Bi-bi Reaction Research Articles

Formalizing the law of diminishing returns in metabolic networks using an electrical analogy.

The way biological systems respond to changes in parameter values caused by mutations is a key issue in evolution and quantitative genetics, as it affects fundamental aspects such as adaptation, selective neutrality, robustness, optimality, evolutionary equilibria, etc. We address this question using the enzyme-flux relationship in a metabolic network as a model of the genotype-phenotype relationship. The lack of a suitable mathematical tool from biochemical theory to investigate this relationship led us to use an analogy between electrical circuits and metabolic networks with uni-uni reactions. We show that a behaviour of diminishing returns, which is commonly observed at various phenotypic levels, is inevitable, irrespective of the complexity of the system. We also present a possible generalization to metabolic networks with both uni-uni and bi-bi reactions.

Synthesis of citronella acetate by lipase catalyzed transesterification in ionic liquid and its kinetics

In this paper, the lipase catalyzed transesterification of citronellol with vinyl acetate was investigated in ionic liquid. Firstly, six kinds of ionic liquids and six kinds of lipases from different sources were screened. The results showed that the lipase from Pseudomonas fluorescens had the best catalytic effect in the ionic liquid [bmimn][TF2]. The factors affecting the transesterification were optimized, and the optimal reaction conditions were as follows: the molar ratio of vinyl acetate to citronellol was 3:1; the reaction temperature was 40 °C; the amount of enzyme was 10 mg/mL; the rotating speed of the shaker was 200 r/min. When the concentration of substrate was below 500 mmol/L, no inhibition of substrate was found, while the inhibition of product was not negligible. The reuse of the lipase was studied, and the results showed that the catalytic activity of the lipase decreased by 20% after 7 times. The kinetic study showed that the reaction was a ping-pong bi-bi reaction mechanism with the inhibition of citronellyl acetate. The reaction kinetics model was established, and the parameters of the model were fitted by MATLAB software. The fitting values and the experimental values were in good agreement, and the relative error was only 7.75%.

Influence of Radial Dimension and Electric Field on a Microwave-Assisted Esterification

Enzymatic synthesis of ethyl hexanoate from hexanoic acid and ethanol in a solvent-free system, carried out at optimum conditions, has been modeled. The process follows a Ping-Pong Bi-Bi reaction rate and it is endothermic, so microwave heating is provided to prevent its shutdown. The mathematical model has been numerically solved with the FEM method. Results show that microwave heating can be successfully used to carry out this kind of reaction.

Phenolic and non-phenolic substrates oxidation by laccase at variable oxygen concentrations: Selection of bisubstrate kinetic models from polarographic data

Using laccase as a case study, an alternative methodology for discrimination of bisubstrate mechanism types, useful for Bi-Bi reactions when water is one of the products, is presented. A purified enzyme from fungal cultures of Ganoderma resinaceum and two reducing substrates: 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP) are used. Kinetic assays of ABTS or DMP oxidation using variable initial concentrations of dioxygen are performed in a closed system and depletion rates of dioxygen are measured using polarography. Seven bisubstrate models based on Michaelis-Menten Bi-Bi mechanisms are applied to the experimental data. Akaike information criterion, a penalized likelihood analysis, and Akaike weights are used for model selection. Results show that the more likely bisubstrate models for ABTS and DMP oxidation are ping-pong and Theorell-Chance, respectively. However, the catalytic efficiency of oxygen reduction to water exhibits similar values, and is compatible with a process relatively independent of reducing substrates and the bisubstrate mechanism type. Finally, Michaelis-Menten constants towards dioxygen, 233 μM with ABTS and 700 μM with DMP, indicate that the soluble electron acceptor available for laccase-catalyzed reactions, is far removed from saturation.

New insights and tools for the elucidation of lipase catalyzed esterification reaction mechanism in n-hexane: The synthesis of ethyl butyrate

The enzyme catalyzed esterification of butyric acid by ethanol under anhydrous conditions in n-hexane, through continuous removal of the formed water, follows a ping pong bi-bi reaction mechanism; immobilized lipase-B from Candida antarctica on acrylic resin (Novozyme 435) was used as biocatalyst. For first time, the kinetic data of an enzymatic bi-substrate reaction generating simultaneous double dead-end substrate inhibitions were processed by surface fitting through multiparametric non-linear equations. Both anhydrous CH3CH2OH and/or CH3CH2OD were employed in an attempt to apply the technique of kinetic isotope effects and validate the selection of the best system among two (ping pong bi-bi and ordered bi-bi), which could describe the aforementioned reaction.

Structure, biochemical and kinetic properties of recombinant Pst2p from Saccharomyces cerevisiae, a FMN-dependent NAD(P)H:quinone oxidoreductase

The genome of the yeast Saccharomyces cerevisiae encodes four flavodoxin-like proteins, namely Lot6p, Pst2p, Rfs1p and Ycp4p. Thus far only Lot6p was characterized in detail demonstrating that the enzyme possesses NAD(P)H:quinone oxidoreductase activity. In the present study, we heterologously expressed PST2 in Escherichia coli and purified the produced protein to conduct a detailed biochemical and structural characterization. Determination of the three-dimensional structure by X-ray crystallography revealed that Pst2p adopts the flavodoxin-like fold and forms tetramers independent of cofactor binding. The lack of electron density for FMN indicated weak binding, which was confirmed by further biochemical analysis yielding a dissociation constant of 20±1μM. The redox potential of FMN bound to Pst2p was determined to −89±3mV and is thus 119mV more positive than that of free FMN indicating that reduced FMN binds ca. five orders of magnitude tighter to Pst2p than oxidized FMN. Due to this rather positive redox potential Pst2p is unable to reduce free FMN or azo dyes as reported for other members of the flavodoxin-like protein family. On the other hand, Pst2p efficiently catalyzes the NAD(P)H dependent two-electron reduction of natural and artificial quinones. The kinetic mechanism follows a ping-pong bi-bi reaction scheme. In vivo experiments with a PST2 knock out and overexpressing strain demonstrated that Pst2p enables yeast cells to cope with quinone-induced damage suggesting a role of the enzyme in managing oxidative stress.

A novel viral thymidylate kinase with dual kinase activity.

Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

Structural comparison between the open and closed forms of citrate synthase from Thermus thermophilus HB8.

The crystal structures of citrate synthase from the thermophilic eubacteria Thermus thermophilus HB8 (TtCS) were determined for an open form at 1.5 Å resolution and for closed form at 2.3 Å resolution, respectively. In the absence of ligands TtCS in the open form was crystalized into a tetragonal form with a single subunit in the asymmetric unit. TtCS was also co-crystallized with citrate and coenzyme-A to form an orthorhombic crystal with two homodimers in the asymmetric unit. Citrate and CoA are found in the active site situated between the large domain and the small domain in all subunit whereas the complex shows two distinct closed conformations, the fully closed form and partially closed form. Structural comparisons are performed to describe conformational changes associated with binding of products of TtCS. Upon binding of citrate, basic residues in the active site move toward citrate and make a hydrogen bond network in the active site, inducing a large-scale rotation of the small domain relative to the large domain. CoA is sandwiched between the small and large domains and then the cysteamine tail is inserted into the active site with a cooperative rotation around mainchain dihedrals in the hinge region connecting helices M and N. According to this rotation these helices are extended to close the active site completely. The considerable flexibility and structural rearrangements in the hinge region are crucial for an ordered bibi reaction in catalysis for microbial CSs.

Delineating the reaction mechanism of reductase domains of Nonribosomal Peptide Synthetases from mycobacteria.

Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order 'bi-bi' reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4'-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (RNRP) and Mycobacterium smegmatis (RGPL) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi-bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3-4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation-π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi-bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology.

Effect of Sulfotransferase Structural Rearrangements in Fulvestrant Sulfation

Fulvestant (FUL) sulfation by SULTs 1A1 and 1E1 is influenced by structural rearrangements in the enzymes elicited by PAPS binding. FUL, a chemotherapeutic used to treat estrogen positive breast cancer, is sulfated by both isoforms in vitro; however, FUL sulfation in vivo may be modulated by PAPS levels. PAPS binding to the SULTs results in structural changes that significantly decrease the volume of the substrate binding pocket resulting in differential binding in the presence or absence of PAPS. FUL shows a random Bi‐Bi reaction mechanism with SULT1E1. In contrast, with SULT1A1 FUL displays an apparent ordered reaction mechanism with FUL binding first. FUL docked competently to SULT1E1 in the presence and absence of bound PAP. FUL docked to SULT1A1 only in the absence of PAPS. SULT1E1 bound FUL with a Kd of 0.2 μM in the presence or absence of PAP, whereas FUL bound SULT1A1 with a Kd of 2.4 μM in the absence of PAP and no binding was detectable in the presence of PAP. PAP was an uncompetitive inhibitor of FUL sulfation by SULT1A1 consistent with an ordered reaction mechanism. In summary, PAPS elicited structural changes in SULT1A1 and 1E1 may significantly affect the kinetics and apparent reaction mechanism of FUL sulfation suggesting that cellular PAPS levels may inhibit FUL sulfation in tissues with high SULT1A1 activity. Supported by PHS grants GM38953 to CNF and CA128897 to SAK.

Steady-state kinetics of <i>Roystonea regia</i> palm tree peroxidase

Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Superdex-200. The novel peroxidase was characterized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The enzyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investigated the steady-state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H2O2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach used expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters KmH2O2, KmAH2, kcat, KSIH2O2, KSIAH2 and of the microscopic rate constants k1 and k3 of the shared three-step peroxidase catalytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the suicide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H2O2 required to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s–1 and 18 s–1.

Molecular determinants of azo reduction activity in the strain Pseudomonas putida MET94

Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi-bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.

Targeting both “Half‐Reactions” of a Glycokinase Enzyme for Drug Discovery

With regard to drug discovery, two glycokinases are of particular interest based upon clinical data; ketohexokinase (EC 2.7.1.3, 1‐3) and glucokinase/hexokinase 4 (EC 2.7.1.2, 4‐8). Yet, modulators for either target have had limited success in pharmaceutical development. Despite, decades of basic and applied research, only a few small molecule activators for glucokinase have made it to phase 1 clinical trial. This could in fact, be due to the basic assay designs used for and the initial steps in drug discovery. Traditionally, glycokinase activities have been monitored by radioactive, enzyme‐linked, fluorescence polarization detection of ATP turnover. Described herein is a novel label‐free means of directly monitoring the bi‐bi reaction for a ketohexokinase where detection of enzyme activity utilizes mass spectroscopy. Here, turnover of ATP and fructose to ADP and fructose 1‐P, respectively, can be monitored directly and simultaneously. The selectivity of analyte detection is maximized by MRM, allowing for detection of each analyte by LCMSMS from both cells and tissues. Basic enzyme characterization is demonstrated using HTMSMS. In addition, the mechanisms of action for two unrelated compounds and the implications of target engagement of pharmaceutically active compounds from “test tube” to tissue samples are discussed.

Trans-sialidase activity of Photobacterium damsela 2,6-sialyltransferase and its application in the synthesis of sialosides

Trans-sialidases catalyze the transfer of a sialic acid from one sialoside to an acceptor to form a new sialoside. alpha2,3-Trans-sialidase activity was initially discovered in the parasitic protozoan Trypanosoma cruzi, and more recently was found in a multifunctional Pasteurella multocida sialyltransferase PmST1. alpha2,8-Trans-sialidase activity was also described for a multifunctional Campylobacter jejuni sialyltransferase CstII. We report here the discovery of the alpha2,6-trans-sialidase activity of a previously reported recombinant truncated bacterial alpha2,6-sialyltransferase from Photobacterium damsela (Delta15Pd2,6ST). This is the first time that the alpha2,6-trans-sialidase activity has ever been identified. Kinetic studies indicate that Delta15Pd2,6ST-catalyzed trans-sialidase reaction follows a ping-pong bi-bi reaction mechanism. Cytidine 5'-monophosphate, the product of sialyltransferase reactions, is not required by the trans-sialidase activity of the enzyme but enhances the trans-sialidase activity modestly as a non-essential activator. Using chemically synthesized Neu5AcalphapNP and LacbetaMU, alpha2,6-linked sialoside Neu5Acalpha2,6LacbetaMU has been obtained in one-step in high yield using the trans-sialidase activity of Delta15Pd2,6ST. In addition to the alpha2,6-trans-sialidase activity, Delta15Pd2,6ST also has alpha2,6-sialidase activity. The multifunctionality is thus a common feature of many bacterial sialyltransferases.

Structural rearrangement of SULT2A1: effects on dehydroepiandrosterone and raloxifene sulfation

BACKGROUND: Human cytosoloic sulfotransferase (SULT) 2A1 is a major hepatic isoform and sulfates hydroxyl groups in structurally diverse sterols and xenobiotics. SULT2A1 crystal structures resolved in the presence and absence of 3',5'-diphosphoadenosine (PAP) or dehydropeiandrosterone (DHEA) suggest a significant rearrangement of the peptide that forms the surface of the active site in the presence of PAP. MATERIALS AND METHODS: Molecular modeling was used to examine the effects of the rearrangement in SULT2A1 associated with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding on the binding of DHEA and raloxifene. The kinetics of DHEA and raloxifene sulfation was analyzed to investigate the effects of the rearrangement on SULT2A1 activity. RESULTS: Molecular models indicate that DHEA is able to bind to SULT2A1 in both conformations (open, without PAP; closed, with PAP) in a catalytic configuration, whereas raloxifene bound in a catalytic conformation only in the open structure. Raloxifene did not bind in the smaller, closed substrate binding pocket. Kinetic analysis of DHEA sulfation was consistent with a random Bi-Bi reaction mechanism, whereas raloxifene sulfation was more indicative of an ordered reaction mechanism with raloxifene binding first. Initial burst kinetics with DHEA yielded similar results after preincubation of SULT2A1 with DHEA or PAPS. Preincubation of SULT2A1 with raloxifene showed a burst of raloxifene sulfate formation with the addition of PAPS. In contrast, little raloxifene sulfate was formed if SULT2A1 was preincubated with PAPS and the reaction initiated with raloxifene. CONCLUSIONS: The structural rearrangements in SULT2A1 caused by PAPS binding can alter the sulfation mechanism and kinetics of different substrates.

Acetate:succinate CoA-transferase in the anaerobic mitochondria of Fasciola hepatica

Fasciola hepatica contains anaerobically functioning mitochondria that produce acetate and propionate, the main endproducts excreted by this parasite. The final reactions in the pathways leading to these endproducts are performed by acetate:succinate CoA-transferase (ASCT) and propionate:succinate CoA-transferase (PSCT), respectively. The enzymes catalysing these essential reactions in anaerobic mitochondria are still not characterized, nor are the corresponding genes identified. Here we describe the identification of the gene that codes for the F. hepatica ASCT. The F. hepatica gene was heterologously expressed and studies on the corresponding enzyme activity showed that the enzyme is indeed a transferase and uses a ping-pong bi-bi reaction mechanism, like most other known CoA-transferases. This F. hepatica CoA-transferase was shown to be a true transferase and not a hydrolase, as it needs an acceptor for optimal activity. Our studies demonstrated that the F. hepatica ASCT can use other CoA-acceptors than succinate, such as propionate, acetate and butyrate, and is in fact a short-chain acyl-CoA-transferase. We further showed that this F. hepatica CoA-transferase can also catalyze the PSCT reaction, which is responsible for the production of propionate. Analysis of the amino acid sequence of F. hepatica clearly indicated the presence of a mitochondrial targeting sequence, and in CHO cells the enzyme is indeed present in the mitochondrial fraction. F. hepatica ASCT is the first ASCT identified in anaerobic mitochondria. It is homologous to the hydrogenosomal ASCT we earlier identified in Trichomonas vaginalis, but not to the ASCT present in the aerobic mitochondria of Trypanosoma brucei.

A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.

Divergence of Cofactor Recognition across Evolution: Coenzyme A Binding in a Prokaryotic Arylamine N-Acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.

Open and Closed Structures of the UDP-glucose Pyrophosphorylase from Leishmania major

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel beta-helix (LbetaH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.

Lot6p from Saccharomyces cerevisiae is a FMN‐dependent reductase with a potential role in quinone detoxification

NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse and rat, and from plant species. Here, we report identification of Lot6p (YLR011wp), the first soluble quinone reductase from the unicellular model organism Saccharomyces cerevisiae. Localization studies using an antibody raised against Lot6p as well as microscopic inspection of Lot6p-GFP demonstrated accumulation of the enzyme in the cytosol of yeast cells. Despite sharing only 23% similarity to type 1 human quinone reductase, Lot6p possesses biochemical properties that are similar to its human counterpart. The enzyme catalyzes a two-electron reduction of a series of natural and artificial quinone substrates at the expense of either NADH or NADPH. The kinetic mechanism follows a ping-pong bi-bi reaction scheme, with K(M) values of 1.6-11 microm for various quinones. Dicoumarol and Cibacron Marine, two well-known inhibitors of the quinone reductase family, bind to Lot6p and inhibit its activity. In vivo experiments demonstrate that the enzymatic activity of Lot6p is consistent with the phenotype of both Deltalot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.