Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid.

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,ß-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,ß-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.

Rational Design, Synthesis, and Mechanism of (3S,4R)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase.

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.

Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase.

The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by ∼60 Šthat are bridged by four Fe4S4 centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4 centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.

The Interaction of Porcine Dihydropyrimidine Dehydrogenase with the Chemotherapy Sensitizer: 5-Ethynyluracil.

Dihydropyrimidine dehydrogenase (DPD) is a complex enzyme that reduces the 5,6-vinylic bond of pyrimidines, uracil, and thymine. 5-Fluorouracil (5FU) is also a substrate for DPD and a common chemotherapeutic agent used to treat numerous cancers. The reduction of 5FU to 5-fluoro-5,6-dihydrouracil negates its toxicity and efficacy. Patients with high DPD activity levels typically have poor outcomes when treated with 5FU. DPD is thus a central mitigating factor in the treatment of a variety of cancers. 5-Ethynyluracil (5EU) covalently inactivates DPD by cross-linking with the active-site general acid cysteine in the pyrimidine binding site. This reaction is dependent on the simultaneous binding of 5EU and nicotinamide adenine dinucleotide phosphate (NADPH). This ternary complex induces DPD to become activated by taking up two electrons from the NADPH. The covalent inactivation of DPD by 5EU occurs concomitantly with this reductive activation with a rate constant of ∼0.2 s-1. This kinact value is correlated with the rate of reduction of one of the two flavin cofactors and the localization of a mobile loop in the pyrimidine active site that places the cysteine that serves as the general acid in catalysis proximal to the 5EU ethynyl group. Efficient cross-linking is reliant on enzyme activation, but this process appears to also have a conformational aspect in that nonreductive NADPH analogues can also induce a partial inactivation. Cross-linking then renders DPD inactive by severing the proton-coupled electron transfer mechanism that transmits electrons 56 Šacross the protein.

Finding Ways to Relax: A Revisionistic Analysis of the Chemistry of E. coli GTP Cyclohydrolase II.

Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-ß phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-ß-pyrimidinone 5'-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity in Escherichia coli (Foor, F., Brown, G. M. J. Biol. Chem., 1975, 250, 9, 3545-3551) and has not been quantitatively evaluated in subsequent works. Using primarily transient state approaches we examined the interaction of RibA from E. coli with the GTP, inosine triphosphate, and PPi. Our data indicate that PPi is a slow substrate for RibA that is cleaved to form two phosphate ions (Pi). A combination of real-time enzymatically coupled Pi reporter assays and end-point 31P NMR revealed that Pi is formed at a catalytically relevant rate in the native reaction of RibA with GTP, redefining the reaction stoichiometry. Furthermore, our data indicate that both PPi and GTP stimulate conformational changes prior to hydrolytic chemistry, and we conclude that the cleavage of PPi bound as a substrate or an intermediate state results in conformational relaxation.

Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase.

The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.

Transient-State Analysis of Porcine Dihydropyrimidine Dehydrogenase Reveals Reductive Activation by NADPH.

Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial step in the catabolism of the pyrimidines uracil and thymine. Crystal structures have revealed an elaborate subunit architecture consisting of two flavin cofactors, apparently linked by four Fe4S4 centers. Analysis of the DPD reaction(s) equilibrium position under anaerobic conditions revealed a reaction that favors dihydropyrimidine formation. Single-turnover analysis shows biphasic kinetics. The serine variant of the candidate general acid, cysteine 671, provided enhanced kinetic resolution for these phases. In the first event, one subunit of the DPD dimer takes up two electrons from NADPH in a reductive activation. Spectrophotometric deconvolution suggests that these electrons reside on one of the two flavins. The fact that oxidation of the enzyme by dioxygen can be suppressed by the addition of pyrimidine is consistent with these electrons residing on the FMN. The second phase involves further oxidation of NADPH and concomitant reduction of the pyrimidine substrate. During this phase no net reduction of DPD cofactors is observed, indicating that the entire cofactor set acts as a wire, transmitting electrons from NADPH to the pyrimidine rapidly. This indicates that the availability of the proton from the C671 general acid controls the transmittance of electrons from NADPH to the pyrimidine. Acid quench and high-performance liquid chromatography product analysis of single-turnover reactions with limiting NADPH confirmed a 2:1 NADPH:pyrimidine stoichiometry for the enzyme, accounting for successive activation and pyrimidine reduction. These data support an alternating subunit model in which one protomer is activated and turns over before the other subunit can be activated and enter catalysis.

NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 µM. The enzyme then binds NADPH with a dissociation constant of 140 µM and reduces the flavin at a rate of 1,350 s-1 Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s-1 The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

Hydrogen movements in the oxidative half-reaction of kynurenine 3-monooxygenase from Pseudomonas fluorescens reveal the mechanism of hydroxylation.

Kynurenine 3-monoxygenase (KMO) catalyzes the conversion of l-kynurenine (L-Kyn) to 3-hydroxykynurenine (3-OHKyn) in the pathway for tryptophan catabolism. We have investigated the effects of pH and deuterium substitution on the oxidative half-reaction of KMO from P. fluorescens (PfKMO). The three phases observed during the oxidative half reaction are formation of the hydroperoxyflavin, hydroxylation and product release. The measured rate constants for these phases proved largely unchanging with pH, suggesting that the KMO active site is insulated from exchange with solvent during catalysis. A solvent inventory study indicated that a solvent isotope effect of 2-3 is observed for the hydroxylation phase and that two or more protons are in flight during this step. An inverse isotope effect of 0.84 ± 0.01 on the rate constant for the hydroxylation step with ring perdeutero-L-Kyn as a substrate indicates a shift from sp2 to sp3 hybridization in the transition state leading to the formation of a non-aromatic intermediate. The pH dependence of transient state data collected for the substrate analog meta-nitrobenzoylalanine indicate that groups proximal to the hydroperoxyflavin are titrated in the range pH 5-8.5 and can be described by a pKa of 8.8. That higher pH values do not slow the rate of hydroxylation precludes that the pKa measured pertains to the proton of the hydroperoxflavin. Together, these observations indicate that the C4a-hydroperoxyflavin has a pKa ≫ 8.5, that a non-aromatic species is the immediate product of hydroxylation and that at least two solvent derived protons are in-flight during oxygen insertion to the substrate aromatic ring. A unifying mechanistic proposal for these observations is proposed.

An improved method for the expression and purification of porcine dihydropyrimidine dehydrogenase.

Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of uracil and thymine bases with electrons derived from NADPH. The mammalian DPD enzyme is a functional homodimer and has an elaborate cofactor arrangement. Two flavin cofactors (FAD and FMN) reside in two active site cavities that are separated by around 60 Å. The flavins are apparently bridged by four Fe4S4 clusters, two of which are provided by the partner protomer of the dimer. The study of DPD has been hampered by modest yield from both native sources and from heterologous expression in E. coli. In addition, minimal active enzyme is obtained when the DPD gene is fused to an N-terminal 6His-tag. This limitation has dictated the use of traditional purification methods that are made more challenging by apparent over-expression of truncated and/or non-active forms of DPD. Here we detail methods of expression and purification that result in a ~4-fold improvement in the yield of active porcine DPD when expressed in E. coli BL21 DE3 cells via the pET plasmid expression system. The addition of ferrous ions and sulfate during induction provide a small increase in purified active enzyme. However, the addition of FAD and FMN during cell lysis results in a substantial increase in activity that also reduces the relative proportion of non-active, high molecular weight protein contaminants. We also describe methods that permit correlation of the flavin content with the amount of active enzyme and thus permit simple, rapid quantitation and evaluation of purified DPD sample.

Ligand binding phenomena that pertain to the metabolic function of renalase.

Renalase catalyzes the oxidation of isomers of ß-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form ß-NAD(P)+. This activity is thought to alleviate inhibition of multiple ß-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 (kred/Kd) 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 ß-NADH and ß-NADPH compete with dinucleotide substrates for access to the active site.

Bacterial Renalase: Structure and Kinetics of an Enzyme with 2- and 6-Dihydro-ß-NAD(P) Oxidase Activity from Pseudomonas phaseolicola.

Despite a lack of convincing in vitro evidence and a number of sound refutations, it is widely accepted that renalase is an enzyme unique to animals that catalyzes the oxidative degradation of catecholamines in blood in order to lower vascular tone. Very recently, we identified isomers of ß-NAD(P)H as substrates for renalase (Beaupre, B. A. et al. (2015) Biochemistry, 54, 795-806). These molecules carry the hydride equivalent on the 2 or 6 position of the nicotinamide base and presumably arise in nonspecific redox reactions of nicotinamide dinucleotides. Renalase serves to rapidly oxidize these isomers to form ß-NAD(P)⁺ and then pass the electrons to dioxygen, forming H2O2. We have also shown that these substrate molecules are highly inhibitory to dehydrogenase enzymes and thus have proposed an intracellular metabolic role for this enzyme. Here, we identify a renalase from an organism without a circulatory system. This bacterial form of renalase has the same substrate specificity profile as that of human renalase but, in terms of binding constant (K(d)), shows a marked preference for substrates derived from ß-NAD⁺. 2-dihydroNAD(P) substrates reduce the enzyme with rate constants (k(red)) that greatly exceed those for 6-dihydroNAD(P) substrates. Taken together, k(red)/K(d) values indicate a minimum 20-fold preference for 2DHNAD. We also offer the first structures of a renalase in complex with catalytically relevant ligands ß-NAD⁺ and ß-NADH (the latter being an analogue of the substrate(s)). These structures show potential electrostatic repulsion interactions with the product and a unique binding orientation for the substrate nicotinamide base that is consistent with the identified activity.

Metabolic function for human renalase: oxidation of isomeric forms of ß-NAD(P)H that are inhibitory to primary metabolism.

Renalase is a recently identified flavoprotein that has been associated with numerous physiological maladies. There remains a prevailing belief that renalase functions as a hormone, imparting an influence on vascular tone and heart rate by oxidizing circulating catecholamines, chiefly epinephrine. This activity, however, has not been convincingly demonstrated in vitro, nor has the stoichiometry of this transformation been shown. In prior work we demonstrated that renalase induced rapid oxidation of low-level contaminants of ß-NAD(P)H solutions ( Beaupre, B. A. et al. (2013) Biochemistry 52 , 8929 - 8937 ; Beaupre, B. A. et al. (2013) J. Am. Chem. Soc . 135 , 13980 - 13987 ). Slow aqueous speciation of ß-NAD(P)H resulted in the production of renalase substrate molecules whose spectrophotometric characteristics and equilibrium fractional accumulation closely matched those reported for α-anomers of NAD(P)H. The fleeting nature of these substrates precluded structural assignment. Here we structurally assign and identify two substrates for renalase. These molecules are 2- and 6-dihydroNAD(P), isomeric forms of ß-NAD(P)H that arise either by nonspecific reduction of ß-NAD(P)(+) or by tautomerization of ß-NAD(P)H (4-dihydroNAD(P)). The pure preparations of these molecules induce rapid reduction of the renalase flavin cofactor (230 s(-1) for 6-dihydroNAD, 850 s(-1) for 2-dihydroNAD) but bind only a few fold more tightly than ß-NADH. We also show that 2- and 6-dihydroNAD(P) are potent inhibitors of primary metabolism dehydrogenases and therefore conclude that the metabolic function of renalase is to oxidize these isomeric NAD(P)H molecules to ß-NAD(P)(+), eliminating the threat they pose to normal respiratory activity.

Renalase does not catalyze the oxidation of catecholamines.

It is widely accepted that the function of human renalase is to oxidize catecholamines in blood. However, this belief is based on experiments that did not account for slow, facile catecholamine autoxidation reactions. Recent evidence has shown that renalase has substrates with which it reacts rapidly. The reaction catalyzed defines renalase as an oxidase, one that harvests two electrons from either 2-dihydroNAD(P) or 6-dihydroNAD(P) to form ß-NAD(P)(+) and hydrogen peroxide. The apparent metabolic purpose of such a reaction is to avoid inhibition of primary dehydrogenase enzymes by these ß-NAD(P)H isomers. This article demonstrates that renalase does not catalyze the oxidation of neurotransmitter catecholamines. Using high-performance liquid chromatography we show that there is no evidence of consumption of epinephrine by renalase. Using time-dependent spectrophotometry we show that the renalase FAD cofactor spectrum is unresponsive to added catecholamines, that adrenochromes are not observed to accumulate in the presence of renalase and that the kinetics of single turnover reactions with 6-dihydroNAD are unaltered by the addition of catecholamines. Lastly we show using an oxygen electrode assay that plasma renalase activity is below the level of detection and only when exogenous renalase and 6-dihydroNAD are added can dioxygen be observed to be consumed.

Kinetics and equilibria of the reductive and oxidative half-reactions of human renalase with α-NADPH.

Renalase is a recently discovered flavoprotein that has been reported to be a hormone produced by the kidney to down-modulate blood pressure and heart rate. The consensus belief has been that renalase oxidizes circulating catecholamine neurotransmitters thereby attenuating vascular tone. However, a convincing in vitro demonstration of this activity has not been made. We have recently discovered that renalase has α-NAD(P)H oxidase/anomerase activity. Unlike most naturally occurring nucleotides, NAD(P)H can accumulate small amounts of the α-anomers that once oxidized are configurationally stable and unable to participate in cellular activity. Thus, anomerization of NAD(P)H would result in a continual loss of cellular redox currency. As such, it appears that the root purpose of renalase is to return α-anomers of nicotinamide dinucleotides to the ß-anomer pool. In this article, we measure the kinetics and equilibria of renalase in turnover with α-NADPH. Renalase is selective for the α-anomer, which binds with a dissociation constant of ∼20±3 µM. This association precedes monophasic two-electron reduction of the FAD cofactor with a rate constant of 40.2±1.3 s(-1). The reduced enzyme then delivers both electrons to dioxygen in a second-order reaction with a rate constant of ∼2900 M(-1) s(-1). Renalase has modest affinity for its ß-NADP+ product (Kd=2.2 mM), and the FAD cofactor has a reduction potential of -155 mV that is unaltered by saturating ß-NADP+. Together these data suggest that the products are formed and released in a kinetically ordered sequence (ß-NADP+ then H2O2), however, the reoxidation of renalase is not contingent on the dissociation of ß-NADP+. Neither the oxidized nor the reduced form of renalase is able to catalyze anomerization, implying that the redox and anomerization chemistries are inextricably linked through a common intermediate.

Renalase is an α-NAD(P)H oxidase/anomerase.

Renalase is a protein hormone secreted into the blood by the kidney that is reported to lower blood pressure and slow heart rate. Since its discovery in 2005, renalase has been the subject of conjecture pertaining to its catalytic function. While it has been widely reported that renalase is the third monoamine oxidase (monoamine oxidase C) that oxidizes circulating catecholamines such as epinephrine, there has been no convincing demonstration of this catalysis in vitro. Renalase is a flavoprotein whose structural topology is similar to known oxidases, lysine demethylases, and monooxygenases, but its active site bears no resemblance to that of any known flavoprotein. We have identified the catalytic activity of renalase as an α-NAD(P)H oxidase/anomerase, whereby low equilibrium concentrations of the α-anomer of NADPH and NADH initiate rapid reduction of the renalase flavin cofactor. The reduced cofactor then reacts with dioxygen to form hydrogen peroxide and releases nicotinamide dinucleotide product in the ß-form. These processes yield an apparent turnover number (0.5 s(-1) in atmospheric dioxygen) that is at least 2 orders of magnitude more rapid than any reported activity with catechol neurotransmitters. This highly novel activity is the first demonstration of a role for naturally occurring α-NAD(P)H anomers in mammalian physiology and the first report of a flavoprotein catalyzing an epimerization reaction.

Structural and Kinetic Analyses Reveal the Dual Inhibition Modes of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic Acid (BCF3).

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the leading cause of death among people with cirrhosis. HCC is typically diagnosed in advanced stages when tumors are resistant to both radio- and chemotherapy. Human ornithine aminotransferase (hOAT) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme involved in glutamine and proline metabolism. Because hOAT is overexpressed in HCC cells and a contributing factor for the uncontrolled cellular division that propagates malignant tumors (Ueno et al. J. Hepatol. 2014, 61, 1080-1087), it is a potential drug target for the treatment of HCC. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic acid (BCF3) has been shown in animal models to slow the progression of HCC by acting as a selective and potent mechanism-based inactivator of OAT (Zigmond et al. ACS Med. Chem. Lett. 2015, 6, 840-844). Previous studies have shown that the BCF3-hOAT reaction has a bifurcation in which only 8% of the inhibitor inactivates the enzyme while the remaining 92% ultimately acts as a substrate and undergoes hydrolysis to regenerate the active PLP form of the enzyme. In this manuscript, the rate-limiting step of the inactivation mechanism was determined by stopped-flow spectrophotometry and time-dependent 19F NMR experiments to be the decay of a long-lived external aldimine species. A crystal structure of this transient complex revealed both the structural basis for fractional irreversible inhibition and the principal mode of inhibition of hOAT by BCF3, which is to trap the enzyme in this transient but quasi-stable external aldimine form.

N5 Is the New C4a: Biochemical Functionalization of Reduced Flavins at the N5 Position.

For three decades the C4a-position of reduced flavins was the known site for covalency within flavoenzymes. The reactivity of this position of the reduced isoalloxazine ring with the dioxygen ground-state triplet established the C4a as a site capable of one-electron chemistry. Within the last two decades new types of reduced flavin reactivity have been documented. These studies reveal that the N5 position is also a protean site of reactivity, that is capable of nucleophilic attack to form covalent bonds with substrates. In addition, though the precise mechanism of dioxygen reactivity is yet to be definitively demonstrated, it is clear that the N5 position is directly involved in substrate oxygenation in some enzymes. In this review we document the lineage of discoveries that identified five unique modes of N5 reactivity that collectively illustrate the versatility of this position of the reduced isoalloxazine ring.