Crystal structure of human renalase, a novel flavoenzyme involved in the pathogenesis of cardiovascular diseases

Renalase is a recently identified flavoprotein (1), highly conserved in vertebrates, with orthologs in other organisms, including lower eukaryotes and bacteria. In humans, renalase is synthetized in kidneys, heart, skeletal muscles, brain and small intestine, being present in blood and urine (2). In mammals, renalase has been shown to regulate blood pressure, sodium and phosphate excretion, and to exert a cardioprotectant action (2). Despite its medical relevance, the mechanism of renalase action is not known at the molecular level (3); based on its moderate sequence similarity to monoamine oxidases (MAOs), it has been proposed to be a catecholamine degrading enzyme. To gain insight into its catalytic activity, we produced human renalase in Escherichia coli and showed that it contains non-covalently bound FAD (4), slightly stabilizes the neutral flavin semiquinone, and reacts slowly with sodium dithionite to yield a flavin adduct, with a Kd of ca. 2 mM. We found that renalase is devoid of any measurable catecholamine oxidase or dehydrogenase activities. Further, we solved the crystal structure of human renalase at 2.5 Å resolution. The protein adopts the p-hydroxybenzoate hydroxylase fold, and is structurally related to MAO-like enzymes. However, renalase is composed of two domains, thus lacking the third domain conserved in the other members of the family. A cavity (228 Å3), facing the re-face of the isoalloxazine, likely representing the active site, opens to the molecular surface. Compared to mono- or poly-amine oxidases, the renalase putative active-site lacks both a conserved Lys, that would interact via a water molecule with the N5 atom of the flavin ring, and the ‘aromatic cage’ expected to bind the substrate amino-group. Although these data do not allow to assign a catalytic activity to renalase yet, our studies represent a reference framework to test hypotheses on the enzyme molecular mechanism of action. References 1)Xu, J., Li, G., Wang, P., Velazquez, H., Yao, X., Li, Y., Wu, Y., Peixoto, A., Crowley, S., Desir, G.V. (2005) J. Clin. Invest. 115, 1275-1280. 2)Desir, G. V. (2011) Curr. Opin. Nephrol. Hypertens. 20, 31-36. 3)Eikelis, N., Hennebry, S.C., Lambert, G.W., Schlaich, M.P. (2011) Kidney Int. 79, 1380. 4)Pandini, V., Ciriello, F., Tedeschi, G., Rossoni, G., Zanetti, G., Aliverti, A. (2010) Protein Expr. Purif. 72, 244-253.