Phosphate Group Of Pyridoxal 5'-phosphate Research Articles

Structural and Functional Analysis of the Pyridoxal Phosphate Homeostasis Protein YggS from Fusobacterium nucleatum.

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.

Preparation and characterization of an imogolite/chitosan hybrid with pyridoxal-5'-phosphate as an interfacial modifier.

Imogolite/chitosan hybrid films were prepared using pyridoxal-5′-phosphate (PLP) as an interfacial modifier. Thermogravimetric analysis and spectroscopic measurements revealed that the phosphate group of PLP was adsorbed on the imogolite. Furthermore, rheological measurements suggested that the PLP-modified imogolites (PLP–imogolite) had strong interactions with chitosan in solution. Moreover, UV absorption of the hybrid film showed that PLP and chitosan formed Schiff base linkages. Therefore, the hybrid films exhibited a significant improvement in their mechanical properties compared to those of pristine chitosan/imogolite hybrid films.

The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum

The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5'-phosphate (PLP) dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer. The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.

Crystal structures of the Chromobacterium violaceumω‐transaminase reveal major structural rearrangements upon binding of coenzyme PLP

The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form • -transaminases and -transaminases bind by dynamic light scattering (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction).

Structure of biosynthetic N‐acetylornithine aminotransferase from Salmonella typhimurium: Studies on substrate specificity and inhibitor binding

Acetylornithine aminotransferase (AcOAT) is one of the key enzymes involved in arginine metabolism and catalyzes the conversion of N-acetylglutamate semialdehyde to N-acetylornithine (AcOrn) in the presence of L-glutamate. It belongs to the Type I subgroup II family of pyridoxal 5'-phosphate (PLP) dependent enzymes. E. coli biosynthetic AcOAT (eAcOAT) also catalyzes the conversion of N-succinyl-L-2-amino-6-oxopimelate to N-succinyl-L,L-diaminopimelate, one of the steps in lysine biosynthesis. In view of the critical role of AcOAT in lysine and arginine biosynthesis, structural studies were initiated on the enzyme from S. typhimurium (sAcOAT). The K(m) and k(cat)/K(m) values determined with the purified sAcOAT suggested that the enzyme had much higher affinity for AcOrn than for ornithine (Orn) and was more efficient than eAcOAT. sAcOAT was inhibited by gabaculine (Gcn) with an inhibition constant (K(i)) of 7 microM and a second-order rate constant (k(2)) of 0.16 mM(-1) s(-1). sAcOAT, crystallized in the unliganded form and in the presence of Gcn or L-glutamate, diffracted to a maximum resolution of 1.90 A and contained a dimer in the asymmetric unit. The structure of unliganded sAcOAT showed significant electron density for PLP in only one of the subunits (subunit A). The asymmetry in PLP binding could be attributed to the ordering of the loop L(alphak-) (betam) in only one subunit (subunit B; the loop from subunit B comes close to the phosphate group of PLP in subunit A). Structural and spectral studies of sAcOAT with Gcn suggested that the enzyme might have a low affinity for PLP-Gcn complex. Comparison of sAcOAT with T. thermophilus AcOAT and human ornithine aminotransferase suggested that the higher specificity of sAcOAT towards AcOrn may not be due to specific changes in the active site residues but could result from minor conformational changes in some of them. This is the first structural report of AcOAT from a mesophilic organism and could serve as a basis for drug design as the enzyme is important for bacterial cell wall biosynthesis.

Structure of the Antitumour Enzyme L-Methionine -Lyase from Pseudomonas putida at 1.8 A Resolution

l-Methionine gamma-lyase (EC 4.4.1.11, MGL_Pp) from Pseudomonas putida is a multifunctional enzyme, which belongs to the gamma-family of pyridoxal-5'-phosphate (PLP) dependent enzymes. In this report, we demonstrate that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method to an R-factor of 20.4% at 1.8 A resolution. Detailed information of the overall structure of MGL_Pp supplies a clear picture of the substrate- and PLP-binding pockets. Tyr59 and Arg61 of neighbouring subunits, which are strongly conserved in other gamma-family enzymes, contact the phosphate group of PLP. These residues are important as the main anchor within the active site. Lys240, Asp241 and Arg61 of one partner monomer and Tyr114 and Cys116 of the other partner monomer form a hydrogen-bond network in the MGL active site which is specific for MGLs. It is also suggested that electrostatic interactions at the subunit interface are involved in the stabilization of the structural conformation. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug.

Structure of P-protein of the glycine cleavage system: implications for nonketotic hyperglycinemia

The crystal structure of the P-protein of the glycine cleavage system from Thermus thermophilus HB8 has been determined. This is the first reported crystal structure of a P-protein, and it reveals that P-proteins do not involve the alpha(2)-type active dimer universally observed in the evolutionarily related pyridoxal 5'-phosphate (PLP)-dependent enzymes. Instead, novel alphabeta-type dimers associate to form an alpha(2)beta(2) tetramer, where the alpha- and beta-subunits are structurally similar and appear to have arisen by gene duplication and subsequent divergence with a loss of one active site. The binding of PLP to the apoenzyme induces large open-closed conformational changes, with residues moving up to 13.5 A. The structure of the complex formed by the holoenzyme bound to an inhibitor, (aminooxy)acetate, suggests residues that may be responsible for substrate recognition. The molecular surface around the lipoamide-binding channel shows conservation of positively charged residues, which are possibly involved in complex formation with the H-protein. These results provide insights into the molecular basis of nonketotic hyperglycinemia.

Crystal Structure of Serine Dehydratase from Rat Liver,

SDH (L-serine dehydratase, EC 4.3.1.17) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent dehydration of L-serine to yield pyruvate and ammonia. Liver SDH plays an important role in gluconeogenesis. Formation of pyruvate by SDH is a two-step reaction in which the hydroxyl group of serine is cleaved to produce aminoacrylate, and then the aminoacrylate is deaminated by nonenzymatic hydrolysis to produce pyruvate. The crystal structure of rat liver apo-SDH was determined by single isomorphous replacement at 2.8 A resolution. The holo-SDH crystallized with O-methylserine (OMS) was also determined at 2.6 A resolution by molecular replacement. SDH is composed of two domains, and each domain has a typical alphabeta-open structure. The active site is located in the cleft between the two domains. The holo-SDH contained PLP-OMS aldimine in the active site, indicating that OMS can form the Schiff base linkage with PLP, but the subsequent dehydration did not occur. Apo-SDH forms a dimer by inserting the small domain into the catalytic cleft of the partner subunit so that the active site is closed. Holo-SDH also forms a dimer by making contacts at the back of the clefts so that the dimerization does not close the catalytic cleft. The phosphate group of PLP is surrounded by a characteristic G-rich sequence ((168)GGGGL(172)) and forms hydrogen bonds with the amide groups of those amino acid residues, suggesting that the phosphate group can be protonated. N(1) of PLP participates in a hydrogen bond with Cys303, and similar hydrogen bonds with N(1) participating are seen in other beta-elimination enzymes. These hydrogen bonding schemes indicate that N(1) is not protonated, and thus, the pyridine ring cannot take a quinone-like structure. These characteristics of the bound PLP suggest that SDH catalysis is not facilitated by forming the resonance-stabilized structure of the PLP-Ser aldimine as seen in aminotransferases. A possible catalytic mechanism involves the phosphate group, surrounded by the characteristic sequence, acting as a general acid to donate a proton to the leaving hydroxyl group of serine.

Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes

Twenty-four structures of pyridoxal-5′-phosphate (PLP)-dependent enzymes that represent five different folds are shown to share a common recognition pattern for the phosphate group of their PLP-ligands. All atoms that interact with the phosphate group of PLP in these proteins are organized within a two-layer structure so that the first interacting layer contains from five to seven atoms and parallel with this is a second layer containing from three to seven interacting atoms. In order to identify features of the phosphate-binding site common to PLP-dependent enzymes, a simple procedure is described that assigns relative positions to all interacting atoms unambiguously, such that the networks of interactions for different proteins can be compared. On the basis of these diagrams for 24 enzyme-cofactor complexes, a detailed comparison of the two-layer structures of PLP-dependent enzymes, with both similar and different folds, was made. A majority of the structurally defined PLP-dependent proteins use the same atom types in analogous “key” positions to bind their PLP-ligands. In some instances, proteins use water molecules when a key position is unoccupied. A similar two-layer recognition pattern extends to protein recognition of at least one other, non-PLP ligand, glucosamine 6-phosphate. We refer to this three-dimensional recognition pattern as the phosphate-binding cup. In general, the phosphate-binding cup provides a very stable anchoring point for PLP. When numerous water molecules occur within the cup, however, then the phosphate group of PLP participates directly in the enzymatic reactions with inorganic phosphate replacing the water molecules of the cup. With glucosamine-6-phosphate synthase, the water molecules of the phosphate-binding cup facilitate the entry of substrate and the exit of product.

Role of Arg-277 in the binding of pyridoxal 5'-phosphate to Trypanosoma brucei ornithine decarboxylase.

The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy. The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A., & Goldsmith, E. J. (1995) Protein Sci. 4, 1291-1304]. Mutation of Arg-277 to Ala increases the K(m) for PLP by 270-fold compared to that of wild-type ODC while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity was measured directly by ultrafiltration; the K(d) for PLP is at least 20-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC also has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necessary for high-affinity PLP binding by ODC. The 31P NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a dianion to both wild-type and R277A ODC. However, the 31P chemical shift for R277A ODC (6.7 ppm) is 0.5 ppm downfield from that observed for the wild-type enzyme, indicating that the environment of the enzyme-bound phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 8.5. Concomitant with these changes are a decreased k(cat) and an altered absorption spectra which arises from bound PLP. PLP bound to wild-type ODC has a 31P chemical shift and a CD signal observable over the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the 31P chemical shift becomes solution-like and the CD signal is abolished. The data suggest that for R277A ODC the rigid PLP binding mode which characterizes the wild-type enzyme is lost at high pH. Thus, multiple interactions between the wild-type active site and PLP maintain the cofactor in a constrained conformation that is essential for efficient catalysis, tempering the consequence of the removal of any single interaction.

Determination of the rates of formation and hydrolysis of the Schiff bases formed by 5′-deoxypyridoxal and poly- l-lysine

The kinetics of the reaction between 5′-deoxypyridoxal (DPL) and poly- l-lysine in aqueous solutions at a variable pH and a constant ionic strength of 0.1 M was studied spectrophotometrically. The rate constants of formation and hydrolysis of the resulting Schiff base and its stability constant at a variable pH were also determined. A comparison of the formation rate constant for the Schiff base with those for the models of pyridoxal 5′-phosphate (PLP) with poly- l-lysine and DPL with n-hexylamine revealed the effect of both the phosphate group of PLP and of the polypeptide chain on intramolecular catalysis in the dehydration of the intermediate carbinolamine formed. The effects of the polypeptide and the phosphate group on the hydrolysis of the Schiff base and hence on the base stability are also demonstrated.

Direct phosphate-phosphate interaction between coenzyme and substrate in the catalytic reaction of glycogen phosphorylase

Glycogen phosphorylase contains firmly bound pyridoxal 5′-phosphate (PLP), and catalyzes the reversible transfer of a glucosyl moiety between glucose-1-phosphate (G-1-P) and α-1,4-glucan. X-ray crystallographic studies revealed that PLP is located in a pocket where the phosphate group of PLP is pointed toward the G-1-P binding site. We have synthesized pyridoxal(5′)diphospho(1)-α-d-glucose, as a model compound for the phosphate-phosphate interaction between PLP and G-1-P, and reconstituted the enzyme with this compound. The resulting enzyme is catalytically inactive in itself, but, in the presence of glucan, the glycosyl moiety of this compound is transferred to the glucan forming a new α-1,4-glucosidic linkage along with the production of pyridoxal 5′-diphosphate. This glucosyltransfer is similar to the normal catalytic reaction in various aspects, although the rate is smaller in the order of three. AMP accelerates the transfer about 24 times compared with the reaction in its absence. We have more recently used pyridoxal(5′)triphospho(1)-α-D-glucose to reconstitute the enzyme. In the presence of glucan, the compound bound to enzyme is gradually degraded to pyridoxal 5′-triphosphate. This reaction is essentially dependent on AMP, and proceeds several times more slowly than the glucosyltransfer from the diphospho compound. These results provide evidence for the direct phosphate-phosphate interaction between the coenzyme and the substrate in the normal enzyme reaction, and seem to reflect a rather wide allowance in regard to this interaction.