A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum

2012 ◽  
Vol 444 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Takayuki Ohnuma ◽  
Tatsuya Fukuda ◽  
Satoshi Dozen ◽  
Yuji Honda ◽  
Motomitsu Kitaoka ◽  
...  
Keyword(s):  
Water Molecule ◽  
Hydrogen Fluoride ◽  
Amino Sugar ◽  
Hydrolytic Activity ◽  
Major Product ◽  
Polymerization Degree ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glycosidic Bonds ◽  
Inverting Mechanism

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)n [a β-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)2 fluoride] in the absence or presence of (GlcNAc)2, (GlcNAc)2 and hydrogen fluoride were found to be produced through the Hehre resynthesis–hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426–1431; (2008) Glycobiology 18, 325–330] of mutating Ser102, which holds a nucleophilic water molecule, and Glu70, which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)6 was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)4 as a major product from α-(GlcNAc)2-F in the presence of (GlcNAc)2. The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F− releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.

scholarly journals Crystal Structures of Pyrophosphatase from Acinetobacter baumannii: Snapshots of Pyrophosphate Binding and Identification of a Phosphorylated Enzyme Intermediate

2019 ◽  
Vol 20 (18) ◽  
pp. 4394
Author(s):  
Yunlong Si ◽  
Xing Wang ◽  
Guosong Yang ◽  
Tong Yang ◽  
Yuying Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Water Molecule ◽  
Acinetobacter Baumannii ◽  
Divalent Cations ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Catalytic Center ◽  
Favorable Reaction ◽  
Enzyme Intermediate

All living things have pyrophosphatases that hydrolyze pyrophosphate and release energy. This energetically favorable reaction drives many energetically unfavorable reactions. An accepted catalytic model of pyrophosphatase shows that a water molecule activated by two divalent cations (M1 and M2) within the catalytic center can attack pyrophosphate in an SN2 mechanism and thus hydrolyze the molecule. However, our co-crystal structure of Acinetobacter baumannii pyrophosphatase with pyrophosphate shows that a water molecule from the solvent may, in fact, be the actual catalytic water. In the co-crystal structure of the wild-type pyrophosphatase with pyrophosphate, the electron density of the catalytic centers of each monomer are different from one another. This indicates that pyrophosphates in the catalytic center are dynamic. Our mass spectroscopy results have identified a highly conserved lysine residue (Lys30) in the catalytic center that is phosphorylated, indicating that the enzyme could form a phosphoryl enzyme intermediate during hydrolysis. Mutation of Lys30 to Arg abolished the activity of the enzyme. In the structure of the apo wild type enzyme, we observed that a Na+ ion is coordinated by residues within a loop proximal to the catalytic center. Therefore, we mutated three key residues within the loop (K143R, P147G, and K149R) and determined Na+ and K+-induced inhibition on their activities. Compared to the wild type enzyme, P147G is most sensitive to these cations, whereas K143R was inactive and K149R showed no change in activity. These data indicate that monovalent cations could play a role in down-regulating pyrophosphatase activity in vivo. Overall, our results reveal new aspects of pyrophosphatase catalysis and could assist in the design of specific inhibitors of Acinetobacter baumannii growth.

scholarly journals Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

2005 ◽  
Vol 71 (10) ◽  
pp. 5823-5827 ◽  
Author(s):  
Kazutoshi Fujii ◽  
Hirotaka Minagawa ◽  
Yoshinobu Terada ◽  
Takeshi Takaha ◽  
Takashi Kuriki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Random Mutagenesis ◽  
Hydrolytic Activity ◽  
Amino Acid Replacement ◽  
Site Directed Mutagenesis ◽  
Efficient Production ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Thermus Aquaticus ◽  
Hydrolytic Activities

ABSTRACT Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of α-1,4 glucans to produce cyclic α-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.

scholarly journals Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

2021 ◽  
Vol 22 (6) ◽  
pp. 3230
Author(s):  
Mireia Castejón-Vilatersana ◽  
Magda Faijes ◽  
Antoni Planas
Keyword(s):  
Hydrolytic Activity ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Type I ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Transglycosylation Activity ◽  
Binding Cleft ◽  
Breast Fed ◽  
Enzyme Variant

The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.

scholarly journals Modulation of the Activity and Regioselectivity of a Glycosidase: Development of a Convenient Tool for the Synthesis of Specific Disaccharides

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5445
Author(s):  
Yari Cabezas-Pérusse ◽  
Franck Daligault ◽  
Vincent Ferrières ◽  
Olivier Tasseau ◽  
Sylvain Tranchimand
Keyword(s):  
Molecular Dynamics ◽  
Molecular Modeling ◽  
Fold Increase ◽  
Building Blocks ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Modeling Methodology ◽  
Glycosidic Bonds ◽  
Predictive Quality ◽  
Good Agreement

The synthesis of disaccharides, particularly those containing hexofuranoside rings, requires a large number of steps by classical chemical means. The use of glycosidases can be an alternative to limit the number of steps, as they catalyze the formation of controlled glycosidic bonds starting from simple and easy to access building blocks; the main drawbacks are the yields, due to the balance between the hydrolysis and transglycosylation of these enzymes, and the enzyme-dependent regioselectivity. To improve the yield of the synthesis of β-d-galactofuranosyl-(1→X)-d-mannopyranosides catalyzed by an arabinofuranosidase, in this study we developed a strategy to mutate, then screen the catalyst, followed by a tailored molecular modeling methodology to rationalize the effects of the identified mutations. Two mutants with a 2.3 to 3.8-fold increase in transglycosylation yield were obtained, and in addition their accumulated regioisomer kinetic profiles were very different from the wild-type enzyme. Those differences were studied in silico by docking and molecular dynamics, and the methodology revealed a good predictive quality in regards with the regioisomer profiles, which is in good agreement with the experimental transglycosylation kinetics. So, by engineering CtAraf51, new biocatalysts were enabled to obtain the attractive central motif from the Leishmania lipophosphoglycan core with a higher yield and regioselectivity.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

2021 ◽  
Vol 11 (12) ◽  
pp. 5521
Author(s):  
Li Liu ◽  
Shuhuai Yu ◽  
Wei Zhao
Keyword(s):  
Ph Value ◽  
Wild Type ◽  
Michaelis Constant ◽  
Wild Type Enzyme ◽  
Maximum Reaction Rate ◽  
E Coli ◽  
Maximum Reaction ◽  
Conversion Rates ◽  
Raoultella Terrigena ◽  
Sucrose Isomerase

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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