Phosphohistidine Intermediate Research Articles

The actions of NME1/NDPK-A and NME2/NDPK-B as protein kinases

Nucleoside diphosphate kinases (NDPKs) are multifunctional proteins encoded by the nme (non-metastatic cells) genes, also called NM23. NDPKs catalyze the transfer of γ-phosphate from nucleoside triphosphates to nucleoside diphosphates by a ping-pong mechanism involving the formation of a high-energy phosphohistidine intermediate. Growing evidence shows that NDPKs, particularly NDPK-B, can additionally act as a protein histidine kinase. Protein kinases and phosphatases that regulate reversible O-phosphorylation of serine, threonine, and tyrosine residues have been studied extensively in many organisms. Interestingly, other phosphoamino acids histidine, lysine, arginine, aspartate, glutamate, and cysteine exist in abundance but remain understudied due to the paucity of suitable methods and antibodies. The N-phosphorylation of histidine by histidine kinases via the two- or multi-component signaling systems is an important mediator in cellular responses in prokaryotes and lower eukaryotes, like yeast, fungi, and plants. However, in vertebrates knowledge of phosphohistidine signaling has lagged far behind and the identity of the protein kinases and protein phosphatases involved is not well established. This article will therefore provide an overview of our current knowledge on protein histidine phosphorylation particularly the role of nm 23 gene products as protein histidine kinases.

Crystallographic and Biochemical Analysis of Rotavirus NSP2 with Nucleotides Reveals a Nucleoside Diphosphate Kinase-Like Activity

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.

Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal b-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.

Nucleoside-diphosphate kinase: Structural and kinetic analysis of reaction pathway and phosphohistidine intermediate

This chapter describes the structural and kinetic analysis of reaction pathway and phosphohistidine intermediate. The structural studies of enzymes have made major contributions to understanding of catalytic mechanisms. Unstable chemical species—such as a Michaelis complex or a covalent intermediate—that form and vanish on the millisecond to second time scale, and the even more elusive transition state that lives a few femto- or picoseconds, may be made accessible to study. In practice, their impact has been marginal and much more structural information on enzyme catalysis has come from experiments, where the time scale of the chemistry has been changed to that of crystallography rather than the converse. There are many ways to slow enzymatic catalysis: by low temperature trapping, by removing catalytic groups through site-directed mutagenesis of the protein or chemical modification of the substrate, by replacing substrates with unreactive analogs of either the ground state or the transition state.

Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.

The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were labeled with (15)N, both specifically at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated (15)N resonances were assigned by correlation to the C2' protons which had been assigned previously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the (1)H-(15)N HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation of the phosphate binding site filled by the O2 phosphate of the substrate, fructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermediate. The phosphohistidine intermediate is characterized by two hydrogen bonds involving the catalytic histidines, His-258 and His-392, which are directly observed at the N1' positions of the imidazole rings. The N1' of phospho-His-258 is protonated ((1)H chemical shift, 14.0 ppm) and hydrogen bonded to the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded ((1)H chemical shift, 13.5 ppm) to the phosphoryl moiety of the phosphohistidine. The existence of a protonated phospho-His-258 intermediate and the observation of a fairly strong hydrogen bond to the same phosphohistidine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as probes of the catalytic site mutation of Glu-327 to alanine revealed that, in addition to its function as the proton donor to fructose-6-phosphate during formation of the transient phosphohistidine intermediate at the N3' of His-258, this residue has a significant role in maintaining the structural integrity of the catalytic site. The (1)H-(15)N HSQC data also provide clear evidence that despite being a surface residue, His-446 has a very acidic pK(a), much less than 6.0. On the basis of these observations a revised mechanism for fructose-2,6-bisphosphatase that is consistent with all of the previously published kinetic data and X-ray crystal structures is proposed. The revised mechanism accounts for the structural and kinetic consequences produced by mutation of the catalytic histidines and Glu-327. It also provides the basis for a hypothetical mechanism of bisphosphatase activation by cAMP-dependent phosphorylation of Ser-32, which is located in the N-terminal kinase domain.

Peroxidase and Phosphatase Activity of Active-site Mutants of Vanadium Chloroperoxidase from the Fungus Curvularia inaequalis: IMPLICATIONS FOR THE CATALYTIC MECHANISMS

Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics.

Identification of YPD1, a gene of Candida albicans which encodes a two-component phosphohistidine intermediate protein.

We have identified the YPD1 phosphohistidine intermediate two-component gene of Candida albicans. YPD1 has an open reading frame of 552 bp. It is located on chromosome 1 and an mRNA specific for YPD1 is detected under both yeast and hyphal growth. YPD1 encodes a protein of 184 amino acids with an estimated molecular mass of 20.5 kDa. A search for similarities with other proteins in databases showed that CaYpd1p exhibits the greatest overall similarity with Ypd1p from Saccharomyces cerevisiae (34.2% identity; 49.4% similarity) as well as with the C-terminus half of a protein from Schizosaccharomyces pombe (Accession No. CAA22174). However, CaYpd1p also shows similarity with other eukaryotic and prokaryotic proteins which function as phosphohistidine intermediates in two-component phospho-relay systems. In these cases, similarity was restricted to the amino acid sequences which surround the conserved histidine residue that is phosphorylated. In addition, CaYPD1 (but not CaYPD1(H69Q)) complements the lack of YPD1 in S. cerevisiae. This observation supports the premise that CaYpd1p also may function as a phosphohistidine intermediate protein in C. albicans.

Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate.

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleoside-diphosphate kinase.

Two human nm23 genes have been identified, designated nm23-H1 and nm23-H2, which encode the 88% identical nucleoside-diphosphate kinase (NDPK) A and NDPK B polypeptides, respectively. The nm23-H1 gene product has been shown to play a functional role in the suppression of tumor metastasis. The Nm23 proteins/NDPK are highly conserved throughout evolution and are implicated in controlling cellular differentiation and development in various species, while the underlying mechanisms remain undefined. Neither the NDPK activity nor the DNA-binding activity, identified recently for NDPK B, can satisfactory explain the regulatory functions of Nm23. The present study provides evidence that purified Nm23 proteins are capable of transferring a phosphate group to other proteins when non-denaturing amounts of urea are present. This novel Nm23/NDPK activity was found to be specific for serine and threonine residues, and the transphosphorylation of substrate proteins occurred stoichiometrically. Because of the absence of a substrate turn-over, the novel function was termed protein phosphotransferase activity instead of protein kinase activity. It is demonstrated that urea stimulates the interaction of NDPK with other proteins. Identical phosphoprotein patterns were obtained using purified NDPK preparations from human, Drosophila, yeast and Dictyostelium in the presence of urea. Partially purified NDPK from human erythrocytes produced a similar phosphorylation pattern independent of urea addition and also acted stoichiometrically. In this preparation, a protein phosphotransferase activity of Nm23 species may possibly be generated and/or stabilized by the interaction with copurified proteins. Using different mutants of Dictyostelium NDPK it was shown that the protein phosphotransferase activity depends on the same active site as the NDPK activity. A phosphotransfer mechanism analogous to that of protein-histidine kinases is proposed, involving a high-energy phosphohistidine intermediate. Furthermore, the novel Nm23 function is compared with an apparently similar protein phosphotransferase activity which was observed previously with partially purified NDPK from different plant species.

Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium

Nucleoside diphosphate kinase (NDP kinase) has a ping-pong mechanism with a phosphohistidine intermediate. Crystals of the enzymes from Dictyostelium discoideum and from Drosophila melanogaster were treated with phosphoramidate, and their X-ray structures were determined at 2.1 and 2.2 A resolution, respectively. The atomic models, refined to R factors below 20%, show no conformation change relative to the free proteins. In both enzymes, the active site histidine was phosphorylated on N delta, and it was the only site of phosphorylation. The phosphate group interacts with the hydroxyl group of Tyr56 and with protein-bound water molecules. Its environment is compared with that of phosphohistidines in succinyl-CoA synthetase and in phosphocarrier proteins. The X-ray structures of phosphorylated NDP kinase and of previously determined complexes with nucleoside diphosphates provide a basis for modeling the Michaelis complex with a nucleoside triphosphate, that of the phosphorylated protein with a nucleoside diphosphate, and the transition state of the phosphate transfer reaction in which the gamma-phosphate is pentacoordinated.

The Crystal Structure of Human Nucleoside Diphosphate Kinase, NM23-H2

The 2.8 Å resolution X-ray structure of NM23-H2 has been determined by molecular replacement using the structure Myxococcus xanthusnucleoside diphosphate (NDP) kinase. NM23-H2 is a human NDP kinase. The enzyme catalyses phosphoryl transfer, binds DNA, and can activate the transcription of the c-myconcogene in vitro. NM23 has also been reported to be a suppressor of metastasis in some types of tumours. Whereas the M. xanthusNDP kinase is a tetramer, NM23-H2 is a hexamer. The folding of NM23-H2 is identical to the folding of other NDP kinases. Two antiparallel helices joined by a turn form one edge of the nucleotide binding cleft. This region moves in a hinge-like fashion in response to substrate binding and crystal packing forces. Additional differences in conformation among the NDP kinases are principally in regions involved in protein–protein contacts within the oligomers. The only protein–protein interaction conserved among all NDP kinases is a dimeric interaction. Several mutations of NM23-H2 have been detected in tumour tissues. These mutations do not involve residues interacting with the substrates, and probably destabilise the enzyme without directly affecting the catalytic activity. Low level phosphorylation of serines has been reported for NM23 both in vitroand in vivo. The structure of the hexamer indicates that two serine residues that have been reported as being phosphorylated, Ser44 and Ser122, are on the surface of the hexamer, and are likely to be phosphorylated by exogenous kinases. In contrast, Ser120 is buried, and is most likely phosphorylated by a direct transfer from the phosphohistidine intermediate of the reaction mechanism.

Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus

The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.