Elucidation of the Mechanisms of Inter-domain Coupling in the Monomeric State of Enzyme I by High-pressure NMR.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1H-15N TROSY and 1H-13C methyl TROSY NMR spectra combined with 15N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme.

Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity.

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer-dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI's monomeric state have yet been hampered by the dimer's high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI's monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.

Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I.

Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.

Influence of the phosphoenolpyruvate:carbohydrate phosphotransferase system on toxin gene expression and virulence in Bacillus anthracis.

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post-translational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr- EI- mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.

Resonance assignment of the 128 kDa enzyme I dimer from Thermoanaerobacter tengcongensis.

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) utilizes phosphoenolpyruvate (PEP) as a source of energy in order to transport sugars across the cellular membrane. PEP binding to EI initiates a phosphorylation cascade that regulates a variety of essential pathways in the metabolism of bacterial cells. Given its central role in controlling bacterial metabolism, EI has been often suggested as a good target for antimicrobial research. Here, we report the 1HN, 15N, 13C', 1Hmethyl, and 13Cmethyl chemical shifts of the 128 kDa homodimer EI from the thermophile Thermoanaerobacter tengcongensis. In total 79% of the expected backbone amide correlations and 80% of the expected methyl TROSY peaks from U-[2H, 13C, 15N], Ileδ1-[13CH3], Val-Leu-[13CH3/12CD3] labeled EI were assigned. The reported assignments will enable future structural studies aimed at illuminating the fundamental mechanisms governing long-range interdomain communication in EI and at indicating new therapeutic strategies to combat bacterial infections.

The Phosphohistidine Phosphatase SixA Targets a Phosphotransferase System.

SixA, a well-conserved protein found in proteobacteria, actinobacteria, and cyanobacteria, is the only reported example of a bacterial phosphohistidine phosphatase. A single protein target of SixA has been reported to date: the Escherichia coli histidine kinase ArcB. The present work analyzes an ArcB-independent growth defect of a sixA deletion in E. coli A screen for suppressors, analysis of various mutants, and phosphorylation assays indicate that SixA modulates phosphorylation of the nitrogen-related phosphotransferase system (PTSNtr). The PTSNtr is a widely conserved bacterial pathway that regulates diverse metabolic processes through the phosphorylation states of its protein components, EINtr, NPr, and EIIANtr, which receive phosphoryl groups on histidine residues. However, a mechanism for dephosphorylating this system has not been reported. The results presented here suggest a model in which SixA removes phosphoryl groups from the PTSNtr by acting on NPr. This work uncovers a new role for the phosphohistidine phosphatase SixA and, through factors that affect SixA expression or activity, may point to additional inputs that regulate the PTSNtrIMPORTANCE One common means to regulate protein activity is through phosphorylation. Protein phosphatases exist to reverse this process, returning the protein to the unphosphorylated form. The vast majority of protein phosphatases that have been identified target phosphoserine, phosphotheronine, and phosphotyrosine. A widely conserved phosphohistidine phosphatase was identified in Escherichia coli 20 years ago but remains relatively understudied. The present work shows that this phosphatase modulates the nitrogen-related phosphotransferase system, a pathway that is regulated by nitrogen and carbon metabolism and affects diverse aspects of bacterial physiology. Until now, there was no known mechanism for removing phosphoryl groups from this pathway.

Identification of protective protein antigens for vaccination against Streptococcus dysgalactiae in cobia (Rachycentron canadum).

Streptococcus dysgalactiae is considered a causative agent of severe infection and economic loss for the cobia industry in Taiwan. In this study, protective antigens of this pathogenic bacterium were identified and screened in cobia (Rachycentron canadum). Outer surface proteins (OMPs) of this pathogen were extracted using mutanolysin digestion. Immunogenic targets were detected by western blot and then subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA (ISA 763 AVG) and rGAPDH + ISA, elicited higher levels of specific antibody responses against the recombinant proteins and had high levels of lysozyme activity. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, while 0% RPS value was found in both ISA injected and control groups. The results presented in the study demonstrate that the GAPDH and PtsA are promising vaccine candidates for preventing S. dysgalactiae disease in cobia.

The oligomerization state of bacterial enzyme I (EI) determines EI's allosteric stimulation or competitive inhibition by α-ketoglutarate.

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by phosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP). The EI phosphorylation state determines the phosphorylation states of all other PTS components and is thought to play a central role in the regulation of several metabolic pathways and to control the biology of bacterial cells at multiple levels, for example, affecting virulence and biofilm formation. Given the pivotal role of EI in bacterial metabolism, an improved understanding of the mechanisms controlling its activity could inform future strategies for bioengineering and antimicrobial design. Here, we report an enzymatic assay, based on Selective Optimized Flip Angle Short Transient (SOFAST) NMR experiments, to investigate the effect of the small-molecule metabolite α-ketoglutarate (αKG) on the kinetics of the EI-catalyzed phosphoryl transfer reaction. We show that at experimental conditions favoring the monomeric form of EI, αKG promotes dimerization and acts as an allosteric stimulator of the enzyme. However, when the oligomerization state of EI is shifted toward the dimeric species, αKG functions as a competitive inhibitor of EI. We developed a kinetic model that fully accounted for the experimental data and indicated that bacterial cells might use the observed interplay between allosteric stimulation and competitive inhibition of EI by αKG to respond to physiological fluctuations in the intracellular environment. We expect that the mechanism for regulating EI activity revealed here is common to several other oligomeric enzymes.

1H, 15N, 13C backbone resonance assignment of the C-terminal domain of enzyme I from Thermoanaerobacter tengcongensis.

Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone 1H, 15N and 13C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the 1H-15N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.

Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh.

Background: Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. Methods: We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Results: Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase. Conclusions: This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

One Year Genome Evolution of Lausannevirus in Allopatric versus Sympatric Conditions.

Amoeba-resisting microorganisms raised a great interest during the last decade. Among them, some large DNA viruses present huge genomes up to 2.5 Mb long, exceeding the size of small bacterial genomes. The rate of genome evolution in terms of mutation, deletion, and gene acquisition in these genomes is yet unknown. Given the suspected high plasticity of viral genomes, the microevolution of the 346 kb genome of Lausannevirus, a member of Megavirales, was studied. Hence, Lausannevirus was co-cultured within the amoeba Acanthamoeba castellanii over one year. Despite a low number of mutations, the virus showed a genome reduction of 3.7% after 12 months. Lausannevirus genome evolution in sympatric conditions was investigated by its co-culture with Estrella lausannensis, an obligate intracellular bacterium, in the amoeba A. castellanii during one year. Cultures were split every 3 months. Genome sequencing revealed that in these conditions both, Lausannevirus and E. lausannensis, show stable genome, presenting no major rearrangement. In fact, after one year they acquired from 2 to 7 and from 4 to 10 mutations per culture for Lausannevirus and E. lausannensis, respectively. Interestingly, different mutations in the endonuclease encoding genes of Lausannevirus were observed in different subcultures, highlighting the importance of this gene product in the replication of Lausannevirus. Conversely, mutations in E. lausannensis were mainly located in a gene encoding for a phosphoenolpyruvate-protein phosphotransferase (PtsI), implicated in sugar metabolism. Moreover, in our conditions and with our analyses we detected no horizontal gene transfer during one year of co-culture.

Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine.

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinß4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

Intron sequence of the taurocyamine kinase gene as a marker to investigate genetic variation of Paragonimus species in Japan and the origins of triploidy in P. westermani.

BACKGROUND: Paragonimiasis is a foodborne parasitic infection caused by lung flukes of the genus Paragonimus. Several species of Paragonimus are endemic in Japan: P. westermani (diploid and triploid) P. miyazakii, P. ohirai and P. iloktsuenensis. The taxonomic status and genetic variability of these lung flukes remains poorly understood. METHODS: The second intron of domain 1 of the taurocyamine kinase gene (TKD1int2) region was used to explore genetic variation and differentiation of diploid and triploid P. westermani, as well as P. miyazakii, P. ohirai and P. iloktsuenensis originating from Japan. RESULTS: We found high levels of intraspecific variation in P. westermani, but only low levels of variation within the other species studied. Haplotype network and phylogenetic tree analyses demonstrated the sister-group relationship of P. ohirai and P. iloktsuenensis and the phylogenetically distant relationship of P. westermani with the other species. All individuals except for triploid P. westermani were homozygous. Each triploid contained at least one allele similar to that seen in most diploids from Chiba and one allele resembling that seen in diploids from Oita. One triploid contained three different sequences. CONCLUSIONS: Our findings suggested that the TKD1int2 region is a suitable marker for use in studying the genetic variation and phylogenetics of Paragonimus species, as well as providing clues to the origins of triploidy in P. westermani.

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering.

Enzyme I (EI) is the first component in the bacterial phosphotransferase system, a signal transduction pathway in which phosphoryl transfer through a series of bimolecular protein-protein interactions is coupled to sugar transport across the membrane. EI is a multidomain, 128-kDa homodimer that has been shown to exist in two conformational states related to one another by two large (50-90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His189 located in the N-terminal domain α/ß (EIN(α/ß)) subdomain to the downstream protein partner bound to the EIN(α) subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EIN(α/ß) subdomain into close proximity to the C-terminal dimerization domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate (PEP) bound to EIC to His189. Here, we investigate the solution conformation of a complex of an active site mutant of EI (H189A) with PEP. Simulated annealing refinement driven simultaneously by solution small angle X-ray scattering and NMR residual dipolar coupling data demonstrates unambiguously that the EI(H189A)-PEP complex exists in a dynamic equilibrium between two approximately equally populated conformational states, one corresponding to the closed structure and the other to a partially closed species. The latter likely represents an intermediate in the open-to-closed transition.

The substrate-free and -bound crystal structures of the duplicated taurocyamine kinase from the human parasite Schistosoma mansoni.

The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg(2+)-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or l-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. An SmTK crystal was soaked with the dead end transition state analog (TSA) components taurocyamine-NO3 (2-)-MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle x-ray scattering model of SmTK-TSA in solution with two closed active sites was generated.

Coupling between overall rotational diffusion and domain motions in proteins and its effect on dielectric spectra.

In this work, we formulate a closed-form solution of the model of a semirigid molecule for the case of fluctuating and reorienting molecular electric dipole moment. We illustrate with numeric calculations the impact of protein domain motions on dielectric spectra using the example of the 128 kDa protein dimer of Enzyme I. We demonstrate that the most drastic effect occurs for situations when the characteristic time of protein domain dynamics is comparable to the time of overall molecular rotational diffusion. We suggest that protein domain motions could be a possible explanation for the high-frequency contribution that accompanies the major relaxation dispersion peak in the dielectric spectra of protein aqueous solutions. We propose that the presented computational methodology could be used for the simultaneous analysis of dielectric spectroscopy and nuclear magnetic resonance data. Proteins 2015; 83:1571-1581. © 2015 Wiley Periodicals, Inc.

Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I.

Enzyme I (EI), the first component of the bacterial phosphotransfer signal transduction system, undergoes one of the largest substrate-induced interdomain rearrangements documented to date. Here we characterize the perturbations generated by two small molecules, the natural substrate phosphoenolpyruvate and the inhibitor α-ketoglutarate, on the structure and dynamics of EI using NMR, small-angle X-ray scattering and biochemical techniques. The results indicate unambiguously that the open-to-closed conformational switch of EI is triggered by complete suppression of micro- to millisecond dynamics within the C-terminal domain of EI. Indeed, we show that a ligand-induced transition from a dynamic to a more rigid conformational state of the C-terminal domain stabilizes the interface between the N- and C-terminal domains observed in the structure of the closed state, thereby promoting the resulting conformational switch and autophosphorylation of EI. The mechanisms described here may be common to several other multidomain proteins and allosteric systems.

Phosphagen kinase in Schistosoma japonicum: II. Determination of amino acid residues essential for substrate catalysis using site-directed mutagenesis.

Phosphagen kinases (PKs) play major roles in the regulation of energy metabolism in animals. Creatine kinase (CK) is the sole PK in vertebrates, whereas several PKs are present in invertebrates. We previously identified a contiguous dimer taurocyamine kinase (TK) from the trematode Schistosoma japonicum (Sj), a causative agent of schistosomiasis. SjTK contiguous dimer is comprised of domain 1 (D1) and domain 2 (D2). In this study, we used SjTK contiguous dimer (SjTKD1D2) or truncated single-domain constructs (SjTKD1 or SjTKD2) and employed site-directed mutagenesis to investigate the enzymatic properties of TK mutants. Mutation in SjTKD1 or SjTKD2 (D1E222G or D2E225G) caused complete loss of activity for the substrate taurocyamine. Likewise, a double mutant (D1E222GD2E225G) in the contiguous dimer (D1D2) exhibited complete loss of activity for the substrate taurocyamine. However, catalytic activity in the contiguous dimer remained in both of D1 inactive mutant (D1D2D1E222G) and D2 inactive mutant (D1D2D2E225G), suggesting that efficient catalysis of SjTKD1D2 is dependent on the activity of D1 and D2. The catalytic efficiency of the mixture of both single domains (WTD1+WTD2) showed same enzymatic properties (Km(Tauro)=0.68;Vmax/Km(Tauro)=137.04) to WTD1D2 (Km(Tauro)=0.47; Vmax/Km(Tauro)=144.30). This result suggests that the contiguous dimeric structure is not essential for the catalytic efficiencies of both domains of SjTK. Vmax/Km(Tauro) of the mixture of wild-type and inactivated domains (78.02 in WTD1+D2E225G and 128.24 in D1E222G+WTD2) were higher than the corresponding mutants (47.25 in D1D2D1E222G and 46.77 in D1D2D2E225G). To identify amino acid residues that are critical for taurocyamine binding, we performed alanine scanning mutagenesis at positions 57-63 on the guanidino specificity (GS) region of the SjTKD1, which is considered to be involved in guanidino-substrate recognition. R63A and R63Y mutants lost activity for taurocyamine, suggesting that these residues are associated with taurocyamine binding. In addition, we investigated the role of Tyr84 in D1 and found an association with substrate alignment. The Y84 residue was replaced with R, H, K, I, A, and G. Although the activities of each mutant were decreased (Vmax=2.36-67.50µmolPi/min/mgprotein), Y84 mutants possess binding affinity for taurocyamine (Km(Tauro)=3.19-10.04mM). The D1Y84R, D1Y84H, D1Y84K, and D1Y84A mutants exhibited low activity for taurocyamine, whereas the D1Y84I and D1Y84G mutants exhibited slightly decreased activity compared with the other Y84 mutants. The D1Y84K mutant lost substrate synergy between taurocyamine and ATP, suggesting that this mutation moves the position of the GS loop, similar to that of lombricine kinase (LK), and interferes with taurocyamine binding. This is the first comprehensive investigation of essential amino acid residues for substrate catalysis in trematode TK.

The role of Y84 on domain 1 and Y87 on domain 2 of Paragonimus westermani taurocyamine kinase: Insights on the substrate binding mechanism of a trematode phosphagen kinase.

The two-domain taurocyamine kinase (TK) from Paragonimus westermani was suggested to have a unique substrate binding mechanism. We performed site-directed mutagenesis on each domain of this TK and compared the kinetic parameters Km(Tc) and Vmax with that of the wild-type to determine putative amino acids involved in substrate recognition and binding. Replacement of Y84 on domain 1 and Y87 on domain 2 with R resulted in the loss of activity for the substrate taurocyamine. Y84E mutant has a dramatic decrease in affinity and activity for taurocyamine while Y87E has completely lost catalytic activity. Substituting H and I on the said positions also resulted in significant changes in activity. Mutation of the residues A59 on the GS region of domain 1 also caused significant decrease in affinity and activity while mutation on the equivalent position on domain 2 resulted in complete loss of activity.

Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

BACKGROUND: Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK) constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK) of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK) gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. CONCLUSION: CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart, creatine.