Pyridoxamine Supplementation Effectively Reverses the Abnormal Phenotypes of Zebrafish Larvae With PNPO Deficiency.

Neonatal epileptic encephalopathy (NEE), as a result of pyridoxine 5'-phosphate oxidase (PNPO) deficiency, is a rare neural disorder characterized by intractable seizures and usually leads to early infant death. The clinical phenotypes do not respond to antiepileptic drugs but are alleviated in most cases by giving large doses of pyridoxal 5'-phosphate (PLP). PLP is the active form of vitamin B6 participating in more than 100 enzymatic pathways.

Inactive mutants of human pyridoxine 5'-phosphate oxidase: a possible role for a noncatalytic pyridoxal 5'-phosphate tight binding site.

Pyridoxal 5'-phosphate (PLP) is a cofactor for many vitamin B6-requiring enzymes that are important for the synthesis of neurotransmitters. Pyridoxine 5'-phosphate oxidase (PNPO) is one of two enzymes that produce PLP. Some 16 known mutations in human PNPO (hPNPO), including R95C and R229W, lead to deficiency of PLP in the cell and have been shown to cause neonatal epileptic encephalopathy (NEE).

Molecular basis of E. coli L-threonine aldolase catalytic inactivation at low pH.

L-Threonine aldolases (TAs), a family of enzymes belonging to the fold-type I pyridoxal 5'-phosphate (PLP) dependent enzymes, play a role in catalyzing the reversible cleavage of l-3-hydroxy-α-amino acids to glycine and the corresponding aldehydes. Threonine aldolases have great biotechnological potential for the syntheses of pharmaceutically relevant drug molecules because of their stereospecificity. The pH-dependency of their catalytic activity, affecting reaction intermediates, led us to study the effect of low-pH on Escherichia coli TA (eTA) structure.

Structure-based mechanism for early PLP-mediated steps of rabbit cytosolic serine hydroxymethyltransferase reaction.

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5'-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates.

Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition.

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B₆, pyridoxal 5'-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B₆ is converted to PLP by PL kinase.

Pyridoxal 5'-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase.

Pyridoxal 5'-phosphate (PLP) is a cofactor for dozens of B(6) requiring enzymes. PLP reacts with apo-B(6) enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6) enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase.

Molecular basis of reduced pyridoxine 5'-phosphate oxidase catalytic activity in neonatal epileptic encephalopathy disorder.

Mutations in pyridoxine 5'-phosphate oxidase are known to cause neonatal epileptic encephalopathy. This disorder has no cure or effective treatment and is often fatal. Pyridoxine 5'-phosphate oxidase catalyzes the oxidation of pyridoxine 5'-phosphate to pyridoxal 5'-phosphate, the active cofactor form of vitamin B(6) required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters.

Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase.

Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5'-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows approximately 2-fold decrease in both PL affinity and activity.

Crystal Structure of human pyridoxal kinase: structural basis of M(+) and M(2+) activation.

Pyridoxal kinase catalyzes the transfer of a phosphate group from ATP to the 5' alcohol of pyridoxine, pyridoxamine, and pyridoxal. In this work, kinetic studies were conducted to examine monovalent cation dependence of human pyridoxal kinase kinetic parameters. The results show that hPLK affinity for ATP and PL is increased manyfold in the presence of K(+) when compared to Na(+); however, the maximal activity of the Na(+) form of the enzyme is more than double the activity in the presence of K(+).

Crystal structure of pyridoxal kinase from the Escherichia coli pdxK gene: implications for the classification of pyridoxal kinases.

The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E.

Properties of human and rabbit cytosolic serine hydroxymethyltransferase are changed by single nucleotide polymorphic mutations.

Serine hydroxymethyltransferase (SHMT) is a key enzyme in the formation and regulation of the folate one-carbon pool. Recent studies on human subjects have shown the existence of two single nucleotide polymorphisms that may be associated with several disease states. One of these mutations results in Ser394 being converted to an Asn (S394N) and the other in the change of Leu474 to a Phe (L474F).

Serine hydroxymethyltransferase revisited.

Recent structural data and the properties of several active site mutants of serine hydroxymethyltransferase have resolved some key questions concerning the catalytic mechanism and broad substrate specificity of this enzyme. In the tetrahydrofolate-dependent conversion of serine to glycine, an early proposed mechanism involved a retroaldol cleavage and a formaldehyde intermediate, while a more recent suggestion posits a direct nucleophilic displacement of the serine hydroxyl by N(5) of tetrahydrofolate, without creation of free formaldehyde. Geometric and chemical difficulties with both options led to a new proposal, a modified retroaldol mechanism in which N(5) of tetrahydrofolate makes a nucleophilic attack on serine C(3) leading to breakage of the C(3)-C(2)-bond of serine rather than the C(3)-hydroxyl bond.

Structure of Escherichia coli pyridoxine 5'-phosphate oxidase in a tetragonal crystal form: insights into the mechanistic pathway of the enzyme.

Escherichia coli pyridoxine 5'-phosphate oxidase (ePNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP) by the FMN oxidation of pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP), forming FMNH(2) and H(2)O(2). The crystal structure of ePNPOx is reported in a tetragonal unit cell at 2.6 A resolution.

Crystal structure of the PdxY Protein from Escherichia coli.

The crystal structure of Escherichia coli PdxY, the protein product of the pdxY gene, has been determined to a 2.2-A resolution. PdxY is a member of the ribokinase superfamily of enzymes and has sequence homology with pyridoxal kinases that phosphorylate pyridoxal at the C-5' hydroxyl.

Expression, purification, and kinetic constants for human and Escherichia coli pyridoxal kinases.

Pyridoxal kinase is an ATP dependent enzyme that phosphorylates pyridoxal, pyridoxine, and pyridoxamine forming their respective 5'-phosphorylated esters. The kinase is a part of the salvage pathway for re-utilizing pyridoxal 5'-phosphate, which serves as a coenzyme for dozens of enzymes involved in amino acid and sugar metabolism. Clones of two pyridoxal kinases from Escherichia coli and one from human were inserted into a pET 22b plasmid and expressed in E.

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme.

Structure and properties of recombinant human pyridoxine 5'-phosphate oxidase.

Pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5'-phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of approximately 0.

Role of proline residues in the folding of serine hydroxymethyltransferase.

Previous studies on the folding mechanism of Escherichia coli serine hydroxymethyltransferase (SHMT) showed that the final rate determining folding step was from an intermediate that contained two fully folded domains with N-terminal segments of approximately 55 residues and interdomain segments of approximately 50 residues that were still solvent exposed and subject to proteolysis. The interdomain segment contains 3 Pro residues near its N terminus and 2 Pro residues near its C terminus. The 5 Pro residues were each mutated to both a Gly and Ala residue, and each mutant SHMT was purified and characterized with respect to kinetic properties, stability, secondary structure, and folding mechanism.

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.

Location of the pteroylpolyglutamate-binding site on rabbit cytosolic serine hydroxymethyltransferase.

Serine hydroxymethyltransferase (SHMT; EC 2.1.2.

Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase.

Pyridoxine-5'-phosphate oxidase catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to an aldehyde, forming pyridoxal 5'-phosphate. A hydrogen atom is removed from C4' during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5'-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle.

Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay.

An enzymatic method for determination of B6 vitamers is presented. In this method pyridoxal 5'-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme. The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH.

The role of serine hydroxymethyltransferase isozymes in one-carbon metabolism in MCF-7 cells as determined by (13)C NMR.

The role of cytosolic and mitochondrial serine hydroxymethyltransferase in supplying one-carbon groups for purine and thymidylate biosynthesis in MCF-7 cells was investigated by observing folate-mediated one-carbon metabolism of l-[3-(13)C]serine, [2-(13)C]glycine, and [(13)C]formate. (13)C NMR was used to follow the incorporation of label into carbons 2 and 8 of purines and the methyl group attached to carbon 5 of thymidylate. The percentage enrichment of the (13)C label in purines was determined from the splitting patterns of the (1)H NMR spectra of C2 and C8 of adenine and C8 of guanine.

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E.