytic efficiency (kcat/Km) but a lower kcat in the oxidation of NADH with respect to that of NADPH. By spectroscopy techniques we found that the binding of either NAD+ or NADP+ to renalase significantly alters the polarity of the environment of the FAD isoalloxazine, and that the nicotinamide moiety of the ligand is essential to elicit such effect. The possible localization of the NAD(P)-binding site in the renalase molecule will be discussed in the framework of the enzyme crystal structure (our data to be published).