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

doi:10.1016/j.jmb.2011.06.010

Journal of Molecular Biology

Volume 411, Issue 2, 12 August 2011, Pages 463-473

https://doi.org/10.1016/j.jmb.2011.06.010 Get rights and content

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 O₂-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 Å³), 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.

Graphical Abstract

Research Highlights

► 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.

Introduction

Renalase was identified in a 2005 study that focused on new links between chronic kidney diseases and their cardiovascular complications.¹ The human renalase gene (RNLS) is located on chromosome 10 and includes 10 exons.² Various isoforms arising from alternative splicing have been reported, two of which (renalase1 and renalase2) are annotated in genome databases (GenBank accession numbers NP_001026879 and NP_060833, respectively). RNLS is expressed in the kidneys, heart, skeletal muscle, brain, and small intestine;³ the main product (renalase1) has been detected also in blood, plasma, and urine.² End-stage renal disease is associated with lowered plasma renalase1 levels, indicating that the kidneys are the source of the circulating protein.¹ Renalase has also been proposed as an early biomarker of acute kidney ischemia.⁴ Intravenous administration of renalase1 was found to decrease blood pressure and heart rate in normal rats,¹ while its subcutaneous injection had an intense and prolonged anti-hypertensive effect on an animal model of salt-sensitive hypertension,4, 5 and its perfusion was found to have a heart-protective effect on a cardiac ischemia mouse model.² Furthermore, RNLS gene inactivation in mouse resulted in increased sympathetic activity, tachycardia, and hypertension.⁶ Two allelic variants of renalase displaying similar frequencies in the human population, carrying Glu or Asp at position 37, are known. The Asp variant was found to correlate significantly with an increased risk of developing essential hypertension and cardiac syndromes.7, 8 It has been proposed that renalase could modulate the intrarenal dopamine system, affecting sodium and phosphate excretion.4, 5, 9 In a rat model of chronic heart failure, lowered blood renal flow is associated with decreases in kidney renalase synthesis and norepinephrine clearance.¹⁰

Despite its potential impact on the treatment of some of the “big killer” diseases in the developed world, an understanding of renalase action at the molecular level has not been reached yet. Stemming from its sequence similarity to flavin-dependent monoamine oxidases (MAOs), it has been suggested that renalase could be a catecholamine-degrading flavoenzyme.1, 11, 12 Evidence has been provided for two possible catalytic mechanisms: O₂-dependent direct oxidation of amine substrate (as in the case of MAOs) or its NADH-dependent degradation, mediated by superoxide radical generation.⁸ However, the turnover rate of renalase seems too low to fully justify its physiological effects; moreover, the actual presence of a catecholamine-degrading activity in blood plasma, other than that supported by semicarbazide-sensitive amine oxidase, has been excluded by Boomsma and Tipton.¹³ (For two recent comprehensive reviews on renalase, the reader is referred to Desir⁴ and Medvedev et al.¹⁴) Renalase is highly conserved in mammals, but orthologs are present in protists (Phytophthora infestans T30-4; 27% sequence identity), cyanobacteria (Cyanothece sp.; 28% identity), and bacteria (Spirosoma linguale; 26% identity), suggesting different biological functions associated with similar folds and possibly similar catalyzed reactions (see Fig. 5a).

With the aim of elucidating its catalytic mechanism, we produced an in vivo folded form of human renalase1 in Escherichia coli.¹⁵ The recombinant protein was found to contain noncovalently bound flavin adenine dinucleotide (FAD), thus providing the first direct evidence that it is a flavoprotein. Here we present a further step towards the elucidation of its structure–function relationships by reporting its three-dimensional structure solved at 2.5 Å resolution. These data, together with kinetic and nucleotide binding studies, provide new hints on the active-site structural organization in this intriguing enzyme.

Section snippets

Redox properties and reactivity of the renalase FAD prosthetic group

Since we report here solely on the properties of human renalase1, we use the term “renalase” throughout this article to indicate this specific isoform, unless otherwise stated. To shed light onto renalase enzymatic activity, we investigated the stability and protonation state of the semiquinone form of its FAD cofactor, which are important criteria for flavoprotein classification.16, 17 Anaerobic renalase solutions were subjected to stepwise photoreduction at different pH values, and the

Discussion

The functional part of the study on renalase described here had two main purposes: (i) to assess the prosthetic group key chemical features that allow flavoprotein classification, and (ii) to analyze the interaction of renalase with nicotinamide dinucleotides, as conceivable cosubstrates of the enzyme. Concerning the first question, here we show that renalase provides a mild stabilization of the neutral form of the flavin semiquinone and that the renalase-bound FAD forms a sulfite adduct,

Materials and Methods

NAD⁺, NADP⁺, NADH, NADPH, 2′-phospho-AMP, 5′-AMP, INT, and WST1 were purchased from Sigma-Aldrich.

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Cited by (63)

Roles and mechanisms of renalase in cardiovascular disease: A promising therapeutic target
2020, Biomedicine and Pharmacotherapy
Citation Excerpt :
β-NADH and β-NADPH are considered cosubstrates of renalase [28]; both can be oxidized by renalase to produce β-NAD(P)＋ by transferring two electrons to the flavin cofactor and converting the configuration of ribose C1 from α to β. The chemistry catalysed by renalase is shown in Fig. 2 [29,30]. This catalytic activity provides detoxification as well as metabolite repair function, and it is also partially inhibited by noncatalytic isomers or products.
Cardiovascular disease (CVD) is prevalent worldwide and remains a leading cause of death. Although substantial progress has been made in the diagnosis and treatment of CVD, the prognosis remains unsatisfactory. Renalase is a newly discovered cytokine that is synthesized by the kidney and then secreted into blood. Numerous studies have suggested the efficacy of renalase in treating CVD by metabolizing catecholamines in the circulatory system. As a new biomarker of heart disease, renalase is normally recognized as a signalling molecule that activates cytoprotective intracellular signals to lower blood pressure, protect ischaemic heart muscle and promote atherosclerotic plaque stability in CVD, which subsequently improves cardiac function. Due to its important regulatory role in the circulatory system, renalase has gradually become a potential target in the treatment of CVD. This review summarizes the structure, mechanism and function of renalase in CVD, thereby providing preclinical evidence for alternative approaches and new prospects in the development of renalase-related drugs against CVD.
Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity
2020, Journal of Molecular Biology
The flavin-dependent amine oxidase (FAO) superfamily consists of over 9000 nonredundant sequences represented in all domains of life. Of the thousands of members identified, only 214 have been functionally annotated to date, and 40 unique structures are represented in the Protein Data Bank. The few functionally characterized members share a catalytic mechanism involving the oxidation of an amine substrate through transfer of a hydride to the FAD cofactor, with differences observed in substrate specificities. Previous studies have focused on comparing a subset of superfamily members. Here, we present a comprehensive analysis of the FAO superfamily based on reaction mechanism and substrate recognition. Using a dataset of 9192 sequences, a sequence similarity network, and subsequently, a genome neighborhood network were constructed, organizing the superfamily into eight subgroups that accord with substrate type. Likewise, through phylogenetic analysis, the evolutionary relationship of subgroups was determined, delineating the divergence between enzymes based on organism, substrate, and mechanism. In addition, using sequences and atomic coordinates of 22 structures from the Protein Data Bank to perform sequence and structural alignments, active-site elements were identified, showing divergence from the canonical aromatic-cage residues to accommodate large substrates. These specificity determinants are held in a structural framework comprising a core domain catalyzing the oxidation of amines with an auxiliary domain for substrate recognition. Overall, analysis of the FAO superfamily reveals a modular fold with cofactor and substrate-binding domains allowing for diversity of recognition via insertion/deletions. This flexibility allows facile evolution of new activities, as shown by reinvention of function between subfamilies.
The enzyme: Renalase
2017, Archives of Biochemistry and Biophysics
Citation Excerpt :
Though initially misidentified, 2- and 6DHNAD(P) molecules are clearly substrates for renalase. These molecules are oxidized with rate constants up to ∼1000 s−1; many orders of magnitude more rapid than observations made with catecholamines with or without added β-NAD(P)H [10,34,49]. Moreover, renalase enzymes have been identified in Pseudomonas species, a form of life that does not require catecholamines or experience hypertension [31].
Within the last two years catalytic substrates for renalase have been identified, some 10 years after its initial discovery. 2- and 6-dihydronicotinamide (2- and 6-DHNAD) isomers of β-NAD(P)H (4-dihydroNAD(P)) are rapidly oxidized by renalase to form β-NAD(P)⁺. The two electrons liberated are then passed to molecular oxygen by the renalase FAD cofactor forming hydrogen peroxide. This activity would appear to serve an intracellular detoxification/metabolite repair function that alleviates inhibition of primary metabolism dehydrogenases by 2- and 6-DHNAD molecules. This activity is supported by the complete structural assignment of the substrates, comprehensive kinetic analyses, defined species specific substrate specificity profiles and X-ray crystal structures that reveal ligand complexation consistent with this activity. This apparently intracellular function for the renalase enzyme is not allied with the majority of the renalase research that holds renalase to be a secreted mammalian protein that functions in blood to elicit a broad array of profound physiological changes. In this review a description of renalase as an enzyme is presented and an argument is offered that its enzymatic function can now reasonably be assumed to be uncoupled from whole organism physiological influences.
Increased serum renalase in peritoneal dialysis patients: Is it related to cardiovascular disease risk?
2017, Nefrologia
Renalase, with possible monoamine oxidase activity, is implicated in degradation of catecholamines; which suggests novel mechanisms of cardiovascular complications in patients with chronic kidney diseases. Epicardial adipose tissue (EAT) has been found to correlate with cardiovascular diseases (CVD) in dialysis patients. The present study aimed to evaluate the association of serum renalase levels with EAT thickness and other CVD risk factors in peritoneal dialysis (PD) patients.
The study included 40 PD patients and 40 healthy controls. All subjects underwent blood pressure and anthropometric measurements. Serum renalase was assessed by using a commercially available assay. Transthoracic echocardiography was used to measure EAT thickness and left ventricular mass index (LVMI) in all subjects.
The median serum renalase level was significantly higher in the PD patients than in the control group [176.5 (100–278.3) vs 122 (53.3–170.0) ng/ml] (p = 0.001). Renalase was positively correlated with C-reactive protein (r = 0.705, p < 0.001) and negatively correlated with RRF (r = −0.511, p = 0.021). No correlation was observed between renalase and EAT thickness or LVMI. There was a strong correlation between EAT thickness and LVMI in both the PD patients and the controls (r = 0.848, p < 0.001 and r = 0.640, p < 0.001 respectively).
This study indicates that renalase is associated with CRP and residual renal function but not with EAT thickness as CVD risk factors in PD patients.
La renalasa, posiblemente con actividad monoaminooxidasa, está implicada en la degradación de catecolaminas, lo que indica nuevos mecanismos de complicaciones cardiovasculares en pacientes con enfermedades renales crónicas. Se ha encontrado que el tejido adiposo epicárdico (TAE) se correlaciona con las enfermedades cardiovasculares (ECV) en pacientes de diálisis. El presente estudio tuvo como objetivo evaluar la asociación de los niveles de renalasa sérica con el espesor del EAT y otros factores de riesgo de ECV en pacientes de diálisis peritoneal (DP).
El estudio incluyó a 40 pacientes de DP y a 40 controles sanos. Se tomaron la presión arterial y las medidas antropométricas de todos los individuos. Se evaluó la renalasa sérica mediante un ensayo disponible comercialmente. Se utilizó la ecocardiografía transtorácica para medir el espesor del TAE y el índice de masa ventricular izquierda (IMVI) en todos los individuos.
La mediana del nivel de renalasa sérica fue significativamente mayor en los pacientes de DP que en el grupo control (176,5 [100-278,3] frente a 122 [5,3-170,0] ng/ml) (p = 0,001). La renalasa se correlacionó positivamente con la proteína C reactiva (r = 0,705; p < 0,001) y negativamente con la FRR (r = -0,511, p = 0,021). No se observó correlación entre la renalasa y el espesor del TAE ni el IMVI. Hubo una fuerte correlación entre el espesor del TAE y el IMVI tanto en los pacientes de DP como en los controles (r = 0,848; p < 0,001 y r = 0,640; p < 0,001, respectivamente).
Este estudio indica que la renalasa está asociada con la proteína C reactiva y la función renal residual, pero no con el espesor del TAE, como factores de riesgo de ECV en pacientes de DP.
Ligand binding phenomena that pertain to the metabolic function of renalase
2016, Archives of Biochemistry and Biophysics
Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P)⁺. This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase. We offer evidence of the potential for primary metabolism inhibition with structures of malate dehydrogenase and lactate dehydrogenase bound to the 6-dihydroNAD isomer. The previously observed preference of renalase from Pseudomonas for NAD-derived substrates over those derived from NADP is accounted for by the structure of the enzyme in complex with NADPH. We also show that nicotinamide nucleosides and mononucleotides reduced in the 2- and 6-positions are renalase substrates, but bind weakly. A seven-fold enhancement of acquisition (k_red/K_d) for 6-dihydronicotinamide riboside was observed for human renalase in the presence of ADP. However, generally the addition of complement ligands, AMP for mononucleotide or ADP for nucleoside substrates, did not enhance the reductive half-reaction. Non-substrate nicotinamide nucleosides or nucleotides bind weakly suggesting that only β-NADH and β-NADPH compete with dinucleotide substrates for access to the active site.
Renalase gene polymorphism is associated with increased blood pressure in preeclampsia
2016, Pregnancy Hypertension
Renalase is a novel enzyme that degrades circulating catecholamines. We aimed to investigate the role of rs2576178 and rs10887800 polymorphisms of the renalase gene in preeclampsia (PE) patients
This case-control study consisted of 110 women with PE and 102 normotensive controls. PCR-RFLP method was used for determination of renalase gene polymorphisms.
Allele frequency and genotype distribution of rs10887800 polymorphism were found statistically significantly higher in women with PE (p < 0.05). Also G allele and GG genotype of rs10887800 polymorphism were found higher in women with severe PE than that of mild PE (p < 0.05). There was no significant difference for rs2576178 polymorphism in terms of allele frequency and genotype distribution (p > 0.05). In PE patients, systolic blood pressure (SBP) means according to rs10887800 genotypes were found statistically significantly higher (GG vs AA; p = 0.001) and (GG vs GA; p = 0.001). Similarly, diastolic blood pressure (DBP) means were found statistically significantly higher in PE patients (GG vs GA: p = 0.001); (GG vs AA: p = 0.004). For rs2576178 polymorphism, SBP means were found as (GG vs AA; p = 0.012, GG vs GA; p > 0.05) in PE patients. DBP means were not significant according to rs2576178 genotypes in PE patients (p > 0.05).
The findings of the present study suggest that blood pressure may be increased by GG genotype and G allele of rs10887800 polymorphism and the polymorphism may increase the susceptibility to PE.

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Journal of Molecular Biology

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

Abstract

Graphical Abstract

Research Highlights

Introduction

Section snippets

Redox properties and reactivity of the renalase FAD prosthetic group

Discussion

Materials and Methods

Kidney Int.

Protein Expression Purif.

Biochim. Biophys. Acta

J. Biol. Chem.

Protein Expression Purif.

Trends Biochem. Sci.

Arch. Biochem. Biophys.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Mol. Biol.

Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure

J. Clin. Invest.

Renalase, a novel soluble FAD-dependent protein, is synthesized in the brain and peripheral nerves

Mol. Psychiatry

Role of renalase in the regulation of blood pressure and the renal dopamine system

Curr. Opin. Nephrol. Hypertens.

Novel insights into the physiology of renalase and its role in hypertension and heart disease

Pediatr. Nephrol.

Renalase deficiency aggravates ischemic myocardial damage

Kidney Int.

Renalase gene is a novel susceptibility gene for essential hypertension: a two-stage association study in northern Han Chinese population

J. Mol. Med.