Phosphoryl Group Transfer Research Articles

Swiveling Domain Mechanism in Pyruvate Phosphate Dikinase,

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.

Synthetic Src-Kinase Domain Inhibitors and Their Structural Requirements

Protein tyrosine kinases catalyze the transfer of phosphoryl groups from ATP to amino acids on proteins and play a fundamental role in signal transduction pathways in mammalian cells. In particular, Src and Src-family are non-receptor tyrosine kinases that regulate cell growth, differentiation, migration, adhesion and apoptosis. Src-family members share common features, with well defined domains. The activation of these enzymes in response to a variety of stimuli leads from a close and inactive conformation to an open and active one, through a balance of phosphorylation and dephosphorylation of the enzyme structure, characterized in different cases by x-ray crystallography. Overexpression, deregulation or mutations of these enzymes have been observed and studied in many diseases, first of all in many human malignancies, such as colon, breast, pancreatic and other cancers. Src-family is also involved in other pathologic situations, such as osteoporosis, cardiovascular diseases, immune system disorders, and, recently, it has been also demonstrated the involvement of Src in prion diseases. Therefore, Src-family is an attractive and fundamental target for the design of new therapeutic agents against different pathologies, in particular cancer and bone diseases. Currently, there is no approved drug acting as Src kinase inhibitor, but new molecules, very potent and selective toward this family of kinases and also in vivo, are continuously synthesized, as demonstrated by the high number of publications and patents in this field. Here, we report several examples of Src kinase domain inhibitors, focusing our attention on chemical structures, structure-activity relationships and mechanism of action.

Phosphorylation Reaction in cAPK Protein Kinase-Free Energy Quantum Mechanical/Molecular Mechanics Simulations

We present results of a theoretical analysis of the phosphorylation reaction in cAMP-dependent protein kinase using a combined quantum mechanical and molecular mechanics (QM/MM) approach. Detailed analysis of the reaction pathway is provided using a novel QM/MM implementation of the nudged elastic band method, finite temperature fluctuations of the protein environment are taken into account using free energy calculations, and an analysis of hydrogen bond interactions is performed on the basis of calculated frequency shifts. The late transfer of the substrate proton to the conserved aspartate (D166), the activation free energy of 15 kcal/mol, and the slight exothermic (-3 kcal/mol) character of the reaction are all consistent with the experimental data. The near attack conformation of D166 in the reactant state is maintained by interactions with threonine-201, asparagine-177, and most notably by a conserved water molecule serving as a strong structural link between the primary metal ion and the D166. The secondary Mg ion acts as a Lewis acid, attacking the beta-gamma bridging oxygen of ATP. This interaction, along with a strong hydrogen bond between the D166 and the substrate, contributes to the stabilization of the transition state. Lys-168 maintains a hydrogen bond to a transferring phosphoryl group throughout a reaction process. This interaction increases in the product state and contributes to its stabilization.

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: Evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.

Active Site Modifications of the Brain Isoform of Creatine Kinase by 4-Hydroxy-2-nonenal Correlate with Reduced Enzyme Activity: Mapping of Modified Sites by Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

Creatine kinase reversibly catalyzes the transfer of the high-energy phosphoryl group from phosphocreatine to MgADP for rapid regeneration of ATP. It is hypothesized that factors which perturb creatine kinase activity, such as reactive oxygen species resulting from oxidative stress, could have a major role in the pathogenesis of diseases, particularly in the brain, where the level of ATP utilization is high. The reactive aldehyde 4-hydroxy-2-nonenal is a major secondary product of lipid peroxidation caused by oxidative stress; the levels of both free and protein-bound 4-hydroxy-2-nonenal are increased in Alzheimer's disease brain. Preliminary reports indicated that creatine kinase had lower activity in Alzheimer's disease brain. In this study, we investigated the structural and functional consequences of reacting the cytosolic brain isoform of creatine kinase with 4-hydroxy-2-nonenal at pathophysiologically relevant concentrations of 4-hydroxy-2-nonenal (10-300 microM). Dose-dependent reduction of enzyme activity was observed and, for the first time, correlated with 4-hydroxy-2-nonenal adduct formation on specific amino acid residues, including the active site residues His66, His191, Cys283, and His296 as determined by Fourier transform-ion cyclotron resonance mass spectrometry.

The X‐ray crystallographic structure and activity analysis of a Pseudomonas‐specific subfamily of the HAD enzyme superfamily evidences a novel biochemical function

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO_2114, from the plant pathogen Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO_2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO_2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO_2114 was confirmed by kinetic assays. To explore PSPTO_2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO_2114 homologs were mapped onto the PSPTO_2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst.

Target Genes and DNA-Binding Sites of the Response Regulator PhoR fromCorynebacterium glutamicum

The two-component signal transduction system PhoRS of Corynebacterium glutamicum is involved in the phosphate (P(i)) starvation response. To analyze the binding of unphosphorylated and phosphorylated PhoR to the promoters of phosphate starvation-inducible (psi) genes, this response regulator and the kinase domain of its cognate sensor, PhoS (MBP-PhoSDelta1-246), were overproduced and purified. MBP-PhoSDelta1-246 showed constitutive autophosphorylation activity, and a rapid phosphoryl group transfer from phosphorylated MBP-PhoSDelta1-246 to PhoR was observed. Gel mobility shift assays revealed that phosphorylation increases the DNA-binding affinity of PhoR. The affinity of PhoR approximately P to different promoters varied and decreased in the order pstSCAB > phoRS > phoC > ushA > porB > ugpA > pitA > nucH and phoH1 > glpQ1. The binding sites in front of pstSCAB and phoRS were localized at positions -194 to -176 and -61 to -43 upstream of the transcriptional start sites, respectively. Alignment of these two 19-bp binding sites revealed a high identity in the 5'-terminal part, but not in the 3'-terminal part. As many OmpR-type response regulators bind to direct repeats, the 19-bp sequence might be interpreted as a loosely conserved 8-bp direct repeat separated by 3 bp. This idea was supported by the fact that the highest binding affinity was observed with a perfect 8-bp direct repeat of the sequence CCTGTGAAaatCCTGTGAA. Inspection of the other target promoters revealed sequences with some similarity to this binding motif, which might represent PhoR binding sites. The in vivo relevance of the PhoR-binding site within the phoRS promoter was supported by reporter gene studies.

Seeing the Process of Histidine Phosphorylation in Human Bisphosphoglycerate Mutase

Bisphosphoglycerate mutase is an erythrocyte-specific enzyme catalyzing a series of intermolecular phosphoryl group transfer reactions. Its main function is to synthesize 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In this paper, we directly observed real-time motion of the enzyme active site and the substrate during phosphoryl transfer. A series of high resolution crystal structures of human bisphosphoglycerate mutase co-crystallized with 2,3-bisphosphoglycerate, representing different time points in the phosphoryl transfer reaction, were solved. These structures not only clarify the argument concerning the substrate binding mode for this enzyme family but also depict the entire process of the key histidine phosphorylation as a "slow movie". It was observed that the enzyme conformation continuously changed during the different states of the reaction. These results provide direct evidence for an "in line" phosphoryl transfer mechanism, and the roles of some key residues in the phosphoryl transfer process are identified.

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein.

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg(2+)-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg(2+) and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg(2+) and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189 approximately P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an alpha-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189 approximately P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.

Cooperation among Microorganisms

Cooperation among Microorganisms

Selective Ion Tracing and MSn Analysis of Peptide Digests from FSBA-Treated Kinases for the Analysis of Protein ATP-Binding Sites

Kinases play a key role in many cellular processes by catalyzing the transfer of phosphoryl groups from ATP to a broad number of substrates, including amino acids on target proteins. The reagent 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA) has been widely used to identify ATP-binding sites in kinases since it reacts with nucleophilic amino acids occurring within these motifs, determining a mass increase of 433 Da. In this study, we present a versatile MS approach that has been developed to recognize labeled peptides generated after enzymatic digestion of FSBA-treated kinases. Using selective ion tracing and MS(2)/MS(3) experiments, we were able to easily identify peptides occurring at protein ATP-binding sites, also affording a complete characterization of the modified amino acids. This methodology may be used in the development of future parent ion scanning-based applications directed to large scale analysis of kinases within complex protein mixtures.

06/01716 Investigation of humic acid N with X-ray photoelectron spectroscopy: Effect of acid hydrolysis and comparison with 15N cross polarization/magic angle spinning nuclear magnetic resonance spectroscopy : Abe, T. et al. Organic Geochemistry, 2005, 36, (11), 1490–1497

Phosphoryl group transfer is central to genetic replication, cellular signalling and many metabolic processes. Understanding the mechanisms of phosphorylation and phosphate ester and anhydride cleavage is key to efforts towards biotechnological and biomedical exploitation of phosphate-handling enzymes. Analogues of phosphate esters and anhydrides are indispensable tools, alongside protein mutagenesis and computational methods, for the dissection of phosphoryl transfer mechanisms. Hydrolysable and non-hydrolysable phosphate analogues have provided insight into the nature and sites of phosphoryl transfer processes. Kinetic isotope effects and crystallography using transition state analogues have painted more detailed pictures of transition states and how enzymes work to stabilise them.

Crystal structure of the apo form of D‐alanine: D‐alanine ligase (Ddl) from Thermus caldophilus: A basis for the substrate‐induced conformational changes

Introduction. D-alanine:D-alanine ligase (Ddl) catalyses the dimerization of D-alanine before its incorporation in peptidoglycan precursors. The synthesis of D-alanine:Dalanine begins with an attack on the first D-alanine by the -phosphate of adenosine triphosphate (ATP) to yield an acylphosphate. That is followed by attack by the amino group of the second D-alanine, which eliminates the phosphate and produces the D-alanine: D-alanine dipeptide. Peptidoglycan biosynthesis has long been an attractive target for antibacterial drugs, such as D-cycloserine, glycopeptide antibiotics (vancomycin and teicoplanins), and -lactams (penicillin and cephalosporins). Vancomycintype antibiotics, for example, bind directly to the D-alanine: D-alanine terminus, thereby inhibiting crosslinking by the transpeptidase. Notably, bacteria that show vancomycin resistance, which develops after prolonged clinical treatment with vancomycin, possess an inactive Ddl and rely on another ligase, D-alanine:D-lactate ligase (Van), which produces D-alanine:D-lactate rather than D-alanine:Dalanine for cell wall synthesis. The switch from D-alanine: D-alanine peptidoglycan termini to D-alanine:D-lactate results in the loss of crucial hydrogen bonding interactions that causes a 1000-fold reduction in vancomycin binding affinity. X-ray crystallographic studies of Ddl and Van have contributed significantly to our understanding of the ligand specificity these two enzymes and suggest that a His residue in Van plays a critical role. A positive charge on the side chain imidazole nitrogen of His would attract the negatively charged lactate to the second substrate binding site at pH values less than 7, but at higher pH values Van would predominantly synthesize D-alanine:D-alanine. In Ddl, a Tyr residue [Tyr216 in Escherichia coli (Eco) DdlB, Tyr232 in Thermus caldophilus (Tca) Ddl] occupies the same spatial position as the His residue, and the hydroxyl group of the Tyr interacts with the COOH-terminal of the second D-alanine substrate. The structure of Eco DdlB complexed with ADP/ phosphorylated phosphinate (PDB ID: 2DLN) or with ADP/phosphorylated phosphonate (PDB ID: 1IOV) has been determined, as have the structures of Leuconostoc mesenteroides (Lme) D-Alanine:D-Lactate ligase complexed with ADP and phosphinophosphate (PDB ID:1EHI) and Enterococcus faecium (Efa) VanA complexed with ADP and phosphinophosphate (PDB ID:1E4E). However, to analyze the reaction mechanisms of these enzymes and their associated conformational changes, it is necessary to know the structures of both the substrate-bound and substrate-free forms of these enzymes. Our aim in the present study, therefore, was to grow crystals of Ddl that diffracted to high resolution in the absence of substrates. Here we report the X-ray structure of TcaDdl resolved to a resolution of 1.9 A and describe the conformational differences of the apo structure, comparing it with the structures of the previously described transition state analogue complex.

Protein kinase D directly phosphorylates histone deacetylase 5 via a random sequential kinetic mechanism

Class II histone deacetylases (HDACs) are signal-responsive repressors of gene transcription. In the heart, class II HDAC5 suppresses expression of genes that govern stress-induced cardiomyocyte growth. Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser 259 and Ser 498), resulting in nuclear export of HDAC5 and de-repression downstream target genes. Although prior studies established a role for protein kinase D (PKD) in the regulation of HDAC5 phosphorylation, it remained unclear whether PKD functions directly or indirectly to control the phosphorylation status of this transcriptional repressor. Here, we demonstrate that PKD catalyzes direct phosphoryl-group transfer to Ser 498 of HDAC5. Each of the three PKD family members, PKD1, PKD2, and PKD3, is capable of phosphorylating HDAC5 ( K m for substrate = 2.07, 3.12, and 1.43 μM, respectively), although PKD2 exhibits highest catalytic efficiency ( k cat/ K m = 6.77 min −1 μM −1). Kinetic studies revealed that the three PKD isozymes phosphorylate HDAC5 through a random sequential mechanism, and that ATP has no effect on association of kinase with peptide substrate. In addition, we demonstrate that ADP competitively inhibits phosphorylation of HDAC5 ( K i = 8.50, 17.54, and 11.98 μM for PKD1, PKD2, and PKD3, respectively). These findings define PKD as an HDAC kinase and thus suggest key roles for PKD family members in the control of chromatin structure and gene expression.

Protein Kinase D Directly Phosphorylates Ser498 of Histone Deacetylase 5 via a Random Sequential Kinetic Mechanism

Class II histone deacetylases (HDACs) are signal-responsive repressors of gene transcription. In the heart, class II HDAC 5 suppresses expression of genes that govern stress-induced cardiomyocyte growth. Signaling via pro-growth G protein coupled receptors (GPCRs) triggers phosphorylation of HDAC5 on two serine residues (Ser259 and Ser498), resulting in nuclear export of HDAC5 and de-repression downstream target genes. Although prior studies established a role for protein kinase D (PKD) in the regulation of HDAC5 phosphorylation, it remained unclear whether PKD functions directly or indirectly to control the phosphorylation status of this transcriptional repressor. Here, we demonstrate that PKD catalyzes direct phosphoryl-group transfer to Ser498 of HDAC5. Each of the three PKD family members, PKD1, PKD2 and PKD3, is capable of phosphorylating HDAC5 (Km for substrate = 2.07, 3.12 and 1.43 uM, respectively), although PKD2 exhibits highest catalytic efficiency (kcat/Km = 6.77 min−1 uM−1). Kinetic studies revealed that the three PKD isozymes phosphorylate HDAC5 through a random sequential mechanism, and that ATP has no effect on association of kinase with peptide substrate. In addition, we demonstrate that ADP competitively inhibits phosphorylation of HDAC5 (Ki = 8.50, 17.54 and 11.98 uM for PKD1, PKD2 and PKD3, respectively), suggesting that PKD is subject to product feedback inhibition. These findings define PKD as an HDAC kinase and thus further support PKD as a key regulator of HDAC phosphorylation and nuclear export.

Loop Movement and Catalysis in Creatine Kinase

Recently the crystal structure of creatine kinase from Torpedocalifornica was determined to 2.1 A. The dimeric structure revealed two different forms in the unit cell: one monomer was bound to a substrate, MgADP, and the other monomer was bound to a transition-state analogue complex composed of MgADP, nitrate and creatine. The most striking difference between the structures is the movement of two loops (comprising residues 60-70 and residues 323-333) into the active site in the transition state structure. This loop movement effectively occludes the active site from solvent, and the loops appear to be locked into place by a salt bridge formed between His66 and Asp326. His66 is of particular interest as it is located within a PGHP motif conserved in all creatine kinases but not found in other guanidino kinases. We have carried out alanine-scanning mutagenesis of each of the residues in the PGHP motif and determined that only the His66 plays a significant role in the creatine kinase reaction. Although neither residue interacts directly with the substrate, the interaction His66 and Asp326 appears to be important in providing the precise alignment of substrates necessary for phosphoryl group transfer. Finally, it is clear that neither His66 nor Asp326 are responsible for the pKs observed in the pH-rate profile for HMCK.

Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

Calcium pump-catalyzed (18)O exchange between inorganic phosphate and water was studied to test the hypothesis that all P-type pumps bind Mg(2+) before P(i) and validate utilization of the rate equation for ordered binding to interpret differences between site-directed mutants and wild-type enzyme. The results were remarkably similar to those obtained earlier with sodium pump (Kasho, V. N., Stengelin, M., Smirnova, I. N., and Faller, L. D. (1997) Biochemistry 36, 8045- 8052). The equation for ordered binding of Mg(2+) before P(i) fit the data best with only a slight chance (0.6%) of P(i) binding to apoenzyme. Therefore, P(i) is the substrate, and Mg(2+) is an obligatory cofactor. The intrinsic Mg(2+) dissociation constant from metalloenzyme (K(M) = 3.5 +/- 0.3 mm) was experimentally indistinguishable from the sodium pump value. However, the half-maximal concentration for P(i) binding to metalloenzyme ((K(p)(')=6.3+/-0.6 mM)) was significantly higher ( approximately 6-fold), and the probability of calcium pump forming phosphoenzyme from bound P(i) (P(c) = 0.04 +/- 0.03) was significantly lower ( approximately 6-fold) than for the sodium pump. From estimates of the rate constants for phosphorylation and dephosphorylation, the calcium pump appears to catalyze phosphoryl group transfer less efficiently than the sodium pump. Ordered binding of Mg(2+) before P(i) implies that both calcium pump and sodium pump form a ternary enzyme.metal.phosphate complex, consistent with molecular structures of other haloacid dehalogenase superfamily members that were crystallized with Mg(2+) and phosphate, or a phosphate analogue, bound.

Arginine kinase in the demosponge Suberites domuncula:regulation of its expression and catalytic activity by silicic acid

In Demospongiae (phylum Porifera) the formation of the siliceous skeleton, composed of spicules, is an energetically expensive reaction. The present study demonstrates that primmorphs from the demosponge Suberites domuncula express the gene for arginine kinase after exposure to exogenous silicic acid. The deduced sponge arginine kinase sequence displays the two characteristic domains of the ATP:guanido phosphotransferases; it can be grouped to the 'usual' mono-domain 40 kDa guanidino kinases (arginine kinases). Phylogenetic studies indicate that the metazoan guanidino kinases evolved from this ancestral sponge enzyme; among them are also the 'unusual' two-domain 80 kDa guanidino kinases. The high expression level of the arginine kinase gene was already measurable 1 day after addition of silicic acid by northern blot, as well as by in situ hybridization analysis. Parallel determinations of enzyme activity confirmed that high levels of arginine kinase are present in primmorphs that had been exposed for 1-5 days to silicic acid. Finally, transmission electron-microscopical studies showed that primmorphs containing high levels of arginine kinase also produce siliceous spicules. These data highlight that silicic acid is an inorganic morphogenetic factor that induces the expression of the arginine kinase, which in turn probably catalyzes the reversible transfer of high-energy phosphoryl groups.

A common docking site for response regulators on the yeast phosphorelay protein YPD1

In Saccharomyces cerevisiae, a multi-component phosphorelay signal transduction pathway mediates cellular responses to environmental stress. A histidine-containing phosphotransfer protein, YPD1, represents a bifurcation point between the SLN1–YPD1–SSK1 pathway responsible for osmotic stress responses and the SLN1–YPD1–SKN7 pathway involved in cell wall biosynthesis and cell cycle control. The phosphorelay protein YPD1 must physically interact with and transfer phosphoryl groups between three homologous response regulator domains, designated SLN1-R1, SSK1-R2, and SKN7-R3. In this comparative study, the molecular basis of interaction was examined between YPD1 and each of the three response regulator domains utilizing alanine scanning mutagenesis combined with a yeast two-hybrid assay. Results from the yeast two-hybrid assay indicate that all three response regulator domains bind to a common area, largely hydrophobic in nature, on the surface of YPD1. We postulate that other YPD1 surface residues surrounding this common docking site are involved in making specific interactions with one or more of the response regulator domains.

Functional domain organization of the potato alpha-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants.

The potato tuber (Solanum tuberosum) GWD (alpha-glucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure-function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E+ATP-->E-P+AMP+P(i) (where P(i) stands for orthophosphate) partial reaction, but not the E-P+glucan-->E+glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.