Cloning and expression of Helicobacter pylori GDP-l-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae.

Helicobacter pylori is a Gram-negative gastric pathogen causing diseases from mild gastric infections to gastric cancer. The difference in clinical outcome has been suggested to be due to strain differences. H. pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions. The O-antigen of H. pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium. These Lewis glycans of H. pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium. In the synthesis of fucosylated structures, GDP-l-fucose is needed as a fucose donor. Here, we cloned the two key enzymes of GDP-l-fucose synthesis, H. pylori gmd coding for GDP-d-mannose dehydratase (GMD), and gmer coding for GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae. The end product of these enzymes, GDP-l-fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl-N-acetyllactosamine to sialyl Lewis X.     

Enzymatic characterization and inhibitor discovery of a new cystathionine {gamma}-synthase from Helicobacter pylori

Cystathionine gamma-synthase (CGS) catalyses the first step of the transsulfuration pathway that converts l-cysteine to l-homocysteine in bacteria, whereas this pathway is absent in human. In this report, we identified a new metB gene from Helicobacter pylori strain SS1, and the recombinant H. pylori Cystathionine gamma-synthase (HpCGS) was successfully cloned, expressed and purified in Escherichia coli system. Enzymatic study of HpCGS indicated that the K(m) and k(cat)/K(m) values against the substrate O-succinyl-l-homoserine (l-OSHS) were 3.02 mM and 98.7 M(-)(1)s(-)(1), respectively. Moreover, four natural products (alpha-lapachone, 9-hydroxy-alpha-lapachone, Paulownin and Yangambin, Fig. 1) were discovered to demonstrate inhibitory activities against HpCGS with IC(50) values of 11 +/- 3, 9 +/- 1, 19 +/- 2 and 27 +/- 6 microM, respectively. All these four inhibitors prevent the binding of l-OSHS to HpCGS in a non-competitive fashion. In vitro antibacterial assays further indicated that these four discovered compounds could highly inhibit the growth of H. pylori and exhibited strong inhibitory specificity against H. pylori related to E. coli.     

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.     

Characterization of a GDP-D-mannose 3',5'-epimerase from rice

The enzymatic characterization of GDP-d-mannose 3',5'-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD(+) in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD(+) on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K(m) and k(cat) were determined to be 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM NAD(+). The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.     

Molecular cloning and characterization of a new peptide deformylase from human pathogenic bacterium Helicobacter pylori

Helicobacter pylori is a gram-negative pathogenic bacterium, which is associated with peptic ulcer disease and gastric cancer. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. In bacteria, peptide deformylase (PDF) catalyzes the removal of a formyl group from the N-termini of nascent polypeptides. Due to its essentiality and absence in mammalian cells, PDF has been considered as an attractive target for the discovery of novel antibiotics. In this work, a new PDF gene (def) from H. pylori strain SS1 was cloned, expressed, and purified in Escherichia coli system. Sequence alignment shows that H. pylori PDF (HpPDF) shares about 40% identity to E. coli PDF (EcPDF). The enzymatic properties of HpPDF demonstrate its relatively high activity toward formyl-Met-Ala-Ser, with K(cat) of 3.4s(-1), K(m) of 1.7 mM, and K(cat) / K(m) of 2000M(-1)s(-1). HpPDF enzyme appears to be fully active at pH between 8.0 and 9.0, and temperature 50 degrees C. The enzyme activity of Co(2+)-containing HpPDF is apparently higher than that of Zn(2+)-containing HpPDF. This present work thereby supplies a potential platform that facilitates the discovery of novel HpPDF inhibitors and further of possible antimicrobial agents against H. pylori.     

WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase

WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the K(m)(NADH) was 14 +/- 0.43 microM and the K(m)(benzoquinone) was 5.8 +/- 0.12 microM. For A. fulgidus WrbA, the K(m)(NADH) was 19 +/- 1.7 microM and the K(m)(benzoquinone) was 37 +/- 3.6 microM. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress.     

Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint

Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes. Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay. cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described. The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409. The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone. The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914. This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1). Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity. (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.     

Biochemical characterization and inhibitor discovery of shikimate dehydrogenase from Helicobacter pylori

Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway. It catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate, and has been developed as a promising target for the discovery of antimicrobial agent. In this report, we identified a new aroE gene encoding SDH from Helicobacter pylori strain SS1. The recombinant H. pylori shikimate dehydrogenase (HpSDH) was cloned, expressed, and purified in Escherichia coli system. The enzymatic characterization of HpSDH demonstrates its activity with k(cat) of 7.7 s(-1) and K(m) of 0.148 mm toward shikimate, k(cat) of 7.1 s(-1) and K(m) of 0.182 mm toward NADP, k(cat) of 5.2 s(-1) and K(m) of 2.9 mm toward NAD. The optimum pH of the enzyme activity is between 8.0 and 9.0, and the optimum temperature is around 60 degrees C. Using high throughput screening against our laboratory chemical library, five compounds, curcumin (1), 3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl 3-chlorobenzoate (2), butyl 2-{[3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl]oxy}propanoate (3), 2-({2-[(2-{[2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl}-1,3-benzothiazol-6-yl)amino]-2-oxoethyl}sulfanyl)-N-(2-naphthyl)acetamide (4), and maesaquinone diacetate (5) were discovered as HpSDH inhibitors with IC(50) values of 15.4, 3.9, 13.4, 2.9, and 3.5 microm, respectively. Further investigation indicates that compounds 1, 2, 3, and 5 demonstrate noncompetitive inhibition pattern, and compound 4 displays competitive inhibition pattern with respect to shikimate. Compounds 1, 4, and 5 display noncompetitive inhibition mode, and compounds 2 and 3 show competitive inhibition mode with respect to NADP. Antibacterial assays demonstrate that compounds 1, 2, and 5 can inhibit the growth of H. pylori with MIC of 16, 16, and 32 microg.mL(-1), respectively. This current work is expected to favor better understanding the features of SDH and provide useful information for the development of novel antibiotics to treat H. pylori-associated infection.     

Characterization of d-lactate dehydrogenase from Pediococcus acidilactici that converts phenylpyruvic acid into phenyllactic acid

The gene coding for D-lactate dehydrogenase (D-LDH) from Pediococcus acidilactici DSM 20284 was cloned and expressed in E. coli. The recombinant enzyme was purified by nickel-affinity chromatography. It converted phenylpyruvic acid (PPA) to 3-phenyllactic acid maximally at 30°C and pH 5.5 with a specific activity of 140 and 422 U/mg for PPA and pyruvate, respectively. The K(m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) for PPA were 2.9 mM, 305 s(-1), and 105 mM(-1) s(-1), respectively.     

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily.

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l-arginine to l-ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity. A translationally fused His(6)-RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase-containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His(6)-RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His(6)-RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.     

Purification and some characteristics of a recombinant dimeric rhizobium meliloti beta-galactosidase expressed in escherichia coli

A recombinant Rhizobium meliloti beta-galactosidase was purified to homogeneity from an Escherichia coli expression system. The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E. coli JM109 cells. The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively). The purified enzyme had an apparent molecular mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer. It was active with both synthetic substrates p-nitrophenyl beta-D-galactopyranoside (PNPG) and o-nitrophenyl beta-D-galactopyranoside (ONPG) with K(m)(PNPG) and K(m)(ONPG) of 1 mM at 25 degrees C. The k(cat)/K(m) ratios for both substrates were approximately 70 mM(-1) sec(-1), indicating no clear preference for either PNPG or ONPG, unlike E. coli beta-galactosidase. After non-denaturing electrophoresis, active beta-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl beta-D-galactopyranoside (BNG) and diazo blue B.     

Over-expression, purification, and characterization of aminopeptidase N from Escherichia coli

The gene from Escherichia coli encoding aminopeptidase N (PepN) was subcloned into pET-26b, and PepN was over-expressed in BL21(DE3) E. coli and purified using Q-Sepharose chromatography. This protocol yielded over 17 mg of purified, recombinant PepN per liter of growth culture under optimum conditions. Gel filtration chromatography revealed that recombinant PepN exists as a monomer. MALDI-TOF mass spectra showed that the enzyme has a molecular mass of 98,750 Da, and steady-state kinetic studies revealed that as-isolated, recombinant PepN exhibits a k(cat) of 354 +/- 11s(-1) and a K(m) of 376 +/- 39 microM when using L-alanine-p-nitroanilide as the substrate. Metal analyses demonstrated that as-isolated, recombinant PepN binds 0.5 and <0.1 equivalents of iron and zinc, respectively. The addition of Zn(II) to recombinant PepN inhibits catalytic activity, while the addition of iron causes a slight decrease or no change in activity. Further metal binding studies revealed that recombinant PepN tightly binds 5 equivalents of iron and <0.1 equivalents of Zn(II). By using this over-expression and purification system, E. coli PepN can now be obtained in quantities necessary for structural characterization and possibly inhibitor design efforts.     

Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) from green bamboo was isolated and cloned from the shell of Bambusa oldhamii. The K(m) of bamboo shell PAL for L-Phe was 476 μM, and the molecular mass of native PAL was estimated as 275 kDa and the molecular mass of a subunit was about 76 kDa, indicating that PAL from bamboo also exists as a tetramer. The optimum temperature for PAL activity was 50°C and the optimal pH 9.0. The identity of the purified bamboo shell PAL was confirmed using Q-TOF tandem MS/MS de novo sequencing. Four PAL genes, designated as BoPAL1 to BoPAL4, were cloned from B. oldhamii. The open reading frames of BoPAL3 and BoPAL4 were 2142 and 2106 bp in size, respectively: BoPAL2-4 contained one intron and two exons, but no intron was found in BoPAL1. BoPAL4 expressed in Escherichia coli possessed both PAL and tyrosine ammonia-lyase activities. While recombinant wild-type PAL proteins had similar biochemical properties to the native bamboo shell PAL, both site-directed mutagenesis of BoPAL1 F133H and BoPAL2 F134H, respectively, showed decreased k(cat)/K(m) values toward L-Phe, whereas BoPAL2 F134H showed a slightly increased k(cat)/K(m) value toward L-Tyr. These data suggest other residues largely control Phe/Tyr substrate specificity. An antibody raised against the purified shell PAL was generated for histochemical studies. In bamboo shell and branch shoots, PAL was localized primarily in sclerenchyma cells.     

Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96

A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P(mxaF)), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S. tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.     

Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.     

Characteristics of a new enantioselective thermostable dipeptidase from Brevibacillus borstelensis BCS-1 and its application to synthesis of a D-amino-acid-containing dipeptide

A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the D-Glu auxotroph Escherichia coli WM335 on a plate containing D-Ala-D-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M(r) of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P(1) and P(1)' site of Ala-Ala revealed that the ratio of the specificity constant (k(cat)/K(m)) for L-enantioselectivity to the P(1) site of Ala-Ala was 23.4 +/- 2.2 [E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(D,D)], while the D-enantioselectivity to the P(1)' site of Ala-Ala was 16.4 +/- 0.5 [E = (k(cat)/K(m))(L,D)/(k(cat)/K(m))(L,L)] at 55 degrees C. The enzyme was stable up to 55 degrees C, and the optimal pH and temperature were 8.5 and 65 degrees C, respectively. The enzyme was able to hydrolyze L-Asp-D-Ala, L-Asp-D-AlaOMe, Z-D-Ala-D-AlaOBzl, and Z-L-Asp-D-AlaOBzl, yet it could not hydrolyze D-Ala-L-Asp, D-Ala-L-Ala, D-AlaNH(2), and L-AlaNH(2.) The enzyme also exhibited beta-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-L-Asp-D-AlaOBzl.     

Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina

Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k_m values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi.     

Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. Using p-nitrophenyl-β-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.     

Properties of the alpha subunit of a Chaperonin from the hyperthermophilic Crenarchaeon Aeropyrum pernix K1

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.