Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients’ fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.
Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.
The crystal structure of a putative dipeptidase (Phdpd) from Pyrococcus horikoshii OT3 was solved using X-ray data at 2.4 Å resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of the α/β fold, and the C-terminal domain consists of five long mixed strands, four helices, and two 310 helices. The structure of Phdpd is quite similar to reported structures of prolidases from P. furiosus (Zn-Pfprol) and P. horikoshii (Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However, Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) of Phdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those of Phdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein with Zn2+ ions is inactive.
Insecticide and nerve agent organophosphorus compounds are potent inhibitors of the serine hydrolase superfamily of enzymes. Nerve agents, such as sarin, soman, tabun and VX exert their toxicity by inhibiting human acetycholinesterase at nerve synapses. Following the initial phosphonylation of the active site serine, the enzyme may reactivate spontaneously or through reaction with an appropriate nucleophilic oxime. Alternatively, the enzyme-nerve agent complex can undergo a secondary process, called “aging”, which dealkylates the nerve agent adduct and results in a product that is highly resistant to reactivation by any known means. Here we report the structures of paraoxon, soman and sarin complexes of group-VIII phospholipase A2 from bovine brain. In each case, the crystal structures indicate a non-aged adduct; a stereoselective preference for binding of the PSCS isomer of soman and the PS isomer of sarin was also noted. The stability of the non-aged complexes was corroborated by trypsin digest and electrospray ionization mass spectrometry, which indicates non-aged complexes are formed with diisopropylfluorophosphate, soman and sarin. The PS stereoselectivity for reaction with sarin was confirmed by reaction of racemic sarin, followed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate each stereoisomer. The PS stereoisomers of soman and sarin are known to be the more toxic stereoisomers, as they react preferentially to inhibit human acetylcholinesterase. The results obtained for non-aged complexes of group-VIII phospholipase A2 are compared to those obtained for other serine hydrolases and discussed to partly explain determinants of OP aging. Furthermore, structural insights can now be exploited to engineer variant versions of this enzyme with enhanced nerve agent binding and hydrolysis functions.
type-Ib PAF-AH; LIS1; group-VIII PLA2; organophosphorus; nerve agent; paraoxon; sarin; soman; non-aged; unaged; aging
Diisopropyl fluorophosphatase (DFPase) effectively hydrolyzes a number of organophosphorus nerve agents, including sarin, cyclohexylsarin, soman and tabun. Neutron diffraction data have been collected from DFPase crystals to 2.2 Å resolution in an effort to gain further insight into the mechanism of this enzyme.
The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 Å resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.
neutron diffraction; DFPase; time-of-flight; phosphotriesterase
We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the kcat/Km value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108°C that increases to 112°C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-Å resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.
Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.
The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of PR and PS stereoisomers at the P-chiral center. The tabun complex displayed only the PR stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents.
phospholipase A2; Lp-PLA2; PAF-AH; organophosphate; nerve agent
The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 Å resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 104-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.
Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme.
Three paralogues of a deblocking aminopeptidase from the hyperthermophilic archeon P. horikoshii have been overexpressed in E. coli, purified and crystallized and exist in different oligomeric states.
The deblocking aminopeptidase (DAP) of Pyrococcus horikoshii is a hyperthermophilic exoprotease that cleaves the N-terminal amino acid of peptide substrates with a putative deblocking activity for acylated peptides. DAP has been found to be homologous to a tetrahedral aminopeptidase from the halophilic Haloarcula marismortui. The latter enzyme is a dodecameric complex and has been revealed to be a self-compartmentalized protease whose central cavity harbouring the catalytic site is accessible through several channels of different size, unlike all other known proteolytic complexes. Three paralogues of DAP have been identified in P. horikoshii, with about 40% identity between them. Each of them has been overexpressed in Escherichia coli, purified and crystallized in the native and selenomethionine-substituted states. The results indicate that they form two kinds of assemblies, of 12 and of 24 subunits, with a molecular weight of ∼400 and ∼800 kDa, respectively. Crystals of the different variants of DAP and in their different oligomeric states diffract up to a resolution of 3 Å.
The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed β-1,4 but not β-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only β-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage β-glucans such as barley β-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (−I, −II, and −III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100°C and 6.0, respectively; a half-life of 40 h at 95°C; and a denaturing temperature of 112°C as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.
Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcus furiosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca2+, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca2+ also affected substrate binding, as evidenced by a decrease in Km for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.
The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100°C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.
2-O-(beta)-Mannosylglycerate, a solute that accumulates in some (hyper)thermophilic organisms, was purified from Pyrococcus furiosus cells, and its effect on enzyme stabilization in vitro was assessed. Enzymes from hyperthermophilic, thermophilic, and mesophilic sources were examined. The thermostabilities of alcohol dehydrogenases from P. furiosus and Bacillus stearothermophilus and of glutamate dehydrogenases from Thermotoga maritima and Clostridium difficile were improved to a significant extent when enzyme solutions were incubated at supraoptimal temperatures in the presence of 2-O-(beta)-mannosylglycerate, but no effect on the thermostability of glutamate dehydrogenase from P. furiosus was detected. On the other hand, there was a remarkable effect on the thermal stabilities of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and bovine liver glutamate dehydrogenase, which were used as model systems to evaluate stabilization of enzymes of mesophilic origin. For all of the enzymes examined and at the highest temperatures tested, 2-O-(beta)-mannosylglycerate was a better thermoprotectant than trehalose. The stabilizing effect exerted by 2-O-(beta)-mannosylglycerate on enzymes suggests a role for this compound as a protein thermostabilizer under physiological conditions. 2-O-(beta)-Mannosylglycerate was also effective in the protection of enzymes against stress imposed by freeze-drying, with its protecting effect being similar to or better than that exerted by trehalose. The data show 2-O-(beta)-mannosylglycerate to be a potential enzyme stabilizer in biotechnological applications.
Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD+ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes autoadenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD+ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90°C and nick-joining activity at 70 to 90°C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP.
Prolidase deficiency (PD) is a rare recessive disorder resulting from mutations in the prolidase gene (PEPD); only 17 causative mutant alleles had been so far characterized. Prolidase is a ubiquitous enzyme that hydrolyses dipeptides with C-terminal proline or hydroxyproline residues and indeed, lack of this enzyme activity causes massive urine excretion of undigested iminodipeptides. The clinical manifestations of PD are widely variable, and include intractable skin ulcers, unusual face, different degree of mental retardation, and recurrent infections. No definitive treatment is at present available.
We report an 8-year girl with a typical PD facies, normal intelligence, and recurrent deep ulcerations complicated by infections. She was found to be compound heterozygous for two novel mutations in PEPD, c.1133delACG and c.1301delT, affecting the C-terminal end of the enzyme where the active site is located. Given her life-threatening course, she underwent allogeneic hematopoietic stem cell transplantation (HSCT) from her HLA-identical brother, confirmed heterozygous for the c.1133delACG allele. Successful engraftment was documented by full-donor chimerism. Posttransplant monitoring of erythrocyte prolidase activity showed that the child had converted to a heterozygous pattern. Reduction of excreted urine dipeptides, evaluated by capillary electrophoresis, supported the effectiveness of the treatment. Unfortunately the patient died on day +92 of invasive fungal infection.
Despite the unfavorable outcome, we provide the first evidence that HSCT has the potential to reverse some of the biochemical features of PD patients. The indication to transplant must be balanced against the clinical manifestation of individual patients.
Electronic supplementary material The online version of this article (doi: 10.1007/8904_2011_62) contains supplementary material, which is available to authorized users.
Many organophosphorus (OP) based compounds are highly toxic and powerful inhibitors of cholinesterases that generate serious environmental and human health concerns. Organothiophosphates with a thiophosphoryl (P=S) functional group constitute a broad class of these widely used pesticides. They are related to the more reactive phosphoryl (P=O) organophosphates, which include very lethal nerve agents and chemical warfare agents, such as, VX, Soman and Sarin. Unfortunately, widespread and frequent commercial use of OP-based compounds in agricultural lands has resulted in their presence as residues in crops, livestock, and poultry products and also led to their migration into aquifers. Thus, the design of new sensors with improved analyte selectivity and sensitivity is of paramount importance in this area. Herein, we review recent advances in the development of fluorescent chemosensors for toxic OP pesticides and related compounds. We also discuss challenges and progress towards the design of future chemosensors with dual modes for signal transduction.
organophosphorus compounds; fluorescent chemosensors; pollutants; dual modes of signal transduction
Human butyrylcholinesterase (BChE) is recognized as the most promising bioscavenger for organophosphorus (OP) warfare nerve agents. The G117H mutant of human BChE has been identified as a potential catalytic bioscavenger with a remarkably improved activity against OP nerve agents such as sarin, but it still does not satisfy the clinical use. For further design of the higher-activity mutants against OP nerve agents, it is essential to understand how the G117H mutation improves the activity. The reaction mechanisms and the free energy profiles for spontaneous reactivation of the wild-type BChE and its G117H mutant phosphorylated by sarin have been explored, in this study, by performing first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations, and the remarkable role of the G117H mutation on the activity has been elucidated. For both the wild-type and G117H mutant enzymes, H438 acts as a general base to initiate the spontaneous reactivation which consists of two reaction steps: the nucleophilic attack at the phosphorus by a water molecule and decomposition of the pentacoordinated phosphorus intermediate. The calculated overall free energy barriers, i.e. 30.2 and 23.9 kcal/mol for the wild-type and G117H mutant, respectively, are in good agreement with available experimental kinetic data. Based on the calculated results, the mutated residue (H117 in the G117H mutant) cannot initiate the spontaneous reactivation as a general base. Instead, it skews the oxyanion hole and makes the phosphorus more open to the nucleophilic water molecule, resulting in remarkable change of the rate-determining step and significantly improved catalytic activity of human BChE.
l-Threonine dehydrogenase from the hyperthermophilic archaeon P. horikoshii was crystallized and preliminary X-ray crystallographic analysis was carried out.
Recombinant l-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii was prepared using an Escherichia coli expression system. The hyperthermostable l-threonine dehydrogenase consists of 348 amino acids with a molecular weight of 37.7 kDa. The enzyme was crystallized by the hanging-drop vapour-diffusion method at 277 K and preliminary X-ray crystallographic analysis was carried out. Diffraction data were collected to 2.20 Å resolution under cryogenic conditions. P. horikoshii
l-threonine dehydrogenase crystals belong to space group I4122, with unit-cell parameters a = b = 143.84, c = 304.13 Å. The presence of three subunits of the enzyme per asymmetric unit was estimsted to give a Matthews coefficient (V
M) of 3.5 Å3 Da−1 and a solvent content of 64.7%(v/v).
archaea; Pyrococcus horikoshii; hyperthermostability; threonine dehydrogenase
The enzyme acetylcholinesterase (AChE) is an important part of cholinergic nervous system, where it stops neurotransmission by hydrolysis of the neurotransmitter acetylcholine. It is sensitive to inhibition by organophosphate and carbamate insecticides, some Alzheimer disease drugs, secondary metabolites such as aflatoxins and nerve agents used in chemical warfare. When immobilized on a sensor (physico-chemical transducer), it can be used for assay of these inhibitors. In the experiments described herein, an AChE- based electrochemical biosensor using screen printed electrode systems was prepared. The biosensor was used for assay of nerve agents such as sarin, soman, tabun and VX. The limits of detection achieved in a measuring protocol lasting ten minutes were 7.41 × 10−12 mol/L for sarin, 6.31 × 10−12 mol/L for soman, 6.17 × 10−11 mol/L for tabun, and 2.19 × 10−11 mol/L for VX, respectively. The assay was reliable, with minor interferences caused by the organic solvents ethanol, methanol, isopropanol and acetonitrile. Isopropanol was chosen as suitable medium for processing lipophilic samples.
biosensor; acetylcholinesterase; sarin; tabun; soman; VX; inhibitor; screen printed electrode; voltammetry; amperometry
According to the structural and biochemical analysis of the stomatin-specific protease 1510-N, two degraded products were produced via acyl-enzyme intermediates. The N-terminal half of the substrate peptide binds to 1510-N more tightly than the C-terminal half of the peptide.
Membrane-bound proteases are involved in various regulatory functions. The N-terminal region of PH1510p (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser–Lys dyad (Ser97 and Lys138), and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511p. In a form of human hemolytic anemia known as hereditary stomatocytosis, the stomatin protein is deficient in the erythrocyte membrane due to mis-trafficking. In order to understand the catalytic mechanism of 1510-N in more detail, here the structural and biochemical analysis of 1510-N is reported. Two degraded products were produced via acyl-enzyme intermediates. 1510-N is a thermostable protease, and thus crystallization after heat treatment of the protease–peptide complex was attempted in order to understand the catalytic mechanism of 1510-N. The structure after heat treatment is almost identical to that with no heat treatment. According to the superposition between the structures with heat treatment and with no heat treatment, the N-terminal half of the peptide is superposed well, whereas the C-terminal half of the peptide is slightly deviated. The N-terminal half of the peptide binds to 1510-N more tightly than the C-terminal half of the peptide. The flexible L2 loops of 1510-N cover the peptide, and are involved in the protease activity.
membrane-bound protease; stomatin; thermostable; substrate peptide; Pyrococcus horikoshii
Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 18.104.22.168) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 22.214.171.124), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50°C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.
A gene encoding for a putative Family inorganic pyrophosphatase
(PPase, EC 126.96.36.199) from the hyperthermophilic archaeon
Pyrococcus horikoshii OT3 was cloned and the
biochemical characteristics of the resulting recombinant protein were
examined. The gene (Accession No. 1907) from P. horikoshii
showed some identity with other Family I inorganic
pyrophosphatases from archaea. The recombinant PPase from P.
horikoshii (PhPPase) has a molecular mass of
24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed
the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum
temperature and pH for PPase activity were 70 °C and 7.5,
respectively. The half-life of heat inactivation was about 50 min at
105 °C. The heat stability of PhPPase was
enhanced in the presence of Mg2+. A divalent cation was
absolutely required for enzyme activity, Mg2+ being most
effective; Zn2+, Co2+ and Mn2+
efficiently supported hydrolytic activity in a narrow range of
concentrations (0.05– 0.5 mM). The Km for
pyrophosphate and Mg2+ were 113 and 303 µM,
respectively; and maximum velocity, Vmax,
was estimated at 930 U mg–1.
This study was designed to test the hypothesis that the acute toxicity of the nerve agents VX, soman and sarin in guinea pigs is age and sex dependent and cannot be fully accounted for by the irreversible inhibition of acetylcholinesterase (AChE). The subcutaneous doses of nerve agents needed to decrease 24-h survival of guinea pigs by 50% (LD50s) were estimated by probit analysis. In all animal groups, the rank order of LD50s was sarin > soman > VX. The LD50 of soman was not influenced by sex or age of the animals. In contrast, the LD50s of VX and sarin were lower in adult male than in age-matched female or younger guinea pigs. A colorimetric assay was used to determine the concentrations of nerve agents that inhibit in vitro 50% of AChE activity (IC50s) in guinea pig brain extracts, plasma, red blood cell and whole blood. A positive correlation between LD50s and IC50s for AChE inhibition would support the hypothesis that AChE inhibition is a major determinant of the acute toxicity of the nerve agents. However, such a positive correlation was found only between LD50s and IC50s for AChE inhibition in brain extracts from neonatal and prepubertal guinea pigs. These results demonstrate for the first time that the lethal potencies of some nerve agents in guinea pigs are age and sex dependent. They also support the contention that mechanisms other than AChE inhibition contribute to the lethality of nerve agents.