Inactivation of gamma-aminobutyric acid aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid does not proceed by the expected aromatization mechanism

Inactivation of pyridoxal 5'-phosphate (PLP)-dependent gamma-aminobutryic acid aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (SADFA) gives pyridoxamine 5'-phosphate, not the expected SADFA-PLP aromatization product. Inactivation appears to proceed by a Michael addition/hydrolysis mechanism instead.     

Vitamin B(6) salvage enzymes: mechanism, structure and regulation

Vitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-enzymes in a "salvage pathway", which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5'-phosphate oxidase. Once it is made, pyridoxal 5'-phosphate is targeted to the dozens of different apo-B(6) enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5'-phosphate to the apo-B(6)-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5'-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5'-phosphate formation. Deficiency of pyridoxal 5'-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5'-phosphate deficiency. Understanding the exact role of vitamin B(6) in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B(6)-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay

Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma.     

Structure and mechanism of Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming pyridoxal 5'-phosphate (PLP). This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli and as a part of the salvage pathway of this coenzyme in both E. coli and mammalian cells. Recent studies have shown that in addition to the active site, PNPOx contains a noncatalytic site that binds PLP tightly. The crystal structures of PNPOx with one and two molecules of PLP bound have been determined. In the active site, the PLP pyridine ring is stacked almost parallel against the re-face of the middle ring of flavin mononucleotide (FMN). A large protein conformational change occurs upon binding of PLP. When the protein is soaked with excess PLP an additional molecule of this cofactor is bound about 11 A from the active site. A possible tunnel exists between the two sites. Site mutants were made of all residues at the active site that make interactions with the substrate. Stereospecificity studies showed that the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon of PMP. The crystal structure and the stereospecificity studies suggest that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.     

Functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate in phosphorylase: a study using pyridoxal-reconstituted enzyme as a model system

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic study was conducted by using competitive inhibitors of phosphite, an activator, and alpha-D-glucopyranose 1-phosphate (glucose-1-P) to study the relationship between the PLP phosphate and the binding of glucose-1-P to phosphorylase. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pKa2 values demonstrated a dependency of KM on pKa2. Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and, possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not effect the glucose-1-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or various amino acid side chains in a proper orientation for effective catalysis. (3) The force between the PLP phosphate and its binding site in phosphorylase is mainly electrostatic; a change of ionization state during catalysis is unlikely. (4) Properties of the central atoms of different anions are important for their effects as either activators or inhibitors of pyridoxal phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine (pyridoxamine) 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to pyridoxal 5'-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx. PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.     

Determination of plasma pyridoxal 5'-phosphate by an enzymatic-high-performance liquid chromatographic procedure

An enzymatic-HPLC procedure for the determination of plasma pyridoxal 5'-phosphate (PLP) has been established. The assay is based on the decarboxylation of L-3,4-dihydroxyphenylalanine using Streptococcus tyrosine decarboxylase apoenzyme, which requires PLP as cofactor. The product of the enzyme reaction, dopamine, is measured by Coulochem electrochemical detection with a series of oxidizing and then reducing electrodes. Trace amounts of PLP in the apoenzyme preparation were removed with the aid of cysteine-sulfinic acid and gel filtration. The detection limit for PLP by this method is 50 pM in plasma.     

Linkage of subunit interactions, structural changes, and energetics of coenzyme binding in tryptophan synthase

The energetics of binding of the coenzyme pyridoxal 5'-phosphate (PLP) to both the apo beta 2 subunit and the apo alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been investigated as a function of pH and temperature by direct microcalorimetric methods. At 25 degrees C, pH 7.5, the binding process proceeds in the time range of minutes and shows a biphasic heat output which permits resolution of the overall reaction into different reaction steps. Binding studies on the coenzyme analogues pyridoxal (PAL), pyridoxine 5'-phosphate (PNP), and pyridoxine (POL) to the protein as well as a comparison of these results with data from studies on PLP binding to epsilon-aminocaproic acid have led to a deconvolution of the complex heat vs. time curves into fast endothermic contributions from electrostatic interaction and Schiff base formation and slow exothermic contributions from the interactions between PLP and the binding domain. The pH-independent, large negative change in heat capacity of about -9.1 kJ/(mol of beta 2 X K) when binding PLP to beta 2 is indicative of major structural changes resulting from complex formation. The much smaller value of delta Cp = -1.7 kJ/(mol of beta 2 X K) for binding of PLP to alpha 2 beta 2 clearly demonstrates the energetic linkage of protein-protein and protein-ligand interactions. Calorimetric titrations of the apo beta 2 subunit with PLP at 35 degrees C have shown that also at this temperature positive cooperativity between the two binding sites occurs. On the basis of these measurements a complete set of site-specific thermodynamic parameters has been established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rv2607 from Mycobacterium tuberculosis is a pyridoxine 5'-phosphate oxidase with unusual substrate specificity

Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx), an enzyme that oxidizes pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) to form pyridoxal 5'-phosphate (PLP). We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat) and K(M) for this reaction were 0.01 s(-1) and 360 μM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.     

Distinct effects of pyridoxal phosphate on NAD- and NADP- linked malic enzymes of Escherichia coli

NADP-linked malic enzyme from Escherichia coli W was inactivated by pyridoxal 5'-phosphate (PLP) following pseudo-first order kinetics. The inactivation was, however, reversed upon addition of an aminothiol, such as penicillamine and cysteamine, whereas the activity was not restored, when the PLP-inactivated enzyme was treated with NaBH4 prior to the addition of aminothiol. The inactivating effect was specific to PLP and no other structural analogs of PLP tested inactivated the enzyme, except that pyridoxal exhibited a similar effect, though to a lesser extent. In contrast, NAD-linked malic enzyme from the same micro-organism was insensitive to PLP, even in the presence of 0.8 M guanidine hydrochloride.     

Molecular modelling studies on the interactions of human DNA topoisomerase IB with pyridoxal-compounds

Candida guilliermondii and human DNA topoisomerases I are inhibited by PL (pyridoxal), PLP (pyridoxal 5'-phosphate) and PLP-AMP (pyridoxal 5'-diphospho-5'-adenosine) (PL相似文献   

Phospholipase C from Bacillus cereus. Evidence for essential lysine residues.

1. Phospholipase C was inactivated by exposure to the three amino-group reagents, ethyl acetamidate, 2,4,6-trinitrobenzensulphonic acid and pyridoxal 5'-phosphate plus reduction. 2. Inactivation by pyridoxal 5'-phosphate showed the characteristics of Schiff's base formation with the enzyme. The pyridoxal 5'-phosphate-treated enzyme after reduction had an absorbance maximum at 325 mm and 6-N-pyridoxyl-lysine was the only fluorescent component after acid hydrolysis. 3. For complete inactivation, 2 mol of pyridoxal 5'-phosphate or 7 mol of 2,4,6-trinitrophenyl were incorporated/mol of enzyme. 4. The two apparently essential lysine residues were much more reactive to pyridoxal 5'-phosphate than the other 19 lysine residues in the enzyme. 5. Binding of phospholipase C to a substrate-based affinity gel caused marked protection against inactivation by pyridoxal 5'-phosphate. For complete inactivation of the gel-bound enzyme, 5 mol of pyridoxal 5'-phosphate were incorporated/mol of enzyme and there was no evidence of two especially reactive lysine residues. 6. On application of pyridoxal 5'-phosphate-treated enzyme (remaining activity 30% of original) to a column of the affinity gel, some material bound and some did not. The latter contained very little enzyme activity and was heavily incorporated with reagent (9.06 mol/mol of enzyme). The former had a specific activity of 34% of that of the control and contained 1.29 mol of reagent/mol of enzyme. 7. Thus phospholipase C appears to contain two lysine residues that are essential for enzyme activity, but probably not for substrate binding.     

Crystal structure analysis of omega-amino acid:pyruvate aminotransferase with a newly developed Weissenberg camera and an imaging plate using synchrotron radiation

The three-dimensional structure of omega-amino acid:pyruvate aminotransferase from Pseudomonas sp. F-126, an isologous alpha 4 tetramer containing pyridoxal 5'-phosphate (PLP) as a cofactor, has been determined at 2.0 A resolution. The diffraction data were collected with a newly developed Weissenberg camera with a Fuji Imaging Plate, using synchrotron radiation. The mean figure-of-merit was 0.57. The subunit is rich in secondary structure and comprises two domains. PLP is located in the large domain. The high homology in the secondary structure between this enzyme and aspartate aminotransferase strongly indicates that these two types of enzymes have evolved from a common ancestor.     

Synthesis of cyclopropane isosteres of the antiepilepsy drug vigabatrin and evaluation of their inhibition of GABA aminotransferase

The antiepilepsy drug vigabatrin (1; 4-aminohex-5-enoic acid; gamma-vinyl GABA) is a mechanism-based inactivator of the pyridoxal 5'-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid aminotransferase (GABA-AT). Inactivation has been shown to proceed by two divergent mechanisms (Nanavati, S. M. and Silverman, R. B. (1991) J. Am. Chem. Soc. 113, 9341-9349), a Michael addition pathway (Scheme 2, pathway a) and an enamine pathway (Scheme 2, pathway b). Analogs of vigabatrin with a cyclopropyl or cyanocyclopropyl functionality in place of the vinyl group (2-5) were synthesized as potential inactivators of GABA-AT that can inactivate the enzyme only through a Michael addition pathway, but they were found to be only weak inhibitors of the enzyme.     

Isolation and characterization of the product of inactivation of gamma-aminobutyric acid aminotransferase by gabaculine.

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid, 1), a naturally occurring neurotoxin isolated from Streptomyces toyocaenis, has been shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) (Rando, R. R. Biochemistry 1977, 16, 4604). Inactivation results from reaction of gabaculine with the pyridoxal 5'-phosphate (PLP) cofactor. Two HPLC systems for isolating this inactivator-PLP adduct are described as well as a detailed characterization of the adduct, including the ultraviolet-visible spectrum, electrospray mass spectra, and NMR spectrum. The same spectral characterization of the chemically synthesized gabaculine-PLP adduct is also reported.     

Identification of Lys190 as the primary binding site for pyridoxal 5'-phosphate in human serum albumin.

The covalent binding of pyridoxal 5'-phosphate (PLP) to human serum albumin (HSA) is important in the regulation of PLP metabolism. In plasma, PLP is bound to HSA at a single high-affinity and at two or more nonspecific sites. To characterize the primary PLP binding site, HSA was incubated with [3H] PLP, and the Schiff base linkage was reduced with potassium borohydride. Tryptic peptides were purified, and the major labeled peptide was sequenced. Amino acid analysis confirmed a homogeneous peptide Leu-Asp-Glu-Leu-Arg-Asp-Glu-Gly-Xaa-Ala-Ser-Ser-Ala-Lys which corresponds to residues 182-195 of HSA. The data indicate that Lys190 is the primary PLP binding site. This Lys residue is distinct from other sites of covalent adduct formation; namely, the primary sites for nonenzymatic glycosylation (Lys525) and acetylation by aspirin (Lys199).     

Influence of pH on the removal of pyridoxal 5'-phosphate from phosphorylase b

A method to break the pyridoxal 5'-phosphate (PLP)-phosphorylase b bond using hydroxylamine and slightly acid pH is put forward and described in the present paper. This method does not involve drastic conditions or deforming reagents. The influence of pH and protein concentration on the removal of PLP from phosphorylase has also been studied, resulting in an order of -0.3 with respect to the enzyme, a value that implies a complex reaction. An additional conclusion is that an increase in the protein concentration entails better protection of the enzyme from attack by hydroxylamine.     

Human brain pyridoxal-5'-phosphate phosphatase (PLPP):protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).