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

Abstract

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.

Is Renalase a Novel Player in Catecholaminergic Signaling? The Mystery of the Catalytic Activity of an Intriguing New Flavoenzyme

“…We found that renalase catalyzes NADH-and were all in the range of 1-2 mM. Taken together, the very low turnover numbers, the low affinity for nucleotides, and the poor selectivity in discriminating among them, strongly suggest that renalase is not a NAD(P)H-dependent enzyme, and that the observed diaphorase activity indicates a physiologically irrelevant side reaction [20]. Finally, we showed that renalase slightly stabilizes the neutral form of FAD and that is able to form a sulfite adduct [20].…”

“…The other two characterized RNLS transcripts (AK296262 and BX648154) encode much shorter deduced polypeptides (233 and 138, respectively). The comparison of the primary structures of the alternatively spliced renalase isoforms in the light of the crystal structure of renalase 1 [20] suggests that, while renalase 2 would probably be a compact globular protein similar to its larger isoform, the other two polypeptides would be unlikely to yield flavin-containing proteins, since they lack essential structural elements for FAD binding (see Chapter 6). Thus, the potential physiological significance of such shortest variants is uncertain.…”

“…Taken together, the very low turnover numbers, the low affinity for nucleotides, and the poor selectivity in discriminating among them, strongly suggest that renalase is not a NAD(P)H-dependent enzyme, and that the observed diaphorase activity indicates a physiologically irrelevant side reaction [20]. Finally, we showed that renalase slightly stabilizes the neutral form of FAD and that is able to form a sulfite adduct [20]. These observations indicate that the reactivity of the FAD prosthetic group of renalase dramatically differs from that of MAO A and B, allowing us to conclude, in agreement with the phylogenetic data (see Chapter 3), that renalase is not a MAOlike enzyme and likely is not even an oxidase.…”

“…The crystal structure of human renalase isoform 1 (PDB ID 3QJ4) was solved at 2.5 Å resolution by molecular replacement using a putative oxidoreductase from Pseudomonas syringae q888a4 (PDB ID 3KKJ) as starting model [20]. The renalase molecule has a compact, elongated, globular shape, with  and  secondary structure content of 26%…”

“…In comparison to most of its structural homologs, renalase lacks a third domain, which in PHBH is named the interface domain [50], and which participates in substrate binding in MAOs and polyamine oxidase [51]. Due to the absence of this structural element, the polar, positively charged cavity of 224 Å 3 that faces the re side of the flavin ring and presumably represents the active site is freely accessible to the solvent trough a large opening [20].…”