Engineering the acceptor specificity of trehalose phosphorylase for the production of trehalose analogs

Trehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐glucopyranoside) is widely used in the food industry, thanks to its protective effect against freezing and dehydration. Analogs of trehalose have the additional benefit that they are not digested and thus do not contribute to our caloric intake. Such trehalose analogs can be produced with the enzyme trehalose phosphorylase, when it is applied in the reverse, synthetic mode. Despite the enzyme's broad acceptor specificity, its catalytic efficiency for alternative monosaccharides is much lower than for glucose. For galactose, this difference is shown here to be caused by a lower K_m whereas the k_cat for both substrates is equal. Consequently, increasing the affinity was attempted by enzyme engineering of the trehalose phosphorylase from Thermoanaerobacter brockii, using both semirational and random mutagenesis. While a semirational approach proved unsuccessful, high‐throughput screening of an error‐prone PCR library resulted in the discovery of three beneficial mutations that lowered K_m two‐ to three‐fold. In addition, it was found that mutation of these positions also leads to an improved catalytic efficiency for mannose and fructose, suggesting their involvement in acceptor promiscuity. Combining the beneficial mutations did not further improve the affinity, and even resulted in a decreased catalytic activity and thermostability. Therefore, enzyme variant R448S is proposed as new biocatalyst for the industrial production of lactotrehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐galactopyranoside). © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

纤维二糖磷酸化酶和纤维寡糖磷酸化酶的研究与应用

自然界中一些厌氧的纤维素降解菌能够产生纤维二糖磷酸化酶(Cellobiose Phosphorylase,CBP)和纤维寡糖磷酸化酶(Cellodextrin Phosphorylase,CDP)磷酸化裂解纤维二糖和纤维寡糖.CBP和CDP属于糖苷水解酶94家族(Glycoside Hydrolase Family 9...     

Engineering of cellobiose phosphorylase for glycoside synthesis

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes.     

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0–8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1?1)-α-d-Manp6P, was synthesized with 51% yield.     

Substrate specificity of Escherichia coli thymidine phosphorylase

Substrate specificity of Escherichia coli thymidine phosphorylase to thymidine derivatives modified at 5' -, 3' -, and 2' ,3' - positions of the sugar moiety was studied. Equilibrium and kinetic constants (K(m), K(I), k(cat)) of the phosphorolysis reaction have been determined for 20 thymidine analogs. The results are compared with X-ray and molecular dynamics data. The most important hydrogen bonds in the enzyme-substrate complex are revealed.     

Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis

Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.     

The acceptor specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases

The in vitro and in vivo specificity of the family of peptide:N-acetylgalactosaminyltransferases (GalNAcT) is analyzed on the basis of the reactivity and/or inhibitory activity of peptides and protein segments. The transferases appear to be multi-substrate enzymes with extended active sites containing a least nine subsites that interact cooperatively with a linear segment of at least nine amino acid residues on the acceptor polypeptide. Functional acceptor sites are located on the surface of the protein and extended conformations (-strand conformation) are preferred. The acceptor specificity of GalNAc-T can be predicted from the primary structure of the acceptor peptide with an accuracy of 70 to 80%. The same GalNAc-T enzymes catalyze the glycosylation of both serine and threonine residues. The higher in vitro catalytic efficiency toward threonine versus serine is the result of enhanced binding as well as increased reaction velocity, both effects being the result of steric interactions between the active site of the enzyme and the methyl group of threonine. Results from substrate binding studies suggest that GalNAc-T catalyzed transfer proceeds via an ordered sequential mechanism.     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases.     

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme

ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5–10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi–Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k_cat(app)/K_m(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23–12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.     

Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein‐engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N‐acetyl‐D‐glucosamine units at their subsite (?2), which is impossible for the wildtype enzyme.     

Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.     

Directed Evolution of the Actinomycete Cytochrome P450 MoxA (CYP105) for Enhanced Activity

Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did.     

Quantitative specificity‐based display library screening identifies determinants of antibody‐epitope binding specificity

Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three‐fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications.     

Improving the quality of industrially important enzymes by directed evolution

Directed evolution is a new process for developing industrially viable biocatalysts. This technique does not require a comprehensive knowledge of the relationships between sequence structure and function of proteins as required by protein engineering. It mimics the process of Darwinian evolution in a test tube combining random mutagenesis and recombination with screening or selection for enzyme variants that have the desired properties. Directed evolution helps in enhancing the enzyme performance both in natural and synthetic environments. This article reviews the process of directed evolution and its application to improve substrate specificity, activity, enantioselectivity and thermal stability.     

Role of sequence evolution and conformational dynamics in the substrate specificity and oligomerization mode of thymidylate kinases

Thymidylate kinase (TMK) is a key enzyme for the synthesis of DNA, making it an important target for the development of anticancer, antibacterial, and antiparasitic drugs. TMK homologs exhibit significant variations in sequence, residue conformation, substrate specificity, and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly, and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on TMK from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three-dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode, and substrate recognition in TMKs and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors.     

Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2′-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P_i) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P_i binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.     

Formation of Fine Oil-in-Water Emulsions by the Gel Emulsification Method with Saccharide and Protein

An emulsification method using a gel-like phase of a saccharide and protein mixture has been developed. In the method, which is called a gel emulsification method, an oil is added to the highly concentrated saccharide solution containing protein to form a clear gel-like phase, which followed by dilution with water to form a fine oil-in-water emulsion. This emulsion was investigated as to its emulsifying activity and emulsion stability as compared with that obtained by high-shear equipment, which was called a homomixer method. The emulsifying activity of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The emulsions prepared by both methods were highly stable in terms of the stability against coalescence. On the other hand, the stability against creaming of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The surface hydrophobicity of the protein and the unfreezable water content in the highly concentrated saccharide solution containing protein were not correlated to the emulsifying properties of the emulsions prepared by the gel emulsification method, which appeared to be dependent on the viscosity of the highly concentrated saccharide solution containing protein.     

Functions,structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)₆-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.