Acidity of persulfides and its modulation by the protein environments in sulfide quinone oxidoreductase and thiosulfate sulfurtransferase.

Persulfides (RSSH/RSS-) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6-6.3) and pH-independent rate constants (3.2-9.0 × 103 M-1 s-1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes was measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated sulfide quinone oxidoreductase appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The TST thiol reacted in the anionic state with thiosulfate, with an apparent pKa of 6.5 ± 0.1. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments.

Disease-causing cystathionine ß-synthase linker mutations impair allosteric regulation.

Cystathionine ß-synthase (CBS) catalyzes the committing step in the transsulfuration pathway, which is important for clearing homocysteine and furnishing cysteine. The transsulfuration pathway also generates H2S, a signaling molecule. CBS is a modular protein with a heme and pyridoxal phosphate-binding catalytic core, which is separated by a linker region from the C-terminal regulatory domain that binds S-adenosylmethionine (AdoMet), an allosteric activator. Recent cryo-EM structures reveal that CBS exists in a fibrillar form and undergoes a dramatic architectural rearrangement between the basal and AdoMet-bound states. CBS is the single most common locus of mutations associated with homocystinuria, and, in this study, we have characterized three clinical variants (K384E/N and M391I), which reside in the linker region. The native fibrillar form is destabilized in the variants, and differences in their limited proteolytic fingerprints also reveal conformational alterations. The crystal structure of the truncated K384N variant, lacking the regulatory domain, reveals that the overall fold of the catalytic core is unperturbed. M391I CBS exhibits a modest (1.4-fold) decrease while the K384E/N variants exhibit a significant (∼8-fold) decrease in basal activity, which is either unresponsive to or inhibited by AdoMet. Pre-steady state kinetic analyses reveal that the K384E/N substitutions exhibit pleiotropic effects and that the differences between them are expressed in the second half reaction, that is, homocysteine binding and reaction with the aminoacrylate intermediate. Together, these studies point to an important role for the linker in stabilizing the higher-order oligomeric structure of CBS and enabling AdoMet-dependent regulation.

Structural perspectives on H2S homeostasis.

The enzymes involved in H2S homeostasis regulate its production from sulfur-containing amino acids and its oxidation to thiosulfate and sulfate. Two gatekeepers in this homeostatic circuit are cystathionine beta-synthase, which commits homocysteine to cysteine, and sulfide quinone oxidoreductase, which commits H2S to oxidation via a mitochondrial pathway. Inborn errors at either locus affect sulfur metabolism, increasing homocysteine-derived H2S synthesis in the case of CBS deficiency and reducing complex IV activity in the case of SQOR deficiency. In this review, we focus on structural perspectives on the reaction mechanisms and regulation of these two enzymes, which are key to understanding H2S homeostasis in health and its dysregulation and potential targeting in disease.

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.

Transient-State Analysis of Human Isocitrate Dehydrogenase I: Accounting for the Interconversion of Active and Non-Active Conformational States.

Human isocitrate dehydrogenase 1 (HsICDH1) is a cytoplasmic homodimeric Mg(II)-dependent enzyme that converts d-isocitrate (D-ICT) and NADP+ to α-ketoglutarate (AKG), CO2, and NADPH. The active sites are formed at the subunit interface and incorporate residues from both protomers. The turnover number titrates hyperbolically from 17.5 s-1 to a minimum of 7 s-1 with an increasing enzyme concentration. As isolated, the enzyme adopts an inactive open conformation and binds NADPH tightly. The open conformation displaces three of the eight residues that bind D-ICT and Mg(II). Enzyme activation occurs with the addition of Mg(II) or D-ICT with a rate constant of 0.12 s-1. The addition of both Mg(II) and D-ICT activates the enzyme with a rate constant of 0.6 s-1 and displaces half of the bound NADPH. This indicates that HsICDH1 may have a half-site mechanism in which the active sites alternate in catalysis. The X-ray crystal structure of the half-site activated complex reveals asymmetry in the homodimer with a single NADPH bound. The structure also indicates a pseudotetramer interface that impedes the egress of NADPH consistent with the suppression of the turnover number at high enzyme concentrations. When the half-site activated form of the enzyme is reacted with NADP+, NADPH forms with a rate constant of 204 s-1 followed by a shift in the NADPH absorption spectrum with a rate constant of 28 s-1. These data indicate the accumulation of two intermediate states. Once D-ICT is exhausted, HsICDH1 relaxes to the inactive open state with a rate constant of ∼3 s-1.

Structurally diverse dehydroshikimate dehydratase variants participate in microbial quinate catabolism.

Quinate and shikimate can be degraded by a number of microbes. Dehydroshikimate dehydratases (DSDs) play a central role in this process, catalyzing the conversion of 3-dehydroshikimate to protocatechuate, a common intermediate of aromatic degradation pathways. DSDs have applications in metabolic engineering for the production of valuable protocatechuate-derived molecules. Although a number of Gram-negative bacteria are known to catabolize quinate and shikimate, only limited information exists on the quinate/shikimate catabolic enzymes found in these organisms. Here, we have functionally and structurally characterized a putative DSD designated QuiC1, which is present in some pseudomonads. The QuiC1 protein is not related by sequence with previously identified DSDs from the Gram-negative genus, Acinetobacter, but instead shows limited sequence identity in its N-terminal half with fungal DSDs. Analysis of a Pseudomonas aeruginosa quiC1 gene knock-out demonstrates that it is important for growth on either quinate or shikimate. The structure of a QuiC1 enzyme from P. putida reveals that the protein is a fusion of two distinct modules: an N-terminal sugar phosphate isomerase-like domain associated with DSD activity and a novel C-terminal hydroxyphenylpyruvate dioxygenase-like domain. The results of this study highlight the considerable diversity of enzymes that participate in quinate/shikimate catabolism in different microbes.

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.