Mechanism of D-Cycloserine Inhibition of D-Amino Acid Transaminase from Haliscomenobacter hydrossis.

D-cycloserine inhibits pyridoxal-5'-phosphate (PLP)-dependent enzymes. Inhibition effect depend on organization of the active site and mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of the enzyme similarly to the substrate (amino acid), and this interaction is predominantly reversible. Several products of the interaction of PLP with D-cycloserine are known. For some enzymes formation of a stable aromatic product - hydroxyisoxazole-pyridoxamine-5'-phosphate at certain pH - leads to irreversible inhibition. The aim of this work was to study the mechanism of D-cycloserine inhibition of the PLP-dependent D-amino acid transaminase from Haliscomenobacter hydrossis. Spectral methods revealed several products of interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and ß-aminooxy-D-alanine, ketimine between pyridoxamine-5'-phosphate and cyclic form of D-cycloserine, and pyridoxamine-5'-phosphate. Formation of hydroxyisoxazole-pyridoxamine-5'-phosphate was not observed. 3D structure of the complex with D-cycloserine was obtained using X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5'-phosphate and D-cycloserine in the cyclic form was found. Ketimine occupied two positions interacting with different active site residues via hydrogen bonds. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and activity of the inhibited transaminase from H. hydrossis could be restored by adding excess of keto substrate or excess of cofactor. The obtained results confirm reversibility of the inhibition by D-cycloserine and interconversion of various adducts of D-cycloserine and PLP.

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Revealing a key inhibitory mechanism of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline via trapping of methylglyoxal.

The inhibitory effects of vitamins (nicotinic acid, pyridoxamine [PM], and l-ascorbic acid) and phenolic acids (ferulic acid and p-coumaric acid) on the formation of 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) were studied in a glycine/glucose/creatinine model system and fried tilapia cakes. The results showed that PM was the most potential inhibitor and the inhibition rates reached 82.72% and 78.54% in model system and fried tilapia cakes, respectively. Detailed formation mechanism of MeIQx was put forward to find the inevitable species in the non-free radical formation mechanism of MeIQx. Dose-dependent analysis of PM on methylglyoxal (MGO ) and MeIQx formation were studied by using model systems and the results showed that MGO and MeIQx were both reduced about 60% in reaction mixtures when the molar ratio of PM to glycine was 1:16, which indicated that MGO is a key intermediate on the pathway of MeIQx formation. Quantum chemistry calculations showed that PM can act as a useful inhibitor to inhibit the formation of MeIQx and react with MGO to form new compounds. A pathway for the inhibitory activity of PM against MeIQx formation was proposed. PRACTICAL APPLICATION: Pyridoxamine was the most effective inhibitor against heterocyclic aromatic amines (HAAs) and could be applied to a variety of food systems. While the inhibitory mechanism is still unclear. Detailed formation mechanism of MeIQx was put forward first and suggested methylglyoxal as an inevitable species in the non-free radical formation mechanism of MeIQx in this study. Pyridoxamine trapping methylglyoxal is likely a key mechanism against the generation of MeIQx was demonstrated by quantum chemistry calculation and experimental demonstration. These findings may provide effective suggestions for reducing HAAs and similar toxicants in daily cuisine.

Structural attributes and substrate specificity of pyridoxal kinase from Leishmania donovani.

The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5'-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5'-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of ß-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors.

PMP-diketopiperazine adducts form at the active site of a PLP dependent enzyme involved in formycin biosynthesis.

ForI is a PLP-dependent enzyme from the biosynthetic pathway of the C-nucleoside antibiotic formycin. Cycloserine is thought to inhibit PLP-dependent enzymes by irreversibly forming a PMP-isoxazole. We now report that ForI forms novel PMP-diketopiperazine derivatives following incubation with both d and l cycloserine. This unexpected result suggests chemical diversity in the chemistry of cycloserine inhibition.

Determination and evaluation of in vitro bioaccessibility of the pyridoxal, pyridoxine, and pyridoxamine forms of vitamin B6 in cereal-based baby foods.

The bioavailability of the pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM) forms of vitamin B6 is different, considering that their bioaccessibility in baby foods is important for infant and young children's nutrition. The aim of this study was to determine and evaluate the bioaccessibility of the PL, PN, and PM forms of vitamin B6 in cereal-based baby foods an in vitro digestive system. In this study, the PL, PN, and PM forms of vitamin B6 were determined using HPLC in 13 cereal-based baby foods. The average bioaccessibility of the PL, PN, and PM forms in gastric pH 1.5 were 53%, 76%, and 50%, respectively. When the gastric pH was 4, the average bioaccessibility of PL, PN, and PM were 38%, 67%, and 36%, respectively. As observed in this study, the bioaccessibility of the PL, PN, and PM forms of vitamin B6 in baby foods is lower in both gastric pHs.

Synthesis of [13C3]-B6 Vitamers Labelled at Three Consecutive Positions Starting from [13C3]-Propionic Acid.

[13C3]-labelled vitamers (PN, PL and PM) of the B6 group were prepared starting from [13C3]-propionic acid. [13C3]-PN was synthesized in ten linear steps with an overall yield of 17%. Hereby, higher alkyl homologues of involved esters showed a positive impact on the reaction outcome of the intermediates in the chosen synthetic route. Oxidation of [13C3]-PN to [13C3]-PL was undertaken using potassium permanganate and methylamine followed by acid hydrolysis of the imine derivative. [13C3]-PM could be prepared from the oxime derivative of [13C3]-PN by hydrogenation with palladium.

Pyridoxamine driven selective turn-off detection of picric acid using glutathione stabilized fluorescent copper nanoclusters and its applications with chemically modified cellulose strips.

The present work reports the interaction of various vitamin B6 cofactors with the red emitting glutathione stabilized copper nanoclusters (GSH-CuNCs). Addition of pyridoxamine (PM) resulted a new turn-on band at 410nm due to the possible adsorption over the surface of GSH-CuNCs. The nano-assembly PM-GSH-CuNCs was applied for the selective detection of nitro-aromatic compounds. Upon addition of picric acid (PA), the fluorescence of PM-GSH-CuNCs was selectively quenched at 410nm and ~ 625nm among the other tested nitro-aromatic compounds. With a linearity range from 9.9µM to 43µM, the concentration of PA can be detected down to 2.74µM. The high selectivity exhibited by the nano-assembly allows to detect PA in real samples like tap water, river water and matchstick. Advantageously, the nano-assembly PM-GSH-CuNCs was chemically adsorbed over the cellulosic strips and applied for the naked-eye detection of PA down to 1µM.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

Rapid determination of lipid peroxidation using a novel pyridoxamine-participating ferrous oxidation-sulfosalicylic acid spectrophotometric method.

A novel method is developed to rapidly analyze lipid peroxidation in edible oils and fatty foods at room temperature, which is called the pyridoxamine-participating ferrous oxidation-sulfosalicylic acid (PFOS) method. The PFOS method evaluates the lipid peroxide value colorimetrically via detecting the pyridoxamine-mediated pigment produced by 5-sulfosalicylic acid and Fe(3+) at 500nm, while the latter is converted from Fe(2+) in the presence of lipid peroxides. The optimized formulation was ethanol (70%, v/v), Fe(2+) (4mmol/L), 5-sulfosalicylic acid (40mmol/L) and pyridoxamine (18mmol/L). The limit of quantitation is 0.087mmol Fe(3+)/L with acceptable reproducibility. In addition, current method has a significant linear correlation with both conventional thiobarbituric acid (R(2)=0.9999) and ferric thiocyanate assays (R(2)=0.9675). This method offers a rapid technique for evaluating lipid peroxidation without heating and sophisticated instrumental procedures. Besides, current method provides a new option to evaluate the lipid peroxidation state and improve the reproducibility of ferrous-oxidation.

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.

Reactivity, Selectivity, and Reaction Mechanisms of Aminoguanidine, Hydralazine, Pyridoxamine, and Carnosine as Sequestering Agents of Reactive Carbonyl Species: A Comparative Study.

Reactive carbonyl species (RCS) are endogenous or exogenous byproducts involved in the pathogenic mechanisms of different oxidative-based disorders. Detoxification of RCS by carbonyl quenchers is a promising therapeutic strategy. Among the most studied quenchers are aminoguanidine, hydralazine, pyridoxamine, and carnosine; their quenching activity towards four RCS (4-hydroxy-trans-2-nonenal, methylglyoxal, glyoxal, and malondialdehyde) was herein analyzed and compared. Their ability to prevent protein carbonylation was evaluated in vitro by using an innovative method based on high-resolution mass spectrometry (HRMS). The reactivity of the compounds was RCS dependent: carnosine efficiently quenched 4-hydroxy-trans-2-nonenal, pyridoxamine was particularly active towards malondialdehyde, aminoguanidine was active towards methylglyoxal and glyoxal, and hydralazine efficiently quenched all RCS. Reaction products were generated in vitro and were characterized by HRMS. Molecular modeling studies revealed that the reactivity was controlled by specific stereoelectronic parameters that could be used for the rational design of improved carbonyl quenchers.

Balancing of B6 Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis.

Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5'-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5'-phosphate (PMP), pyridoxine 5'-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism.

PdxH proteins of mycobacteria are typical members of the classical pyridoxine/pyridoxamine 5'-phosphate oxidase family.

Pyridoxal 5'-phosphate (PLP) biosynthesis is essential for the survival and virulence of Mycobacterium tuberculosis (Mtb). PLP functions as a cofactor for 58 putative PLP-binding proteins encoded by the Mtb genome and could also act as a potential antioxidant. De novo biosynthesis of PLP in Mtb takes place through the 'deoxyxylulose 5'-phosphate (DXP)-independent' pathway, whereas PdxH enzymes, possessing pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPOx) activity, are involved in the PLP salvage pathway. In this study, we demonstrate that the annotated PdxH enzymes from various mycobacterial species are bona fide members of the classical PNPOx enzyme family, capable of producing PLP using both pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) substrates.

Effect and mechanism of pyridoxamine on the lipid peroxidation and stability of polyunsaturated fatty acids in beef patties.

BACKGROUND: Little is known about how vitamins can affect the peroxidation and stability of polyunsaturated fatty acids in cooked foods. Thus the effects of 15 vitamins on toxic malondialdehyde (MDA) formation in cooked beef patties were examined with the application of solid phase extraction and thiobarbituric acid (TBA) analysis by HPLC-DAD. The polyunsaturated fatty acid profiles in cooked beef patties treated with some vitamins were further compared with that of control sample (no vitamin addition) by GC-MS analysis. RESULTS: Pyridoxamine, pyridoxine, retinoic acid, α-tocopherol and L-ascorbic acid exhibited significant effects lowering the amount of MDA. It was further discovered that retinoic acid, α-tocopherol and l-ascorbic acid could help preserve polyunsaturated fatty acids, while pyridoxamine addition actually showed no effect upon the retention of most of the tested polyunsaturated fatty acids, even lowering the content of arachidonic acid. Further LC-MS analysis demonstrated that pyridoxamine could directly react with MDA via an addition reaction. The reaction involves a nucleophilic attack of pyridoxamine's free amine group on one of the aldehyde functional groups of MDA to form a new adduct, and may accelerate lipid peroxidation with the loss of more polyunsaturated fatty acids. CONCLUSION: Some vitamins may directly participate in lipid peroxidation and affect food quality. © 2015 Society of Chemical Industry.

Identification of novel thermostable taurine-pyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis.

ω-Transaminases (ω-TAs) are one of the most popular candidate enzymes in the biosynthesis of chiral amines. Determination of yet unidentified ω-TAs is important to broaden their potential for synthetic application. Taurine-pyruvate TA (TPTA, EC 2.6.1.77) is an ω-TA belonging to class III of TAs. In this study, we cloned a novel thermostable TPTA from Geobacillus thermodenitrificans (TPTAgth) and overexpressed it in Escherichia coli. The enzyme showed the highest activity at pH 9.0 and 65 °C, with remarkable thermostability and tolerance toward organic solvents. Its K M and v max values for taurine were 5.3 mM and 0.28 µmol s(-1) mg(-1), respectively. Determination of substrate tolerance indicated its broad donor and acceptor ranges for unnatural substrates. Notably, the enzyme showed relatively good activity toward ketoses, suggesting its potential for catalyzing the asymmetric synthesis of chiral amino alcohols. The active site of TPTAgth was identified by performing protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking. The protein sequence and structure of TPTAgth were similar to those of TAs belonging to the 3N5M subfamily. Its active site was found to be its special large pocket and substrate tunnel. In addition, TPTAgth showed a unique mechanism of sulfonate/α-carboxylate recognition contributed by Arg163 and Gln160. We also determined the protein sequence fingerprint of TPTAs in the 3N5M subfamily, which involved Arg163 and Gln160 and seven additional residues from 413 to 419 and lacked Phe/Tyr22, Phe85, and Arg409.

Scavenging 4-Oxo-2-nonenal.

4-Oxo-2-nonenal (ONE), a product of cellular lipid oxidation, reacts nonspecifically with the lysine residues of proteins and is generated in increased amounts during degenerative diseases and cancer. We show that pyridoxamine, salicylamine, and related 2-aminomethylphenols react with ONE, to form pyrrolo[2,1-b][1,3]oxazines with the participation of both the amino and the phenolic groups. 2-Aminomethylphenols react with ONE as well as with the Michael adducts of ONE much more rapidly than lysine, suggesting their use for therapeutically scavenging ONE.

5'-O-Alkylpyridoxamines: Lipophilic Analogues of Pyridoxamine Are Potent Scavengers of 1,2-Dicarbonyls.

Pyridoxamine (PM) is a prospective drug for the treatment of diabetic complications. In order to make zwitterionic PM more lipophilic and improve its tissue distribution, PM derivatives containing medium length alkyl groups on the hydroxymethyl side chain were prepared. The synthesis of these alkylpyridoxamines (alkyl-PMs) starting from pyridoxine offers high yields and is amenable to bulk preparations. Interestingly, alkyl-PMs were found to react with methylglyoxal (MGO), a major toxic product of glucose metabolism and autoxidation, several orders of magnitude faster than PM. This suggests the formation of nonionic pyrido-1,3-oxazine as the key step in the reaction of PM with MGO. Since the primary target of MGO in proteins is the guanidine side chain of arginine, alkyl-PMs were shown to be more effective than PM in reducing the modification of N-α-benzoylarginine by MGO. Alkyl-PMs in the presence of MGO also protected the enzymatic activity of lysozyme that contains several arginine residues next to its active site. Alkyl-PMs can be expected to trap MGO and other toxic 1,2-carbonyl compounds more effectively than PM, especially in lipophilic tissue environments, thus protecting macromolecules from functional damage. This suggests potential therapeutic uses for alkyl-PMs in diabetes and other diseases characterized by the elevated levels of toxic dicarbonyl compounds.

Theoretical study on carbon-carbon short contact of ∼2.3 Å: intermediate state between nonbonding and σ-covalent bonding.

An unusual intermolecular carbon-carbon short contact, observed previously in the crystal structure of the copper complex of pyridoxal-5-phosphate- pyridoxamine-5-phospate Schiff base, was investigated from a standpoint of quantum chemistry by DFT calculations with plane wave basis sets. The DFT-optimized structure qualitatively reproduced the short contact (2.6-2.8 Å) of the intermolecular carbon-carbon pairs for the dimer of the copper complexes in the unit cell, compared to that (∼2.3 Å) of the X-ray diffraction data. By the occupied and unoccupied orbitals, the dimer showed the in-phase and out-of-phase interactions along the direction of the intermolecular distance. The dimer of the copper complexes was confirmed as the stable intermediate between nonbonding and σ-covalent bonding by the electronic energy curve along the distance of the monomers.