Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

The Agrobacterium sp. β-glucosidase (Abg) is a retaining β-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using α-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using α-d-galactopyranosyl fluoride and 4-nitrophenyl β-d-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6. The Agrobacterium sp. β-glucosidase (Abg) is a retaining β-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using α-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using α-d-galactopyranosyl fluoride and 4-nitrophenyl β-d-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6. Oligosaccharides have considerable potential as therapeutics because of the numerous medicinally relevant physiological events that involve glycoconjugates (1Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (5004) Google Scholar, 2Jacob G.S. Curr. Opin. Struct. Biol. 1995; 5: 605-611Crossref PubMed Scopus (279) Google Scholar, 3Dwek R.A. Chem. Rev. 1996; 96: 683-720Crossref PubMed Scopus (2860) Google Scholar). To expand our understanding of the various roles of oligosaccharides found in important cellular events, more efficient and selective synthetic protocols must be developed for the preparation of oligosaccharides. Classical chemical synthesis is often impractical for the synthesis of complex oligosaccharides because of the need for selective and labor-intensive protection-deprotection steps and difficulties in directing product stereochemistry. To overcome these limitations, enzymatic syntheses using glycosidases or glycosyl transferases have rapidly gained prominence (4Crout D.H. Vic G. Curr. Opin. Chem. Biol. 1998; 2: 98-111Crossref PubMed Scopus (249) Google Scholar, 5Koeller K.M. Wong C.H. Chem. Rev. 2000; 100: 4465-4494Crossref PubMed Scopus (456) Google Scholar, 6Withers S.G. Carbohydr. Polymers. 2001; 44: 325-337Crossref Scopus (170) Google Scholar). In recent years, the glycosynthase approach developed in this laboratory has added a new dimension to the enzymatic preparation of oligosaccharides (7Williams S.J. Withers S.G. Aust. J. Chem. 2002; 55: 3-12Crossref Google Scholar, 8Jakeman D.L. Withers S.G. Trends Glycosci. Glycotechnol. 2002; 14: 13-25Crossref Scopus (44) Google Scholar, 9Perugino G. Trincone A. Rossi M. Moracci M. Trends Biotechnol. 2004; 22: 31-37Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Glycosynthases are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue. These mutants catalyze the formation of glycosidic bonds when glycosyl fluorides with anomeric configuration opposite to that of the original substrate, thereby mimicking the glycosyl enzyme intermediate, are employed as substrates. The modified enzyme catalyzes the nucleophilic displacement of the fluoride via attack by a hydroxyl group on an added glycosyl acceptor, generating a new glycosidic bond with the same stereochemistry as the normal substrate. The reactions catalyzed by glycosynthases are highly amenable to industrial syntheses because of the high yields of products (70–95%), the relatively inexpensive and easily prepared substrates, and the high stabilities of the enzymes involved (9Perugino G. Trincone A. Rossi M. Moracci M. Trends Biotechnol. 2004; 22: 31-37Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Since the first report on a glycosynthase in 1998 (10Mackenzie L.F. Wang Q. Warren R.A.J. Withers S.G. J. Am. Chem. Soc. 1998; 120: 5583-5584Crossref Scopus (473) Google Scholar), successful glycosynthases have been developed from 11 glycosidases belonging to seven different glycoside hydrolases (GH) 1The abbreviations used are: GH, glycoside hydrolases; Abg, Agrobacterium sp. β-glucosidase; Cel5A, β-1, 4-glucanase from C. fimi; CelB, P. furiosus β-glucosidase; Taβ-Gly, T. aggregans β-glycosidase; α-GalF, α-d-galactopyranosyl fluoride; α-GlcF, α-d-glucopyranosyl fluoride; α-ManF, α-d-mannopyranosyl fluoride; α-XylF, α-d-xylopyranosyl fluoride; MU-Glc, 4-methylumbelliferyl β-d-glucopyranoside; pNP-Gal, 4-nitrophenyl β-d-galactopyranoside; pNP-Glc, 4-nitrophenyl β-d-glucopyranoside; pNP-Man, 4-nitrophenyl β-d-mannopyranoside; pNP-Xyl, 4-nitrophenyl β-d-xylopyranoside. families (9Perugino G. Trincone A. Rossi M. Moracci M. Trends Biotechnol. 2004; 22: 31-37Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). With the exception of a unique α-glycosynthase from Schizosaccharomyces pombe (11Okuyama M. Mori H. Watanabe K. Kimura A. Chiba S. Biosci. Biotechnol. Biochem. 2002; 66: 928-933Crossref PubMed Scopus (84) Google Scholar) all glycosynthases have originated from retaining β-glycosidases, and employ α-glycosyl fluorides as donors. Recently, β-glycosynthases were shown to use activated β-glycosides as donors at low pH in the presence of external nucleophiles, such as acetate or formate (12Perugino G. Trincone A. Giordano A. van der Oost J. Kaper T. Rossi M. Moracci M. Biochemistry. 2003; 42: 8484-8493Crossref PubMed Scopus (45) Google Scholar, 13Trincone A. Giordano A. Perugino G. Rossi M. Moracci M. Bioorg. Med. Chem. Lett. 2003; 13: 4039-4042Crossref PubMed Scopus (23) Google Scholar). This rescue of activity with external nucleophiles permits transglycosylation reactions to be performed via the formation of an intermediate α-glycosyl acetate or formate. The glycosynthase from Agrobacterium sp. β-glucosidase (Abg) was the first glycosynthase to be reported (10Mackenzie L.F. Wang Q. Warren R.A.J. Withers S.G. J. Am. Chem. Soc. 1998; 120: 5583-5584Crossref Scopus (473) Google Scholar), and its utility has been extended to numerous applications (10Mackenzie L.F. Wang Q. Warren R.A.J. Withers S.G. J. Am. Chem. Soc. 1998; 120: 5583-5584Crossref Scopus (473) Google Scholar, 14Howard S. Withers S.G. Biochemistry. 1998; 37: 3858-3864Crossref PubMed Scopus (27) Google Scholar, 15Johnson P.E. Brun E. MacKenzie L.F. Withers S.G. McIntosh L.P. J. Mol. Biol. 1999; 287: 609-625Crossref PubMed Scopus (51) Google Scholar, 16Brun E. Brumer H. MacKenzie L.F. Withers S.G. McIntosh L.P. J. Biomol. NMR. 2001; 21: 67-68Crossref PubMed Scopus (7) Google Scholar, 17Tolborg J.F. Petersen L. Jensen K.J. Mayer C. Jakeman D.L. Warren R.A.J. Withers S.G. J. Org. Chem. 2002; 67: 4143-4149Crossref PubMed Scopus (80) Google Scholar). The first improvement of Abg glycosynthase activity was achieved by replacing alanine with serine at the nucleophile position 2The amino acid sequence numbering system used here is that which has been used throughout for this protein in which the N-terminal methionine (which is processed off in the mature protein) is not counted. This results in a numbering difference of 1 compared to that listed in GenBank™ (accession number M19033), thus the nucleophile of Abg is referred to as the 358th amino acid residue rather than the 359th (52Wakarchuk W.W. Greenberg N.M. Kilburn D.G. Miller R.C.J. Warren R.A.J. J. Bacteriol. 1998; 170: 301-307Crossref Google Scholar). (18Mayer C. Zechel D.L. Reid S.P. Warren R.A.J. Withers S.G. FEBS Lett. 2000; 466: 40-44Crossref PubMed Scopus (120) Google Scholar). Subsequently, using an “on-plate” screening method based on a coupled enzyme assay with an endocellulase, an improved new glycosynthase, AbgE358G with Gly at the nucleophile position, was discovered from a library of mutations at the catalytic nucleophile position (19Mayer C. Jakeman D.L. Mah M. Karjala G. Gal L. Warren R.A.J. Withers S.G. Chem. Biol. 2001; 8: 437-443Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, the reactions catalyzed by these improved Abg glycosynthases are still slow relative to wild type glycosidase activities, requiring relatively large quantities of mutant enzyme and/or extended incubation times (9Perugino G. Trincone A. Rossi M. Moracci M. Trends Biotechnol. 2004; 22: 31-37Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 17Tolborg J.F. Petersen L. Jensen K.J. Mayer C. Jakeman D.L. Warren R.A.J. Withers S.G. J. Org. Chem. 2002; 67: 4143-4149Crossref PubMed Scopus (80) Google Scholar, 18Mayer C. Zechel D.L. Reid S.P. Warren R.A.J. Withers S.G. FEBS Lett. 2000; 466: 40-44Crossref PubMed Scopus (120) Google Scholar). This has stimulated attempts to generate more active glycosynthase mutants. In this report, we present a modified screening method and its use on a mutated gene library to identify a mutant enzyme, designated 2F6, containing three additional mutations and a 27-fold enhancement in catalytic efficiency relative to the parental glycosynthase (AbgE358G) after two rounds of mutagenesis and screening. The enhanced activity also allows 2F6 to use other sugars as donors and acceptors. We also propose a structural basis for these activity changes based upon a suitable homology model. Construction of pGSVIII as a Screening Vector—The gene encoding the catalytic domain of cellulase D (Cel5A) from Cellulomonas fimi (20Meinke A. Gilkes N.R. Kilburn D.G. Miller R.C. Warren R.A.J. J. Bacteriol. 1993; 175: 1910-1918Crossref PubMed Google Scholar) was amplified by PCR using 1 μm and the of the of (19Mayer C. Jakeman D.L. Mah M. Karjala G. Gal L. Warren R.A.J. Withers S.G. Chem. Biol. 2001; 8: 437-443Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and of in of PCR at at and at were performed in a PCR The PCR product was with and and which the J. Biol. Chem. Full Text PDF PubMed Google Scholar), the and the gene from The was designated as pGSVIII and was used for the of the catalytic domain of and Abg glycosynthase mutants in this and Construction of mutagenesis of the gene was performed using in the presence of M. S. P. Biochem. 1995; PubMed Scopus Google Scholar). The glycosynthase gene was amplified from with the and the The of 1 of and 1 of and μm of and of in a were of for 1 for 1 and for The PCR products were with and from using a and with pGSVIII that been with the The mutant library was were on containing of and encoding of Abg are in the two in the To the the of the 1D12 gene with was with that of the 2F6 gene containing the mutation using The was designated as was by the of the of the gene with for that of the 2F6 gene containing the of Abg mutagenesis at the of and of AbgE358G were using a The used in the library were and and were used in and were used in PCR products were by the on a of PCR product was for a second at at and at using and were added to a of μm and the PCR was for a The PCR product was pGSVIII using and and were used for a library of and respectively. and were used for a was used as a in all three Screening for the were to To the the were on a with The were to a were for was with of α-d-glucopyranosyl fluoride and 1 4-methylumbelliferyl β-d-glucopyranoside and at The from active were a and the were from and containing of The screened were at with at to for of were to new and the of the were at was by the at using a of protein with was added to and the were for at to the the were with an of assay α-GlcF, MU-Glc, of in pH and the of was at using a with activity than that of the in the screening were and their improved were using the same as used for the screening. of Abg glycosynthase mutants were from E. the on pGSVIII by using as (18Mayer C. Zechel D.L. Reid S.P. Warren R.A.J. Withers S.G. FEBS Lett. 2000; 466: 40-44Crossref PubMed Scopus (120) Google Scholar). The and the of enzyme were using an The used in enzyme was to were determined by at using an of L. G. T. 1995; PubMed Scopus Google Scholar). of fluoride with a was used to fluoride at enzymatic were for the of the glycosyl The of donor or acceptor was and that of the was to and The of 2F6 for was determined from the of the of at of from 1 to at a of Scholar) was used to by of and synthesis of PCR and the of were by the and in the at the of of Abg and the mutants were using the at the Biochem. Soc. 1996; PubMed Scopus Google Scholar). of were used as The J. G. H. P.E. 2000; PubMed Scopus Google Scholar) for these are J. M. J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar), S. T. J. J. Struct. Biol. 2000; PubMed Scopus Google Scholar), Moracci M. M. Rossi M. J. Mol. Biol. PubMed Scopus Google Scholar), and FEBS Lett. 1999; PubMed Scopus Google Scholar). and of the structure were using the PubMed Scopus Google Scholar). The were with P. J. Google Scholar) and using PubMed Scopus Google Scholar). of the Screening the efficiency of formation of mutant the developed screening method (19Mayer C. Jakeman D.L. Mah M. Karjala G. Gal L. Warren R.A.J. Withers S.G. Chem. Biol. 2001; 8: 437-443Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) was modified to been to use E. as a and to employ because the of the gene was the two for and the glycosynthase to be to these enzymes in and because also a the two to be to the improved glycosynthase after screening. To overcome these limitations, the was to a J. Biol. Chem. Full Text PDF PubMed Google Scholar), and a system was In the the gene for was to a by a second which originated from with a glycosynthase mutant are a and also in E. mutants by the screened be directly using and PCR mutagenesis using and was used to generate of glycosynthase mutants M. S. P. Biochem. 1995; PubMed Scopus Google Scholar). In the first to the of the mutant were amplified at various from to 1 using the gene as a of the in a mutant library in the presence of glycosynthase activity in the and sequence of in the library a mutation of which is an mutagenesis for amino acid screening from the mutant only mutant was of 1D12 amino acid This in times activity than that of AbgE358G in the screening assay The gene encoding 1D12 was used as a for generating the second PCR library the same as the first additional improved 2F6, times more activity than 1D12, was after screening of the second The new mutant three mutations compared with the amino acid of its A19T and E358G, and two additional and To of mutations and of these were two new mutants, (A19T, E358G, and and (A19T, E358G, and were prepared by using In of these mutants were more active than 1D12 and times for and thus all three mutations in the do to the glycosynthase In an to the three mutated were by mutagenesis K. J. Mol. 1999; PubMed Scopus Google Scholar) using the gene as a The screening of from of the three mutagenesis new mutation that the which an was as an improved mutant times than but its activity was than that of 1D12 the A19T mutation of the improved transglycosylation activity of two screened AbgE358G mutants, we the of these mutants with α-d-galactopyranosyl fluoride as a donor and 4-nitrophenyl β-d-glucopyranoside as an The use of as a donor for that the of fluoride to a because the with an not as an the at a of all three glycosynthases The catalytic of two screened mutants were by and than that of AbgE358G The for with the A19T mutation from to but with the two additional mutations and M407V) to the of Abg glycosynthase was improved 27-fold relative to that of AbgE358G this acid and of Abg glycosynthase acid from not from from from Q248R, from C. Jakeman D.L. Mah M. Karjala G. Gal L. Warren R.A.J. Withers S.G. Chem. Biol. 2001; 8: 437-443Abstract Full Text Full Text PDF PubMed Scopus (98) Google not from 18Mayer C. Zechel D.L. Reid S.P. Warren R.A.J. Withers S.G. FEBS Lett. 2000; 466: 40-44Crossref PubMed Scopus (120) Google in a new the of the mutations on the acceptor of the glycosynthase, were determined for various acceptor sugars that have an equatorial hydroxyl group at the using a donor The of for all acceptor sugars more than times relative to that of AbgE358G In the of pNP-Glc, AbgE358G and 2F6 but 2F6 was than AbgE358G of for AbgE358G and 2F6 were and In the of 4-nitrophenyl was for AbgE358G with the for 2F6 times than that of for and not for 2F6 with 4-nitrophenyl were not the from to of the of AbgE358G and 2F6 for various with at high not not in a new glycosynthase for new donor substrates, α-d-xylopyranosyl fluoride and α-d-mannopyranosyl fluoride were AbgE358G activity for the of with with and a of was determined for 2F6 the same which to a was not an of of and of and respectively. In the of α-ManF, with an C2-hydroxyl the of 2F6 by only relative to AbgE358G The catalytic efficiency (kcat/Km) for by because of the of 2F6 for for AbgE358G and 2F6 were and a relatively improvement compared with α-glycosyl fluorides with an equatorial C2-hydroxyl of the of AbgE358G and 2F6 for various donors and at not not in a new the mutations enhanced the model of AbgE358G and 2F6 were using the protein Biochem. Soc. 1996; PubMed Scopus Google Scholar). on the model all of the three mutations are from the to in the the anomeric of the and of A19T and M407V and respectively, is found on the of Abg is that the mutations exert their effects by changing the conformation of the active rather than by directly with substrates. is of the to the of enzymes J. Curr. Opin. Chem. Biol. 1999; 3: PubMed Scopus Google Scholar, Chem. 1998; Scopus Google Scholar, T. A. Biotechnol. 2001; 55: PubMed Scopus Google Scholar). The of the approach on the efficient formation of a random mutant library and a highly and screening In the reported (19Mayer C. Jakeman D.L. Mah M. Karjala G. Gal L. Warren R.A.J. Withers S.G. Chem. Biol. 2001; 8: 437-443Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), we developed an enzyme assay based on a system the of active glycosynthases from a mutant In this we modified the system to a system to to the for high donor a a was added J. Bacteriol. PubMed Scopus Google Scholar). easily the and the oligosaccharides the glycosynthase activity were not but in for screening of active However, as a this assay was still not because of the of on and the of compared with the glycosynthase in the present The for such an is but of from C. fimi in E. has been reported (20Meinke A. Gilkes N.R. Kilburn D.G. Miller R.C. Warren R.A.J. J. Bacteriol. 1993; 175: 1910-1918Crossref PubMed Google Scholar, Gilkes N.R. Kilburn D.G. Warren R.A.J. Miller R.C. 55: PubMed Google Scholar). To these a assay was for that been was for by to by of the by the at of enzyme (Cel5A) to that this was not two rounds of random mutagenesis and we have a improved Abg glycosynthase mutant with a and that only three amino acid These mutations to improved Abg glycosynthase activity for a of donor and acceptor sugars and of the for donor a for the type Abg has high for pNP-Glc, 4-nitrophenyl and 4-nitrophenyl but low for and Withers S.G. Biochemistry. PubMed Scopus Google Scholar). These results are with the of and Withers Withers S.G. Biochemistry. 1995; PubMed Scopus Google Scholar) and that the of Abg is more by interactions with the equatorial C2-hydroxyl group compared with the and the C2-hydroxyl group was shown to at and to for and Withers S.G. Biochemistry. 1995; PubMed Scopus Google Scholar). In of the group the In this the upon to 2F6 are for substrates containing an equatorial C2-hydroxyl such as α-GalF, α-GlcF, and the improvement for was the structural changes by the mutations to 2F6 in improved interactions with the equatorial C2-hydroxyl group of the substrate. In the of a the model structure using of enzymes is in understanding the roles of the various mutations in the of enzymes We to the of the three mutations in 2F6 using the structure with the of from is in the of in AbgE358G to or improved with the the enhancement We that the hydroxyl group of in 1D12 and in glycosynthase activity with the group of in 1D12 a enhancement of by a of active the reported of the enzyme from S. T. J. J. Struct. 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Soc. 2003; PubMed Scopus Google is an expected consequence of the A19T and M407V and The of in 1 highly amino acid Trp-404, Glu-411, and in These in the important roles in substrates K. FEBS Lett. 2001; PubMed Scopus Google Scholar, C. J. Biochem. 2002; PubMed Scopus Google Scholar). in AbgE358G is in the the and the and Glu-411, Trp-412, and Trp-404, which are components of the and interact with the equatorial and of are at the of this The of is by and This is found in β-glucosidase seems that an important in directing and with the of to a in the the of A19T to allows the group of A19T to the of these changes in the from at the of the to by A19T and M407V to the active of 2F6 to a suitable to on which is not a for wild type In the of the the for improvement is as this residue is found on the protein the activity of 2F6 be expected not only to product yields and to times but also to the synthetic be in the of an enzyme to our group has also to a from and in families and J. L. S. 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