Calpain II-like proteinase of scallop (Patinopecten yessoensis) striated adductor muscle.

1. A Ca(2+)-dependent cysteine proteinase was purified from scallop striated adductor muscle by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephacryl S-300. 2. The enzyme is of Mr approximately 200,000, composed of two Mr 100,000 subunits. 3. The enzyme is a cysteine proteinase with optimum activity at pH 6.8 and about 18 degrees C. In addition, it requires 1.7 mM Ca2+ for half-maximal activity and more than 10 mM Ca2+ for maximal activity. Thus the enzyme can be classified as calpain II.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Purification and characterization of hepatic lipase from Todarodes pacificus

Lipase was purified from squid (Todarodes pacificus) liver in an attempt to investigate the possibility of applying the enzyme for biotechnological applications. Crude extract of squid liver was initially fractionated by the batch type ion exchange chromatography. The fraction containing lipase activity was further purified with an octyl-Sepharose column. Finally, lipase was purified by eluting active protein from a non-dissociating polyacrylamide gel after zymographic analysis. Molecular weight of the purified enzyme was determined to be 27 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at a temperature range of 35-40 degrees C and at pH 8.0. The activity was almost completely inhibited at 1 mM concentration of Hg(2+) or Cu(2+) ion. Partial amino acid sequence of the enzyme was also determined.     

Two distinct poly(A) polymerases isolated from the cytoplasm of Ehrlich ascites tumour cells

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE-cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+-dependent poly(A) polymerase (ATP:RNA adenylyltransferase) and (b) a ribosome-associated enzyme defined tentatively as ATP(UTP): RNA nucleotidyltransferase. The cytosol poly(A) polymerase is strictly Mn2+-dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin. The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10--20 mM Mg2+. The enzymes uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.     

New effective sources of the Staphylococcus simulans lysostaphin

The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.     

依赖于DNA的RNA聚合酶与体外转录的研究 Ⅰ.酵母变异株20B-12 RNA聚合酶A和C的纯化及鉴定

从酵母变异株20B-12经过超声波处理、硫酸铵沉淀、DEAE纤维素和磷酸纤维素层析等步骤,纯化依赖于DNA的RNA聚合酶A和C,得到聚丙烯酰胺凝胶电泳均一的条带。其中不含DNase、RNase 和蛋白酶活力,无内源DNA。测定了 RNA 聚合酶A和C对α-鹅膏荤碱的敏感性。酶A在α-鹅膏荤碱为400μg/ml时,活性受到抑制,而酶C在该浓度时,几乎不受抑制。(NH_4)_2SO_4对酶A的最适浓度为20mM,对酶C有二个最适浓度,分别为40mM和240mM。无二价金属离子Mn~(2+)或Mg~(2+),酶A和C几乎无活力。两种酶最适Mn~(2+)浓度均为2.5mM,Mg~(2+)浓度均为5mM。两种酶以热变性小牛胸腺DNA为模板,测活性均较天然小牛胸腺DNA为模板时高。     

Purification and properties of a DNA ligase from sea urchin embryos

DNA ligase was purified about 2,000-fold from blastulae of sea urchin, Hemicentrotus pulcherrimus, by means of 1 M KCl-extraction, phosphocellulose, DEAE-cellulose, Sepharose CL-6B, and double-stranded DNA cellulose column chromatography. The purified DNA ligase had a molecular weight of 80,000 (determined by Sephadex G-150) and a sedimentation coefficient of 4.1S (by glycerol gradient centrifugation). The purified enzyme required ATP and Mg2+ (or Mn2+) as cofactors for activity, and was inhibited by N-ethylmaleimide. Apparent Km values for ATP, Mg2+, and Mn2+ were 4 microM, 2.7 mM, and 0.3 mM, respectively.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.     

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: vanadate nad a dithioerythritol-dependent inhibitor.

A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. The inhibition is half maximally reversed by 250 micrometer epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 micrometer epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.     

Purification and Characterization of Extracellular Phytase from a Marine Yeast <Emphasis Type="Italic">Kodamaea ohmeri</Emphasis> BG3

The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65°C, respectively. The enzyme was stimulated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺ and Co²⁺ (at a concentrations of 5.0 mM), but it was inhibited by Cu²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Zn²⁺ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline (at concentrations of 1.0 mM and 5.0 mM). The K _m, V _max, and K _cat values of the purified enzyme for phytate were 1.45 mM, 0.083 μmol/ml · min, and 0.93 s^-1, respectively.     

Purification and characterization of the sesquiterpene cyclase trichodiene synthetase from Fusarium sporotrichioides

The sesquiterpene cyclase, trichodiene synthetase, has been purified from a supernatant fraction of Fusarium sporotrichioides by hydrophobic interaction, anion exchange, and gel filtration chromatography. Purified enzyme had a specific activity 15-fold higher than that previously reported for preparations of terpene cyclases. Molecular weight determinations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography indicated the enzyme to be a dimer with a subunit of Mr 45,000. The requirement of Mg2+ (Km 0.1 mM) for activity could be partially substituted with Mn2+ at a concentration of 0.01 mM, but higher concentrations of Mn2+ were inhibitory. Maximum activity was observed between pH 6.75 and pH 7.75. The Km for farnesyl pyrophosphate was 0.065 microM.     

Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate

A novel extra-cellular lipase from Bacillus coagulans MTCC-6375 was purified 76.4-fold by DEAE anion exchange and Octyl Sepharose chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 103 kDa. The lipase was optimally active at 45 degrees C and retained approximately 50% of its original activity after 20 min of incubation at 55 degrees C. The enzyme was optimally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+, and Fe3+ at 1mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions resulted in a maximal increase in lipase activity but Zn2+ and Co2+ ions showed an antagonistic effect on this enzyme. EDTA at 150 mM concentration inhibited the activity of lipase but Hg2+ or Al3+ (10mM) restored most of the activity of EDTA-quenched lipase. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was more specific to p-nitrophenyl esters of 8 (pNPC) and 16 (pNPP) carbon chain length esters. The lipase had a Vmax and Km of 0.44 mmol mg(-1)min(-1) and 28 mM for hydrolysis of pNPP, and 0.7 mmol mg(-1)min(-1) and 32 mM for hydrolysis of pNPC, respectively.     

Human serum biotinidase is a thiol-type enzyme

The effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively studied by using a newly developed HPLC assay method. This HPLC method directly measures the product (p-aminobenzoate, PAB), and is not interfered with by sulfhydryl-reactive agents. Further, because the substrate solution of this HPLC assay method contains only substrate (biotin 4-amidobenzoate) and phosphate buffer, accurate studies on the effects of sulfhydryl blocking reagents on the purified enzyme could be performed. Biotinidase activities in human sera (n = 83) were always enhanced by 2-mercaptoethanol (ME). The optimum concentration was found to be 1 mM. The degree of activation was variable (100 to 400% of the original) depending on the serum sample. Sulfhydryl blocking reagents such as organic mercurials were tested on fresh serum and purified enzyme. Mercuric agents were found to inhibit the activity of fresh serum and purified enzyme at 0.05 and 0.005 mM, respectively. Sulfhydryl alkylating agents, N-ethylmaleimide (NEM) and dithiobis(2-nitro)benzoic acid (DTNB), inhibited 100 and 64% of the activity of the purified enzyme at 0.1 and 1.0 mM, respectively. However, lower concentrations (less than 5 nM) of organic mercurials and mercuric ion exhibited a slight enhancement (20-30%) of the activity of the purified enzyme. These results indicate the presence of an essential sulfhydryl residue at the active center. The enzyme contains 2.5 sulfhydryls per molecule, as determined by using Ellman's assay method. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) did not inhibit the enzyme activity at 0.05 mM or higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.     

The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action

Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.     

Purification and some properties of an alkaline proteinase from rat skeletal muscle.

1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.     

Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.     

Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.