Verne Schirch

Interaction of folylpolyglutamates with enzymes in one-carbon metabolism

Of all the coenzymes, tetrahydrofolate exhibits the most structural diversity. The relationship of these structural forms to physiological function is under intense study by numerous research groups. In textbooks, tetrahydrofolate (tetrahydropteroylmonoglutamate) is shown as the coenzyme of one-carbon metabolism, but it has been known for several decades that the physiologically active forms of the coenzyme contain from 4 to 7 glutamyl residues linked by amide bonds through the gamma-carboxyl group. These glutamyl residues do not serve a direct function in transferring the one-carbon group. The tetrahydrofolylpolyglutamates were originally thought to be simply storage forms of the coenzyme, but studies now show that the polyglutamate chain of the coenzyme affects the transport properties of the coenzyme, alters the kinetic properties of many enzymes in one-carbon metabolism, and results in channeling of the coenzyme between several enzymes. In general, the dissociation constants of this group of enzymes for the tetrahydrofolylpolyglutamates are very low, in the 0.1 to 1 microM range. The concentration of the coenzyme in the cell appears to be similar to the concentration of folate-utilizing enzymes, suggesting that the concentration of unbound coenzyme in the cell may be very low. Several of the enzymes in one-carbon metabolism are either multifunctional proteins or multienzyme complexes. An active area of research is to determine if there is a functional relationship between these multifunctional enzymes and the polyglutamate portion of the coenzyme.

The metabolic role of leucovorin

Interest in determining if leucovorin, known chemically as 5-formyltetra-hydrofolate, plays a role in one-carbon metabolism is reemerging. While investigations in the 1940s suggested it was an important donor of one-carbon units in folate-mediated biosynthetic reactions, studies between the 1950s and 1980s disproved this hypothesis and dismissed its presence in biological systems as artifactual. Recently, new data has focused attention on the possible biological function of this compound that is widely used in cancer chemotherapy.

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 ∼0.2 sec−1 and Km values in the low micromolar range for both pyridoxine 5′phosphate and pyridoxamine 5′‐phosphate. Pyridoxal 5′‐phosphate is an effective product inhibitor. The three‐dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5′‐phosphate tightly on each subunit.

Crystal structure of human pyridoxal kinase: Structural basis of M + and M2+ 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+. Other monovalent cations, Li+, Cs+, and Rb+ do not show significant activity. We have determined the crystal structure of hPLK in the unliganded form, and in complex with MgATP to 2.0 and 2.2 Å resolution, respectively. Overall, the two structures show similar open conformation, and likely represent the catalytically idle state. The crystal structure of the MgATP complex also reveals Mg2+ and Na+ acting in tandem to anchor the ATP at the active site. Interestingly, the active site of hPLK acts as a sink to bind several molecules of MPD. The features of monovalent and divalent metal cation binding, active site structure, and vitamin B6 specificity are discussed in terms of the kinetic and structural studies, and are compared with those of the sheep and Escherichia coli enzymes.

Structural Studies on Folding Intermediates of Serine Hydroxymethyltransferase Using Fluorescence Resonance Energy Transfer

Previous studies have demonstrated that the in vitro folding pathway of Escherichia coli serine hydroxymethyltransferase has both monomer and dimer intermediates that are stable for periods of minutes to hours at 4°C (Cai K., Schirch, D., and Schirch, V. (1995) J. Biol. Chem. 270, 19294-19299). Single Trp mutant enzymes were constructed and used in combination with other methods to show that on the folding pathway of this enzyme two domains rapidly fold to form a monomer in which the amino-terminal 55 amino acid residues and a segment around the active site region of Lys229 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate-determining conformational change in which the unstructured segments assume their native state (Cai, K., and Schirch, V. (1996) J. Biol. Chem. 271, 2987-2994). To further assess the kinetics and structural details of the intermediates during folding, fluorescence energy transfer and fluorescence anisotropy measurements were made of the three Trp residues and pyridoxal 5′-phosphate, attached covalently to the active site by reduction to a secondary amine by sodium cyanoborohydride. These studies confirmed that the basic kinetic folding pathway remained the same in the reduced enzyme as compared to the earlier studies with the apoenzyme. Both equilibrium and kinetic intermediates were identified and their structural characteristics determined. The results show that the active site Lys229-bound pyridoxyl 5′-phosphate remains more than 50 angstroms from any Trp residues until the final rate-determining conformational change when it approaches each Trp residue at the same rate. The environment of each Trp residue and the pyridoxyl phosphate in both an equilibrium folding intermediate and a kinetic folding intermediate are described.

Structural Studies on Folding Intermediates of Serine Hydroxymethyltransferase Using Single Tryptophan Mutants

Previous studies showed that during the in vitro folding of Escherichia coli serine hydroxymethyltransferase at 4°C, both monomer and dimer intermediates accumulated and were stable for periods of minutes to hours (Cai, K., Schirch, D., and Schirch, V.(1995) J. Biol. Chem. 270, 19294-19299). To obtain structural information on these intermediates, two of the three Trp residues in the protein were changed to Phe to generate a set of three single Trp mutant enzymes. These mutant enzymes were purified and characterized and shown to retain essentially all of the properties of the wild-type enzyme. The fluorescence and circular dichroism measurements of each mutant enzyme were studied under unfolding-refolding equilibrium conditions and during refolding. In addition, the sensitivity of the protein to digestion by subtilisin during refolding was investigated. The results of these studies show that the unfolded enzyme has two domains that rapidly fold to form a monomer in which the first 55 amino acids and a segment between residues 225 and 276 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate determining conformational change in which the unstructured segments assume their native state.

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 B6 required by more than 140 different catalytic activities, including enzymes involved in amino acid metabolism and biosynthesis of neurotransmitters. Our aim is to elucidate the mechanism by which a homozygous missense mutation (R229W) in the oxidase, linked to neonatal epileptic encephalopathy, leads to reduced oxidase activity. The R229W variant is ∼850-fold less efficient than the wild-type enzyme due to an ∼192-fold decrease in pyridoxine 5′-phosphate affinity and an ∼4.5-fold decrease in catalytic activity. There is also an ∼50-fold reduction in the affinity of the R229W variant for the FMN cofactor. A 2.5 Å crystal structure of the R229W variant shows that the substitution of Arg-229 at the FMN binding site has led to a loss of hydrogen-bond and/or salt-bridge interactions between FMN and Arg-229 and Ser-175. Additionally, the mutation has led to an alteration of the configuration of a β-strand-loop-β-strand structure at the active site, resulting in loss of two critical hydrogen-bond interactions involving residues His-227 and Arg-225, which are important for substrate binding and orientation for catalysis. These results provide a molecular basis for the phenotype associated with the R229W mutation, as well as providing a foundation for understanding the pathophysiological consequences of pyridoxine 5′-phosphate oxidase mutations.

Synthesis of (6S)-5-formyltetrahydropteroyl-polyglutamates and interconversion to other reduced pteroylpolyglutamate derivatives

5-Formyltetrahydropteroylpolyglutamates can be synthesized and purified directly from dihydropteroylpolyglutamates in a single-step procedure without purification of intermediates and with yields greater than 90%. The procedure involves a coupled enzymatic synthesis of 10-formyltetrahydropteroylpolyglutamates using the enzymes dihydrofolate reductase, serine hydroxymethyltransferase, and C1-tetrahydrofolate synthase with catalytic amounts of NADPH. The 10-formyltetrahydropteroylpolyglutamates are subsequently converted to 5-formyltetrahydropteroylpolyglutamates at 90 degrees C with near quantitative yields. 5-Formyltetrahydropteroylpolyglutamates are the only stable reduced derivatives of tetrahydropteroylpolyglutamates and can be purified and stored indefinitely without decomposition. Additionally, 5-formyltetrahydropteroylpolyglutamates can be readily converted to other derivatives of tetrahydropteroylpolyglutamates with yields greater than 95%. Also described is the synthesis of tetrahydropteroylglutamates labeled at C-11 with either 14C or 13C. Rapid purification procedures for serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from frozen rabbit livers are presented.

Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase

Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B6 requiring enzymes. PLP reacts with apo-B6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B6 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. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.

Mechanism for the Coupling of ATP Hydrolysis to the Conversion of 5-Formyltetrahydrofolate to 5,10-Methenyltetrahydrofolate

5,10-Methenyltetrahydrofolate synthetase catalyzes the irreversible conversion of 5-formyl-tetrahydropteroylpolyglutamates (5-CHO-H4PteGlun) to 5,10-methenyltetrahydropteroylpolyglutamates (5, 10-CH+-H4PteGlun). The equilibrium of the nonenzymatic reaction, which equilibrates slowly in the absence of enzyme, greatly favors 5-CHO-H4PteGlun. The enzyme couples the reaction to the hydrolysis of ATP shifting the equilibrium to favor 5,10-CH+-H4PteGlun. Substrate-dependent non-equilibrium isotope exchange of ADP into ATP was observed, suggesting the formation of a phosphorylated intermediate of 5-CHO-H4PteGlunduring the enzyme-catalyzed reaction. The competitive inhibitor 5-formyltetrahydrohomofolate also supported the ADP to ATP exchange, suggesting that this molecule could also form a phosphorylated intermediate. The initial rates of the ADP-ATP exchange with saturating ADP were about 70 s− for both compounds, while the kcat values for product formation were 5 s− for 5-CHO-H4PteGlun and 0.005 s− for 5-formyltetrahydrohomofolate. Starting with 5-[18O]CHO-H4PteGlun, it was shown by 31P NMR that the formyl oxygen of the substrate was transferred to the product phosphate during the reaction. This further supports the existence of a phosphorylated intermediate. The formyl group of 5-CHO-H4PteGlun is known to be an equilibrium mixture of two rotamers. Stopped-flow analysis of the enzymatic reaction showed that only one of the rotamers serves as a substrate for the enzyme.