Pyridoxamine treatment ameliorates large artery stiffening and cerebral artery endothelial dysfunction in old mice.

Age-related increases in large artery stiffness are associated with cerebrovascular dysfunction and cognitive impairment. Pyridoxamine treatment prevents large artery stiffening with advancing age, but the effects of pyridoxamine treatment on the cerebral vasculature or cognition is unknown. The purpose of this study was to investigate the effects of pyridoxamine on blood pressure, large artery stiffness, cerebral artery function, and cognitive function in old mice. Old male C57BL/6 mice consumed either pyridoxamine (2 g/L) or vehicle control in drinking water for ∼7.5 months and were compared with young male C57BL/6 mice. From pre- to post-treatment, systolic blood pressure increased in old control mice, but was maintained in pyridoxamine treated mice. Large artery stiffness decreased in pyridoxamine-treated mice but was unaffected in control mice. Pyridoxamine-treated mice had greater cerebral artery endothelium-dependent dilation compared with old control mice, and not different from young mice. Old control mice had impaired cognitive function; however, pyridoxamine only partially preserved cognitive function in old mice. In summary, pyridoxamine treatment in old mice prevented age-related increases in blood pressure, reduced large artery stiffness, preserved cerebral artery endothelial function, and partially preserved cognitive function. Taken together, these results suggest that pyridoxamine treatment may limit vascular aging.

A promising antifibrotic drug, pyridoxamine attenuates thioacetamide-induced liver fibrosis by combating oxidative stress, advanced glycation end products, and balancing matrix metalloproteinases.

Liver fibrosis is a common chronic hepatic disease. This study was done to examine the effect of pyridoxamine against thioacetamide-induced hepatic fibrosis. Animals were divided into four groups (1) control group; (2) Thioacetamide group (200 mg/kg, i.p.) twice a week for eight weeks; (3) Pyridoxamine-treated group treated with pyridoxamine (100 mg/kg/day, i.p.) for eight weeks; (4) Thioacetamide and pyridoxamine group, in which pyridoxamine was given (100 mg/kg/day, i.p.) during thioacetamide injections. Thioacetamide treatment resulted in hepatic dysfunction manifested by increased serum levels of bilirubin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Oxidative stress was noted by increased hepatic lipid peroxidation and decreased glutathione (GSH). Increased concentrations of total nitrite/nitrate, advanced glycation end products (AGEs), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), matrix metalloproteinases (MMP-2&9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were noticed in hepatic tissues. Immunostaining sections also revealed overexpression of MMP-2, MMP-9 and collagen IV. Liver fibrosis was confirmed by severe histopathological changes. Pyridoxamine improved the assessed parameters. Moreover, histopathological and immunohistological studies supported the ability of pyridoxamine to reduce liver fibrosis. The findings of the present study provide evidence that pyridoxamine is a novel target for the treatment of liver fibrosis.

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.

Characterization and functional insights into the Entamoeba histolytica pyridoxal kinase, an enzyme essential for its survival.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for more than 140 enzymes. This coenzyme plays a pivotal role in catalysis of various enzymatic reactions that are critical for the survival of organisms. Entamoeba histolytica depends on the uptake of pyridoxal (PL), a B6 vitamer from the external environment which is then phosphorylated by pyridoxal kinase (EhPLK) to form PLP via the salvage pathway. E. histolytica cannot synthesise vitamin B6de-novo, and also lacks pyridoxine 5'-phosphate oxidase, a salvage pathway enzyme required to produce PLP from pyridoxine phosphate (PNP) and pyridoxamine phosphate (PMP). Analysing the importance of PLK in E. histolytica, we have determined the high-resolution crystal structures of the dimeric pyridoxal kinase in apo, ADP-bound, and PLP-bound states. These structures provided a snapshot of the transition state and help in understanding the reaction mechanism in greater detail. The EhPLK structure significantly differed from the human homologue at its PLP binding site, and the phylogenetic study also revealed its divergence from human PLK. Further, gene regulation of EhPLK using sense and antisense RNA showed that any change in optimal level is harmful to the pathogen. Biochemical and in vivo studies unveiled EhPLK to be essential for this pathogen, while the molecular differences with human PLK structure can be exploited for the structure-guided design of EhPLK inhibitors.

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.

Pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency in zebrafish results in fatal seizures and metabolic aberrations.

Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo-/-) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo-/- zebrafish develop seizures resulting in only 38% of pnpo-/- zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo-/- zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.

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.

Bioconversion of pyridoxine to pyridoxamine through pyridoxal using a Rhodococcus expression system.

Pyridoxamine, which is a form of vitamin B6, is a promising candidate for a prophylactic and/or remedy for diabetic complications. Pyridoxamine is chemically synthesized by an oxidative method in manufacturing. However, pyridoxamine production by bioconversion, which is generally preferable for environmental and energetic aspects, has been little investigated. Therefore, I aimed to produce pyridoxamine from pyridoxine, which is a readily and economically available starting material, by bioconversion using a Rhodococcus expression system. I found in the bioconversion of pyridoxine to pyridoxal, approximately 450 mM pyridoxal was produced from 500 mM pyridoxine using recombinant Rhodococcus erythropolis expressing the pyridoxine 4-oxidase gene derived from Mesorhizobium loti. Next, in the bioconversion of pyridoxal to pyridoxamine using recombinant R. erythropolis expressing the pyridoxamine-pyruvate aminotransferase gene derived from M. loti, the bioconversion rate was approximately 80% under the same conditions as pyridoxal production. Finally, in the bioconversion of pyridoxine to pyridoxamine through pyridoxal using recombinant R. erythropolis coexpressing the genes for pyridoxine 4-oxidase and pyridoxamine-pyruvate aminotransferase, the bioconversion rate was approximately 75%. Based on these findings, pyridoxamine production by bioconversion using a Rhodococcus expression system may be of interest for future industrial applications.

Involvement of Pyridoxine/Pyridoxamine 5'-Phosphate Oxidase (PDX3) in Ethylene-Induced Auxin Biosynthesis in the Arabidopsis Root.

As sessile organisms, plants have evolved to adjust their growth and development to environmental changes. It has been well documented that the crosstalk between different plant hormones plays important roles in the coordination of growth and development of the plant. Here, we describe a novel recessive mutant, mildly insensitive to ethylene (mine), which displayed insensitivity to the ethylene precursor, ACC (1-aminocyclopropane-1-carboxylic acid), in the root under the dark-grown conditions. By contrast, mine roots exhibited a normal growth response to exogenous IAA (indole-3-acetic acid). Thus, it appears that the growth responses of mine to ACC and IAA resemble those of weak ethylene insensitive (wei) mutants. To understand the molecular events underlying the crosstalk between ethylene and auxin in the root, we identified the MINE locus and found that the MINE gene encodes the pyridoxine 5'-phosphate (PNP)/pyridoxamine 5'-phosphate (PMP) oxidase, PDX3. Our results revealed that MINE/PDX3 likely plays a role in the conversion of the auxin precursor tryptophan to indole-3-pyruvic acid in the auxin biosynthesis pathway, in which TAA1 (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1) and its related genes (TRYPTOPHAN AMINOTRANSFERASE RELATED 1 and 2; TAR1 and TAR2) are involved. Considering that TAA1 and TARs belong to a subgroup of PLP (pyridoxal-5'-phosphate)-dependent enzymes, we propose that PLP produced by MINE/PDX3 acts as a cofactor in TAA1/TAR-dependent auxin biosynthesis induced by ethylene, which in turn influences the crosstalk between ethylene and auxin in the Arabidopsis root.

Study of pyridoxamine against glycation and reactive oxygen species production in human serum albumin as model protein: An in vitro & ex vivo approach.

Hyperglycaemia is considered to be a driving factor for advanced glycated end products (AGEs). Inhibiting the process of glycation play an important role in reducing the diabetes related complications. We have explored the glucose mediated glycation and antiglycation activity of pyridoxamine using human serum albumin (HSA). Protein was incubated with glucose for 28 days at physiological temperature to achieve glycation. Antiglycation activity was assessed by the estimation of carbonyl content, free lysine and AGE specific fluorescence. Molecular docking was used to study the interaction of pyridoxamine with HSA and to get a detailed understanding of binding sites and binding energy. Glycation was reduced by pyridoxamine to commendable levels which was evident by the quantification of free lysine and carbonyl content. Pyridoxamine treatment also prevented the loss in secondary structure induced by glycation. It has also emerged as the quencher of reactive oxygen species which lead to the protection of DNA from oxidative damage. Pyridoxamine was found to be located at subdomain IIA of HSA with binding energy of -5.6 kcal/mol. These results are high points in the antiglycation activity of pyridoxamine. Its antioxidant nature and antiglycation activity are proof of its potential in preventing disease progression in diabetes.

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.

Metabolic profile alterations in the postmortem brains of patients with schizophrenia using capillary electrophoresis-mass spectrometry.

OBJECTIVE: We aimed to find the alterations in the profiles of low-molecular-weight metabolites in the brains of schizophrenia patients that may reflect the pathophysiology of the disorder. METHOD: Human postmortem brain tissues from the frontal cortex (15 schizophrenia patients and 15 controls) and the hippocampus (14 schizophrenia patients and 15 controls) were obtained from the Stanley Foundation Neuropathology Consortium. We analyzed ~300 metabolites, using capillary electrophoresis with time-of-flight mass spectrometry. RESULTS: In the frontal cortex, the mean levels of 29 metabolites were significantly different between the schizophrenia and control groups. In the hippocampus, only a dipeptide, glycylglycine was significantly (p≤0.001, nominal p-value) increased in schizophrenia. Glycylglycine was also significantly (p=0.007) increased in the frontal cortex of schizophrenia. The pathway analyses revealed that several metabolic pathways including KEGG "Central carbon metabolism in cancer" and "Protein digestion and absorption" were commonly affected in the frontal cortex and the hippocampus of schizophrenia patients. CONCLUSION: These findings point out alterations in glucose metabolism and proteolysis in the brains of schizophrenia.

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.

Molecular dynamics simulations of apo, holo, and inactivator bound GABA-at reveal the role of active site residues in PLP dependent enzymes.

The pyridoxal 5-phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ-aminobutyric acid aminotransferase (GABA-AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA-AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA-AT. This is a consequence of a pKa shift triggered by a strong charge-charge interaction with an ionic "diad" formed by Asp298 and His190 that would help the activation of the first half-reaction of the catalytic mechanism in GABA-AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π-π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half-reaction in GABA-AT: the regeneration of PLP-bound GABA-AT (i.e., the holoenzyme). Proteins 2016; 84:875-891. © 2016 Wiley Periodicals, Inc.

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.

Tobacco NUP1 transports both tobacco alkaloids and vitamin B6.

The purine permeases (PUPs) constitute a large plasma membrane-localized transporter family in plants that mediates the proton-coupled uptake of nucleotide bases and their derivatives, such as adenine, cytokinins, and caffeine. A Nicotiana tabacum (tobacco) PUP-family transporter, nicotine uptake permease 1 (NtNUP1), was previously shown to transport tobacco alkaloids and to affect both nicotine biosynthesis and root growth in tobacco plants. Since Arabidopsis PUP1, which belongs to the same subclade as NtNUP1, was recently reported to transport pyridoxine and its derivatives (vitamin B6), it was of interest to examine whether NtNUP1 could also transport these substrates. Direct uptake measurements in the yeast Saccharomyces cerevisiae demonstrated that NtNUP1 efficiently promoted the uptake of pyridoxamine, pyridoxine, anatabine, and nicotine. The naturally occurring (S)-isomer of nicotine was preferentially transported over the (R)-isomer. Transport studies using tobacco BY-2 cell lines overexpressing NtNUP1 or PUP1 showed that NtNUP1, similar to PUP1, transported various compounds containing a pyridine ring, but that the two transporters had distinct substrate preferences. Therefore, the previously reported effects of NtNUP1 on tobacco physiology might involve bioactive metabolites other than tobacco alkaloids.

Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.

Structural and mechanistic insights into the kynurenine aminotransferase-mediated excretion of kynurenic acid.

Kynurenine aminotransferase (KAT) is a homodimeric pyridoxal protein that mediates the catalytic conversion of kynurenine (KYN) to kynurenic acid (KYA), an endogenous N-methyl-d-aspartate (NMDA) receptor antagonist. KAT is involved in the biosynthesis of glutamic and aspartic acid, functions as a neurotransmitter for the NMDA receptor in mammals, and is regulated by allosteric mechanisms. Its importance in various diseases such as schizophrenia makes KAT a highly attractive drug target. Here, we present the crystal structure of the Pyrococcus horikoshii KAT (PhKAT) in complex with pyridoxamine phosphates (PMP), KYN, and KYA. Surprisingly, the PMP was bound to the LYS-269 of phKAT by forming a covalent hydrazine bond. This crystal structure clearly shows that an amino group of KYN was transaminated to PLP, which forms a Schiff's base with the LYS-269 of the KYN. Thus, our structure confirms that the PMPs represent an intermediate state during the KAT reaction. Thus, PhKAT catalyzes the sequential conversion of KYN to KYA via the formation of an intermediate 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD), which is spontaneously converted to KYA in the absence of an amino group acceptor. Furthermore, we identified the two entry and exit sites of the PhKAT homodimer for KYN and KYA, respectively. The structural data on PhKAT presented in this manuscript contributes to further the understanding of transaminase enzyme reaction mechanisms.

The structure of NtdA, a sugar aminotransferase involved in the kanosamine biosynthetic pathway in Bacillus subtilis, reveals a new subclass of aminotransferases.

NtdA from Bacillus subtilis is a sugar aminotransferase that catalyzes the pyridoxal phosphate-dependent equatorial transamination of 3-oxo-α-D-glucose 6-phosphate to form α-D-kanosamine 6-phosphate. The crystal structure of NtdA shows that NtdA shares the common aspartate aminotransferase fold (Type 1) with residues from both monomers forming the active site. The crystal structures of NtdA alone, co-crystallized with the product α-D-kanosamine 6-phosphate, and incubated with the amine donor glutamate reveal three key structures in the mechanistic pathway of NtdA. The structure of NtdA alone reveals the internal aldimine form of NtdA with the cofactor pyridoxal phosphate covalently attached to Lys-247. The addition of glutamate results in formation of pyridoxamine phosphate. Co-crystallization with kanosamine 6-phosphate results in the formation of the external aldimine. Only α-D-kanosamine 6-phosphate is observed in the active site of NtdA, not the ß-anomer. A comparison of the structure and sequence of NtdA with other sugar aminotransferases enables us to propose that the VIß family of aminotransferases should be divided into subfamilies based on the catalytic lysine motif.

Enzymatic transamination of pyridoxamine in tobacco plants.

Vitamin B6 (VB6) comprises a group of pyridine compounds that are involved in a surprisingly high diversity of biochemical reactions. Humans and animals depend largely on plants for their VB6 nutrition. Many studies have focused on biosynthesis of VB6 and comparatively little is known about VB6 metabolic conversion in plants. Recently, we have found that an efficient conversion pathway between pyridoxal (PL) and pyridoxamine (PM) is present in tobacco, but the catalytic enzyme remains an unsolved mystery. In this study, enzymes catalyzing the transamination of PM were purified from tobacco leaves and characterized. Our results suggest that a specific PM-pyruvate aminotranferase dominates the reversible transamination of PM in tobacco, and also show that the apo form of glutamic-oxaloacetic aminotranferase from tobacco, but not the holoenzyme, is able to catalyze the analogous transamination reaction between PM and either oxaloacetate or α-ketoglutarate. PM-pyruvate aminotranferase is involved in a degradation pathway for VB6 compounds in bacteria. Therefore, our study raises questions about whether the degradation pathway of VB6 exists in plants.