SEARCH · PubMed Health

Results for “FLAVOBACTERIUM”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources

PubMed1998

New pathway of heparan sulphate degradation. Iduronate sulphatase and N-sulphoglucosamine 6-sulphatase act on the polymer chain prior to depolymerisation by a N-sulpho-glucosaminidase and glycuronidases in the mollusc Tagelus gibbus.

It has previously been shown that in the mollusc Anomalocardia brasiliana the desulphation of chondroitin sulphate precedes its depolymerisation by beta-glucuronidase and beta-N-acetylgalactosaminidase (Sousa Jr. et al. J. Biol. Chem. 1990;265:20150-20155). This led us to investigate whether in molluscs, sulphatases also act on heparan sulphate before its depolymerisation by glycosidases. Radioactively labelled [35S]heparan sulphate was extensively degraded by enzyme extracts prepared from the mollusc Tagelus gibbus. Several enzymes acting in concert degrade the compound to inorganic sulphate, glucosamine N-sulphate, N-acetylglucosamine-6 sulphate and other oligosaccharide products. These results indicate the presence of iduronate sulphatase, N-sulphoglucosamine 6-sulphatase alpha-N-sulphoglucosaminidase, beta-glucuronidase and alpha-L-iduronidase. The di- and mono-saccharide composition of the oligosaccharides were analysed with the aid of heparitinase II from Flavobacterium heparinum. These analyses led to the characterisation of two sulphatases that act on the polymer chain removing sulphates from the C-2 position of iduronic acid residues and the C-6 position of the glucosamine moieties, respectively. The different enzymes were partially fractionated by ion exchange chromatography and molecular sieving. These results led to the proposition of a new pathway of degradation of heparan sulphate where sulphatases act directly on the polymer chain which is then depolymerised by several glycosidases.

Acetylglucosaminidase↗

PubMed1992

A partial phylogenetic analysis of the "flavobacter-bacteroides" phylum: basis for taxonomic restructuring.

On the basis of small subunit rRNA sequence analyses five major subgroups within the flavobacteria-bacteroides phylum have been defined. These are tentatively designated the cytophaga subgroup (comprising largely Cytophaga species), the flavobacter subgroup (comprising the true flavobacteria and the polyphyletic genus Weeksella), the bacteroides subgroup (comprising the bacteroides and certain cytophaga-like bacteria), the sphingobacter subgroup (which contains the known sphingolipid-producing members of the phylum), and the saprospira subgroup (comprising particular species of Flexibacter, Flavobacterium, Haliscomenobacter, and, of course, the genus Saprospira). These groupings are given not only by evolutionary distance analysis, but can be defined and distinguished on the basis of a simple small subunit rRNA signatures.

Bacteroides↗

PubMed2001

Diffuse lamellar keratitis: isolation of endotoxin and demonstration of the inflammatory potential in a rabbit laser in situ keratomileusis model.

PURPOSE: To systematically examine sources of endotoxin contamination in eye centers as a potential cause of diffuse lamellar keratitis (DLK) and to demonstrate the inflammatory potential of endotoxin in a rabbit model of laser in situ keratomileusis (LASIK) surgery. SETTING: University of Calgary, Calgary, Alberta, Canada. METHODS: In this prospective study, all water sources that routinely come in contact with LASIK instruments, including sterilizer reservoirs, eyedrops, microkeratome blades, and cleaning solutions, were examined for endotoxins at 5 eye centers. Bacterial cultures were performed on water samples from 5 sterilizer reservoirs. A LASIK flap was created in 8 rabbit eyes using an Automated Corneal Shaper microkeratome (Bausch & Lomb). The flaps were reflected, and a dose of endotoxin at various concentrations was placed on the interface. After 1 minute, the flap was irrigated and repositioned. The rabbit eyes were examined daily with a slitlamp biomicroscope for 3 days for the development of DLK, which was classified on a scale from grade 1 to 4 (mild to severe). The rabbits were killed at the conclusion of the study, and the interfaces were stained to rule out infectious etiologies. RESULTS: Endotoxin was detected in significant concentrations in tap water, filtered and distilled water, instrument washbasins, and sterilizer reservoirs at all 5 centers. The cultures of the water samples taken from the sterilizer reservoirs ranged from no growth to the presence of >100 colony-forming units of Flavobacterium and Pseudomonas aeruginosa. Endotoxins caused DLK-like interface inflammation in all eyes tested. Examination of stained scrapings showed no microorganisms in the interface of the rabbit eyes. CONCLUSION: Endotoxin contamination was detected in water sources that routinely come in contact with LASIK instruments. Endotoxins were capable of inducing interface inflammation in a rabbit model and may therefore be a significant factor in epidemic DLK.

Animals↗

PubMed2000

Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations.

The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp. L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2. Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants. Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings. The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd. Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly. The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A. lipoferum 137. Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots. The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements.

Arthrobacter↗

PubMed1996

Isolation and characterization of a new arsenic methylating bacterium from soil.

An arsenic resistant and arsenic methylating bacterium belonging to the Flavobacterium-Cytophaga group was isolated from soil with an arsenic content of 1.5 ppm. The growth of the bacterium is enhanced in the presence of As compounds in concentrations up to 200 ppm in the cultural media with a stronger effect of As(V) than of As(III) compounds. As a volatile product of the methylation of both NaH2AsO3 and NaH2AsO4 exclusively, Me3As was formed and detected by mass spectrometry. Quantitative aspects of the methylation were studied with GC/MS. The intracellular accumulation of arsenic in the methylating strain was compared with two non methylating strains from the same soil.

Arsenic↗

PubMed2001

Active site of chondroitin AC lyase revealed by the structure of enzyme-oligosaccharide complexes and mutagenesis.

The crystal structures of Flavobacterium heparinium chondroitin AC lyase (chondroitinase AC; EC 4.2.2.5) bound to dermatan sulfate hexasaccharide (DS(hexa)), tetrasaccharide (DS(tetra)), and hyaluronic acid tetrasaccharide (HA(tetra)) have been refined at 2.0, 2.0, and 2.1 A resolution, respectively. The structure of the Tyr234Phe mutant of AC lyase bound to a chondroitin sulfate tetrasaccharide (CS(tetra)) has also been determined to 2.3 A resolution. For each of these complexes, four (DS(hexa) and CS(tetra)) or two (DS(tetra) and HA(tetra)) ordered sugars are visible in electron density maps. The lyase AC DS(hexa) and CS(tetra) complexes reveal binding at four subsites, -2, -1, +1, and +2, within a narrow and shallow protein channel. We suggest that subsites -2 and -1 together represent the substrate recognition area, +1 is the catalytic subsite and +1 and +2 together represent the product release area. The putative catalytic site is located between the substrate recognition area and the product release area, carrying out catalysis at the +1 subsite. Four residues near the catalytic site, His225, Tyr234, Arg288, and Glu371 together form a catalytic tetrad. The mutations His225Ala, Tyr234Phe, Arg288Ala, and Arg292Ala, revealed residual activity for only the Arg292Ala mutant. Structural data indicate that Arg292 is primarily involved in recognition of the N-acetyl and sulfate moieties of galactosamine, but does not participate directly in catalysis. Candidates for the general base, removing the proton attached to C-5 of the glucuronic acid at the +1 subsite, are Tyr234, which could be transiently deprotonated during catalysis, or His225. Tyrosine 234 is a candidate to protonate the leaving group. Arginine 288 likely contributes to charge neutralization and stabilization of the enolate anion intermediate during catalysis.

Amino Acid Sequence↗

PubMed1994

Crystal structure of endo-beta-N-acetylglucosaminidase F1, an alpha/beta-barrel enzyme adapted for a complex substrate.

Endo-beta-N-acetylglucosaminidase F1 (Endo F1) is an endoglycosidase, secreted by Flavobacterium meningosepticum, that cleaves asparagine-linked oligosaccharides after the first N-acetylglucosamine residue. The enzyme is selective for high-mannose oligosaccharide chains. The crystal structure of Endo F1 has been determined at 2.0-A resolution. The molecular fold consists of a highly irregular alpha/beta-barrel, a commonly observed motif consisting of a cyclic 8-fold repeat of beta-strand/loop/alpha-helix units with an eight-stranded parallel beta-barrel at the center. Endo F1 lacks two of the alpha-helices, those of units 5 and 6. Instead, the links after beta-strands 5 and 6 consist of a short turn followed by a section in an extended conformation that replaces the helix and a long loop at the bottom of the molecule. The absence of any excursion on top of the molecule following beta-strands 5 and 6 results in a pronounced depression in the rim of the barrel. This depression forms one end of a shallow cleft that runs across the surface of the molecule, over the core of the beta-barrel to the area between the loops of units 1 and 2. The active site residues, Asp130 and Glu132, are located at the carboxyl end of beta-strand 4 and extend into this cleft. These residues are surrounded by several tyrosine residues. The cleft area formed by loops 1 and 2 is lined with polar residues, mainly asparagines. The latter area is thought to be responsible for oligosaccharide binding and recognition while the protein moiety of the substrate would be located outside the molecule but adjacent to the area of loops 5 and 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1985

Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F.

Endo-beta-N-acetylglucosaminidase F (Endo F) and peptide:N-glycosidase F (PNGase F) were purified from cultures of Flavobacterium meningosepticum by ammonium sulfate precipitation followed by gel filtration on TSK HW-55(S). This system separated the two enzymes and provided PNGase F in a high state of purity, but the basis for the resolution appeared to be hydrophobic interaction and not molecular size. Studies using purified Endo F and PNGase F with defined glycopeptides demonstrated that Endo F was somewhat similar to Endo H in that it hydrolyzed many, but not all, high-mannose and hybrid oligosaccharides, as well as complex biantennary oligosaccharides. PNGase F, in contrast, hydrolyzed all classes of asparagine-linked glycans examined, provided both the alpha-amino and carboxyl groups of the asparagine residue were in peptide linkage. Deglycosylation studies with PNGase F revealed that many proteins in their native conformation were susceptible to this enzyme but that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.

Amidohydrolases↗

PubMed1985

Deglycosylation of alpha 1-proteinase inhibitor by endo-beta-N-acetylglucosaminidase F.

The glycosidase endo-beta-N-acetylglucosaminidase F (endo F) from Flavobacterium meningosepticum was used for the deglycosylation of rat alpha 1-proteinase inhibitor (alpha 1 PI). alpha 1 PI containing three oligosaccharide side chains of the complex type was isolated from rat serum or from the medium of rat hepatocyte primary cultures. High-mannose-type alpha 1 PI or hybrid-type alpha 1 PI was isolated from the media of hepatocytes treated with 1-deoxymannojirimycin or swainsonine, respectively. The susceptibility of complex-type alpha 1 PI to endo F was studied in the presence of various detergents. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate and octyl glucopyranoside turned out to be most effective. In the absence of detergents, digestion of alpha 1 PI with high concentrations of endo F and/or long times of incubation led to the formation of alpha 1 PI with one and two oligosaccharide side chains. In the presence of 0.5% octyl glucopyranoside, the major cleavage products were unglycosylated alpha 1 PI and alpha 1 PI carrying one carbohydrate side chain. In contrast to the complex-type alpha 1 PI, the high-mannose type can be totally deglycosylated by endo F even in the absence of detergents. The susceptibility of the hybrid-type alpha 1 PI to endo F is between that of the complex and the high-mannose types.

Acetylglucosaminidase↗

PubMed1990

Examination of the substrate specificity of heparin and heparan sulfate lyases.

We have examined the activities of different preparations of heparin and heparan sulfate lyases from Flavobacterium heparinum. The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis. Heparinase (EC 4.2.2.7) purified in our laboratory and a so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure----4)-alpha-D-GlcNp2S(6R)(1----4)-alpha-L-IdoAp2S( 1----(where R = O-sulfo or OH). Oligosaccharide mapping with two different commercial preparations of heparan sulfate lyase [heparitinase (EC 4.2.2.8)] indicated close similarities in their depolymerization of heparan sulfate. Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid:----4)-alpha-D-GlcNpR(1----4)-beta-D-GlcAp(1----(where R = N-acetamido or N-sulfo). The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin. A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III. When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.

Carbohydrate Conformation↗

PubMed2000

Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III.

The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically. Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate. Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III. Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)). The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified. As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme. Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity.

Animals↗

PubMedDate not supplied

Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae.

The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.

Aryldialkylphosphatase↗

PubMed2004

Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil.

Microbial populations responsible for the oxidation and reduction of As were examined in unsaturated (aerobic) soil columns treated with 75 microM arsenite [As(III)] or 250 microM arsenate [As(V)]. Arsenite [As(III)] was rapidly oxidized to As(V) via microbial activity, whereas no apparent reduction of As(V) was observed in the column experiments. Eight aerobic heterotrophic bacteria with varying As redox phenotypes were isolated from the same columns. Three isolates, identified as Agrobacterium tumefaciens-, Pseudomonas fluorescens-, and Variovorax paradoxus-like organisms (based on 16S sequence), were As(III) oxidizers, and all were detected in community DNA fingerprints generated by PCR coupled with denaturing gradient gel electrophoresis. The five other isolates were identified (16S gene sequence) as A. tumefaciens, Flavobacterium sp., Microbacterium sp., and two Arthrobacter sp. -like organisms and were shown to rapidly reduce As(V) under aerobic conditions. Although the two A. tumefaciens-like isolates exhibited opposite As redox activity,their 16S rDNA sequences (approximately 1400 bp) were 100% identical, and both were shown to contain putative arsC genes. Our results support the hypothesis that bacteria capable of either oxidizing As(III) or reducing As(V) coexist and are ubiquitous in soil environments, suggesting that the relative abundance and metabolic activity of specific microbial populations plays an important role in the speciation of inorganic As in soil pore waters.

Agrobacterium tumefaciens↗

PubMed2001

Oral colonization of aerobic and facultatively anaerobic gram-negative rods and cocci in irradiated, dentate, xerostomic individuals.

This study aimed at investigating the oral colonization of aerobic and facultatively anaerobic gram-negative rods and cocci in head- and neck-irradiated, dentate, xerostomic individuals. Subjects were recruited from a nasopharyngeal carcinoma clinic and were segregated into group A: <60 years (n=25, 48+/-6 years, 5+/-5 years post-irradiation) and group B: >or=60 years (n=8, 67+/-4 years, 2+/-2 years post-irradiation) and were compared with age- and sex-matched normal individuals, group C: <60 years (n=20, 44+/-12 years) and group D: >or=60 years (n=10, 70+/-3 years). Selective culture of the oral rinse samples was carried out to isolate, quantify and speciate (using API 20E kit) aerobic and facultatively anaerobic gram-negative rods and cocci recovery. All test subjects were put under comprehensive oral and preventive care for 3 months, and 12 group A and 5 group B subjects were recalled for reassessment of aerobic and facultatively anaerobic gram-negative rods and cocci colonization. All identical isolates, pre- and post-hygienic care, were phenotypically (Vitek, Hazelwood, MA and antibiogram profile) and genotypically (pulsed-field gel electrophoresis) evaluated. The aerobic and facultatively anaerobic gram-negative rods and cocci isolated from the first round oral rinse samples included: Acinetobacter, Neisseria, Chryseomonas, Flavimonas, Pseudomonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, Flavobacterium and Weeksella species. The aerobic and facultatively anaerobic gram-negative rods and cocci isolation rate was high for irradiated individuals, and they were 64/25% and 100/80% for groups A/C and B/D, respectively. Recovery of aerobic and facultatively anaerobic gram-negative rods and cocci and Klebsiella pneumoniae subsp. pneumoniae in oral rinse samples were found to be significantly more prevalent in the irradiated subjects (groups A and B). Enterobacteriaceae were more frequently isolated from oral rinse samples of aged irradiated subjects (group B vs D, P<0.05), where the quantity of Citrobacter freundii (colony-forming units/ml oral rinse) was also significantly elevated. The isolation rate of aerobic and facultatively anaerobic gram-negative rods and cocci after hygienic care remained unchanged; 3 of 12 and 3 of 5 of the recalled subjects from groups A and B, respectively, harbored same aerobic and facultatively anaerobic gram-negative rods and cocci species. However, only two pairs of K. pneumoniae subsp. pneumoniae, sequentially isolated from same patients in group B, were found to be identical by pulsed-field gel electrophoresis. This may be due to reinfection of the microbes from the same source or permanent colonization. In conclusion, irradiation-induced xerostomia seems to favor frequent, repeated, transient intraoral colonization of aerobic and facultatively anaerobic gram-negative rods and cocci.

Acinetobacter↗

PubMed2002

Isolation and characterization of a feather-degrading bacterium from the poultry processing industry.

A Flavobacterium sp. producing a high keratinolytic activity was isolated from a poultry industry after growth on selective feather meal agar. This bacterium grew on feather meal broth, producing keratinase, and was also capable of complete degradation of raw feathers. The proteolytic activity was assessed in the presence of specific protease inhibitors. The crude enzyme showed mainly metalloprotease character. This novel isolate would have potential biotechnological use in processes involving keratin hydrolysis.

Animals↗

PubMed1968

Novel heparin degradation products. Isolation and characterization of novel disaccharides and oligosaccharides produced from heparin by bacterial degradation.

1. Heparin was degraded by enzymes of adapted Flavobacterium heparinum. Several degradation products were separated by combined Sephadex-gel filtration and paper chromatography, and chemically analysed. 2. These products were identified as glucosamine 2,6-disulphate, saturated disaccharides constituted of uronic acid and glucosamine and containing two and three sulphate residues, and tetra- and hexa-saccharides with the same basic disaccharide units. 3. The implications of these findings with respect to the present knowledge of heparin structure and its enzymic degradation are discussed.

Chromatography↗

PubMed1973

Studies on beta-glucanases. Some properties of a bacterial endo-beta-(1 leads to 3)-glucanase system.

A commercial enzyme preparation, originally obtained from a Flavobacterium(Cytophaga), was fractionated by continuous electrophoresis, giving a protein fraction which hydrolysed laminarin, carboxymethylpachyman, barley beta-glucan, lichenin and cellodextrin in random fashion. This enzymic activity was not very stable. Ion-exchange chromatography and molecular-sieve chromatography on Bio-Gel P-60 showed that this activity was due to two specific beta-glucanases, an endo-beta-(1-->3)-glucanase and an endo-beta-(1-->4)-glucanase. The two enzymes occur in both high- and low-molecular-weight forms, the latter endo-beta-(1-->3)-glucanase having a molecular weight of about 16000.

Chromatography, Ion Exchange↗

PubMed2001

Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247.

A family 3 beta-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the k(cat) values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (beta=-0.85) for the leaving-group-dependent portion (pK(a) of leaving phenols >7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta-D-glucopyanoside, o-nitrophenyl beta-D-glucopyanoside and p-cyanophenyl beta-D-glucopyanoside as substrates were 1.17+/-0.02, 1.19+/-0.02 and 1.04+/-0.02 respectively. These results support an S(N)1-like mechanism for the deglucosylation step and an S(N)2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (k(cat)/K(m)) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

Aspartic Acid↗