The molecular basis of the nonprocessive elongation mechanism in levansucrases

Abstract

Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (−1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity (Km) values to the wildtype (WT) Km value but had a lower turnover number and transfructosylation/hydrolysis ratio. Of importance, compared with the WT, the variants progressively yielded smaller-sized low-molecular-weight levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular-weight levans. Furthermore, an additional oligosaccharide-binding site 20 Å away from the catalytic pocket was identified, and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs. Levansucrases (LSs) synthesize levan, a β2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (−1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity (Km) values to the wildtype (WT) Km value but had a lower turnover number and transfructosylation/hydrolysis ratio. Of importance, compared with the WT, the variants progressively yielded smaller-sized low-molecular-weight levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular-weight levans. Furthermore, an additional oligosaccharide-binding site 20 Å away from the catalytic pocket was identified, and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs. Levans are carbohydrates such as β2-6 fructose oligo- and polysaccharides that have broad applications as prebiotic, anticancer, antibacterial, antioxidant, anti-inflammatory, and antiobesity agents (1Porras-Domínguez J.R. Ávila-Fernández Á. Rodríguez-Alegría M.E. Miranda-Molina A. Escalante A. González-Cervantes R. Olvera C. López Munguía A. Levan-type FOS production using a Bacillus licheniformis endolevanase.Process Biochem. 2014; 49: 783-790Crossref Scopus (43) Google Scholar, 2Byun B.Y. Lee S.J. Mah J.H. Antipathogenic activity and preservative effect of levan (β-2,6-fructan), a multifunctional polysaccharide.Int. J. Food Sci. Technol. 2014; 49: 238-245Crossref Scopus (28) Google Scholar, 3Abdel-Fattah Aza M. Gamal-Eldeen A.M. Helmy W.A. Esawy M.A. Antitumor and antioxidant activities of levan and its derivative from the isolate Bacillus subtilis NRC1aza.Carbohydr. Polym. 2012; 89: 314-322Crossref PubMed Scopus (74) Google Scholar, 4Kang S.A. Hong K.H. Kim S.H. Lee K.H. Chang B.I. Kim C.H. Jang K.H. Choue R.W. Anti-obesity and hypolipidemic effects of dietary levan in high fat diet-induced obese rats.J. Microbiol. Biotechnol. 2004; 14: 796-804Google Scholar, 5van den Ende W. Coopman M. Clerens S. Vergauwen R. le Roy K. Lammens W. van Laere A. Unexpected presence of graminan- and levan-type fructans in the evergreen frost-hardy eudicot pachysandra terminalis (buxaceae): purification, cloning, and functional analysis of a 6-SST/6-SFT enzyme.Plant Physiol. 2011; 155: 603-614Crossref PubMed Scopus (40) Google Scholar, 6Sinai Y. Leibovici J. Wolman M. Effects of route and schedule of administration of high-molecular levan on the growth of AKR lymphoma.Cancer Res. 1976; 36: 1593-1597PubMed Google Scholar, 7Sedgwick A.D. Rutman A. Sin Y.M. Mackay A.R. Willoughby D.A. The effects of levan on the acute inflammatory response.Br. J. Exp. Pathol. 1984; 65: 215-222PubMed Google Scholar). Owing to this wide diversity of applications, the development of methods by which to produce these relevant saccharides is of great interest for several industrial sectors, such as the food, cosmetics, and pharmaceutical industries. In nature, levans are produced from sucrose by levansucrases (LSs), which are enzymes produced primarily by bacteria. LSs (EC 2.4.1.10) catalyze the transfer of the fructosyl moiety from sucrose to an acceptor molecule. In this enzymatic reaction, the sucrose glycosidic linkage is broken, resulting in the formation of a covalent fructosyl-enzyme intermediate and the release of glucose (8Meng G. Fütterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.Nat. Struct. PubMed Scopus Google Scholar). The fructosyl moiety is from the enzyme to an acceptor from which of The the acceptor is a growing levan the acceptor is a the enzyme the sucrose enzymes produce enzymes of with a lower of levan J. of levan from a of to PubMed Scopus Google Scholar). was that the LS from Bacillus subtilis (SacB) is of with molecular elongation M. J. A. Olvera C. by enzyme levan elongation in Bacillus subtilis PubMed Scopus Google Scholar). high-molecular-weight levan is produced a which of of fructose a growing by the enzyme the elongation is the a low-molecular-weight levan is synthesized by a nonprocessive in which the fructose are to growing but are and the to their affinity for the enzyme M. J. A. Olvera C. by enzyme levan elongation in Bacillus subtilis PubMed Scopus Google Scholar). that the for elongation the crystallographic of LSs from Bacillus and have determined (8Meng G. Fütterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.Nat. Struct. PubMed Scopus Google Scholar, C. M. structure of from the J. PubMed Scopus Google Scholar, A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar, J. M. S. R. S. The structure of a of the products of sucrose the Struct. PubMed Scopus (40) Google Scholar, R. S. The structure of from a pocket for J. Sci. Scopus Google Scholar). of these a with a in which the of The structure a central cavity that is the site the substrate the of the the catalytic is are and In residues and have identified as the and the is the (8Meng G. Fütterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.Nat. Struct. PubMed Scopus Google Scholar). of have in with sucrose and (8Meng G. Fütterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.Nat. Struct. PubMed Scopus Google Scholar, G. Fütterer K. substrate in the of Bacillus subtilis Struct. PubMed Scopus Google Scholar). were the site, the the substrate-binding subsites (−1, +1, and The and in with the fructosyl from the residues in with the that the are and (8Meng G. Fütterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.Nat. Struct. PubMed Scopus Google Scholar). Furthermore, residues and were identified as of the as are for a with the from G. Fütterer K. substrate in the of Bacillus subtilis Struct. PubMed Scopus Google Scholar). mutagenesis on LSs have that of residues and of subtilis which are on the of the catalytic the enzyme catalytic and by the polymerization and these residues the of levans A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar, C. Y. R. J. M. mutagenesis of from Bacillus licheniformis for Microbiol. Biotechnol. PubMed Scopus Google Scholar, R. S. and in the site of Bacillus licheniformis production of PubMed Scopus Google Scholar, S. S. R. K. Levan-type fructooligosaccharide production using Bacillus licheniformis on J. Sci. Technol. Scholar, R. K. S. of Bacillus licheniformis for of the of levan-type J. PubMed Scopus Google Scholar). the by these residues have identified, and the of additional subsites as in the elongation of levans is to the of the enzymes with intermediate products to identify the levan involved in us to these enzymes to products with size-defined In this we the structure of an of in complex with levan-type which are intermediate products in the synthesis of levans from sucrose A. Olvera C. the transfer the synthesis of molecular levan by Bacillus subtilis PubMed Scopus Google Scholar). The of the residues belonging to the subsites (−1, +1, and with this acceptor were and subsites and the of residues belonging to the acceptor subsites in the levan elongation mechanism by the effect of their on the and of the In a oligosaccharide-binding site site in the of the catalytic pocket and its potential in the polymerization The structure of an of in the presence of a levan-type FOS a of polymerization of was determined by molecular using the of wildtype (WT) LS from subtilis (8Meng G. Fütterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.Nat. Struct. PubMed Scopus Google and to a of of a Å The of and residues to The using is structure is to that of with an of Å using a and in which are from the and oligosaccharide the enzyme was with an the of fructose by β2-6 glycosidic were the site of site was for the fructose and glucose in the of the high of these which were to the this is to as Furthermore, the for a oligosaccharide was 20 Å from the catalytic pocket site The additional were with levanhexaose and in and and are fructose with β2-6 glycosidic The structure is in of with the of LSs from G. and an of Å using and of with LS an of Å using a LSs from the Bacillus the central pocket of fructose by β2-6 glycosidic of the molecule were with a The levanhexaose molecule is with its the of the site pocket and its of the central The levanhexaose is to the enzyme by with the residues in the catalytic to the to the and the of the with residues with and and with and levanhexaose of have in complex with sucrose and in complex with and have (8Meng G. Fütterer K. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.Nat. Struct. PubMed Scopus Google Scholar, G. Fütterer K. substrate in the of Bacillus subtilis Struct. PubMed Scopus Google Scholar). In the structure of LS determined with the sucrose fructose and the site J. M. S. R. S. The structure of a of the products of sucrose the Struct. PubMed Scopus (40) Google Scholar). The LS with sucrose and identified the substrate-binding +1, and to the of for subsites in J. PubMed Scopus Google Scholar). In the the oligosaccharide is as a a and is with its in an to the fructosyl of sucrose and in their which us to the +1, +2, +3, and with the fructose the site and with the fructose that the The complex that the is with to the sucrose and in which the fructose of the are in similar is by residues and and catalytic residues and the the levanhexaose fructose is in with the sucrose and glucose that in these such as by the of and in of the and In the first the Å the of in to the away from the but an interaction with fructosyl a molecule in this as of the is in the with sucrose and a for the of In the of a of Å the of was the in the with sucrose and Of and a in with the of similar to that of the and their by and away from their in The in the of these residues with fructosyl with the of and and and with was the of these residues in the catalytic the in the of the are with In the the with residues and and the of G. Fütterer K. substrate in the of Bacillus subtilis Struct. PubMed Scopus Google Scholar). In the of the the fructose by with the of and the fructosyl and with the of and and the of and The crystallographic structure of the complex us to for the first time, the of subsites that in the of the catalytic cavity the is to the are identified In this that the of and in the the a with fructosyl and the a is important to that is to a interaction with which is an important that in subsites and in are that the of and the of and that In the the with the fructose of the In this of residues and which and Å and the of away from their in the were the enzyme and the fructose of the that from the catalytic Å away from the fructose was of interest its the in a that the interaction of Of in to the presence of the oligosaccharide the site, of the complex a oligosaccharide that was as a levanhexaose molecule a site site is on the enzyme 20 Å from the of the catalytic The oligosaccharide is to the enzyme by to the of to the of and and to In the oligosaccharide a to Furthermore, are additional the oligosaccharide and the in the The interaction of the site is by the five residues that the fructose the of the oligosaccharide a with the and with the of and the of and the number of five with the of and In fructosyl with the and the of and are in the of and in with the with Å the of and Å the of with the and the and five with In this fructosyl moiety with the of and as as with the and a molecule of the with and is to a a of fructosyl with the and the with the with and with a molecule of the in the fructosyl with a molecule and the of and is in β2-6 fructose on the site, to the fructosyl and of the levanhexaose in which are the with the number of with the The have high to to the and the of as in A. the functional of the site in levan variants were by which are involved in the and acceptor subsites and an The was to the the of the residues and the without affecting the In the of we additional to and to to the interaction with which is an important for sucrose A. R. S. J. oligosaccharide mutagenesis and of a from Bacillus J. PubMed Scopus Google Scholar, S. Y. Y. W. M. W. S. M. in of from the enzyme activities and 2011; PubMed Scopus Google Scholar). and were and and their were determined The variants showed with that of the for the and in the substrate affinity (Km) in variants and showed a in the value compared with that of the from to Of the variants of values by to the A. that the by in the a in the enzyme catalytic which was with the from the site of the The of the variants was in which a levan molecular sucrose and M. J. A. Olvera C. by enzyme levan elongation in Bacillus subtilis PubMed Scopus Google Scholar). analysis showed for and the formation of a and the of the products to for the of and to for the In variants and synthesized levans. In the of levans were The with oligosaccharide the effect on the of the products The had a similar to that of the WT, with a in the of the products in and showed products with a of to which the products were with a and 20 in In the products with a of levan-type compared with the the produced products to and was the FOS the variants in this as of to in the and variants showed of to and the and identified to A. Olvera C. the transfer the synthesis of molecular levan by Bacillus subtilis PubMed Scopus Google Scholar). is for additional products with a of to were but in In of the a in was in the had an similar to that of the which was to a of levans. In and and of the showed values and in the enzyme effect of the enzyme on the M. J. A. Olvera C. by enzyme levan elongation in Bacillus subtilis PubMed Scopus Google these were by a enzyme an effect on levan synthesis in which to an production of levans effect was in the with of and and as as the of products for and Furthermore, oligosaccharide similar to the enzyme were a in the in the were for variants compared with that of the and in the were for variants compared with that the enzyme for the first time, the of an LS with a levan-type The levanhexaose in the site the catalytic pocket from the and from the cavity the In this the oligosaccharide a substrate to the activity for J.R. Olvera C. Rodríguez-Alegría M.E. M. López Munguía A. activity of Bacillus subtilis PubMed Scopus Google the time, the the of a fructosyl transfer and its In the to an intermediate that is as acceptor substrate in nonprocessive levan synthesis from sucrose A. Olvera C. the transfer the synthesis of molecular levan by Bacillus subtilis PubMed Scopus Google Scholar). this the of a levan-type the catalytic pocket the acceptor subsites +1, +2, +3, and in an The complex showed which is for a fructosyl moiety and is by residues in LSs and of the residues in this site have the of mutagenesis resulting in enzyme activity A. R. S. J. oligosaccharide mutagenesis and of a from Bacillus J. PubMed Scopus Google Scholar, S. Y. Y. W. M. W. S. M. in of from the enzyme activities and 2011; PubMed Scopus Google Scholar, K. analysis of catalytic residues of the by the of Biochem. PubMed Scopus Google Scholar, M.E. M. Olvera C. A. in Bacillus subtilis affecting its PubMed Scopus Google Scholar, S. van in of from the of products J. PubMed Scopus Google Scholar, M.E. A. M.E. J. Olvera C. A. production by a Scopus Google Scholar, M.E. J.R. Rodríguez-Alegría M.E. A. J. Munguía of the on Bacillus subtilis and levan J. PubMed Scopus Google Scholar). +1, the complex residues and that with the and with which is an in the of the R. and activities of Bacillus subtilis by site-directed J. PubMed Scopus Google these residues to have as in and In the an acceptor with and with fructosyl of the a In an in which with the and is by the and with fructosyl to acceptor the catalytic as a to the of a molecule that the transfer catalytic an in the of acceptor and in and acceptor the of to the enzymatic activity and the synthesis of levans by the formation of with a of to were similar effect was in residues in LSs from and Bacillus licheniformis A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar, R. K. S. of Bacillus licheniformis for of the of levan-type J. PubMed Scopus Google which are enzymes that levans with M.E. J. J. J. J. J. A. J. of a an Sci. PubMed Google Scholar, S. R. K. M. of from Bacillus licheniformis and synthesis of levan J. PubMed Scopus Google Scholar). the formation of levans by a of this in levan was identified and residues involved in with the moiety in the In this we a of and with residues and their participation in the of acceptor with a of The of the of in with the effect of the of the to in which the of the to in to with A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar). in a relevant in LS activity A. R. S. J. oligosaccharide mutagenesis and of a from Bacillus J. PubMed Scopus Google which is to its participation in the have the participation of in the polymerization of and its to the products to A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar, R. S. and in the site of Bacillus licheniformis production of PubMed Scopus Google Scholar, R. K. S. of Bacillus licheniformis for of the of levan-type J. PubMed Scopus Google Scholar, R. R. M. S. A. K. J. of by substrate and enzyme Biotechnol. PubMed Scopus Google Scholar). that the showed an its effect to the of sucrose as an acceptor affecting the formation of and levans the of the elongation The complex the of a that residues and with the a molecule. The effect of the interaction that the of to to the and of LS M.E. J. J. J. J. J. A. J. of a an Sci. PubMed Google Scholar). In the mutagenesis on and showed that these are residues in the elongation of levans, which with and acceptor of with and with have the of the acceptor in this affecting the catalytic and the of products to of the of with effect on the formation of levans. in the of the additional the of this in the of which is an involved in the of sucrose in subsites and A. R. S. J. oligosaccharide mutagenesis and of a from Bacillus J. PubMed Scopus Google Scholar, S. Y. Y. W. M. W. S. M. in of from the enzyme activities and 2011; PubMed Scopus Google Scholar). Of which is by levans with of to a as the to and an as the to to mutagenesis these for the of the products synthesized by this The identified the which the of and a interaction was that the of this for residues the and of LS A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar, M.E. J. J. J. J. J. A. J. of a an Sci. PubMed Google Scholar). to and have with the of formation and the synthesis of of to to fructose A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar, S. S. R. K. Levan-type fructooligosaccharide production using Bacillus licheniformis on J. Sci. Technol. Scholar). the in and the oligosaccharide we that the synthesis of a levan results in a molecular is that the formation of levan by was high enzyme which with the we which is but is the fructose of the levanhexaose molecule in the the of to the formation of levans but the of the levans, its participation in the elongation of In this the in LS was to in enzyme M.E. J. J. J. J. J. A. J. of a an Sci. PubMed Google Scholar). In of the enzyme in this with a that a derivative to an the synthesis of levans M.E. J. J. J. J. J. A. J. of a an Sci. PubMed Google Scholar). LSs from subtilis and have the of their products and the the levanhexaose that the complex to in levan elongation in The in residues that with levanhexaose that were in this showed an effect on the ability to synthesize levans, which are products with the nonprocessive elongation mechanism M. J. A. Olvera C. by enzyme levan elongation in Bacillus subtilis PubMed Scopus Google Scholar, A. Olvera C. the transfer the synthesis of molecular levan by Bacillus subtilis PubMed Scopus Google Scholar). In the ability to these products to a was to the of the Of the of residues belonging to the +2, +3, and subsites the ability to produce levans of the of the In this by high enzyme which is a of we that the acceptor subsites +2, +3, and as by the to the the nonprocessive synthesis of levans, the synthesis of products the participation of an additional the acceptor involved in the nonprocessive mechanism by the synthesis of products with and in a that the in the of the we a with the mechanism for the nonprocessive from that the elongation as a that is the a by the acceptor subsites M. G. M. J. R. C. M. G. of the the resulting PubMed Scopus Google Scholar). for the subsites the acceptor in the catalytic pocket and its of to an in with the covalent fructosyl-enzyme intermediate and the levan The nonprocessive synthesis of is a in the of the by LSs. In LSs of such as from and the nonprocessive to the of J. C. R. A. G. R. and of by a with J. PubMed Scopus Google Scholar, A. J. fructooligosaccharide synthesis using the from effect of Scopus Google Scholar, C. M. van den Ende W. from first enzyme and Microbiol. Biotechnol. PubMed Scopus Google Scholar, A.D. C. of produced by from PubMed Scopus Google Scholar, R. M. M. S. of the from the its from the J. PubMed Scopus Google Scholar). In a of levans have in LSs of in the such as the LSs of subtilis and and licheniformis A. M. J. synthesis of the from Bacillus is by 2011; PubMed Scopus Google Scholar, S. R. K. M. of from Bacillus licheniformis and synthesis of levan J. PubMed Scopus Google Scholar, A. of by from Bacillus subtilis Biochem. Scopus Google Scholar, M. K. of synthesis of levan by Bacillus 49: Google Scholar, S. The molecular structure of and high molecular levans synthesized by Biochem. PubMed Scopus Google Scholar). The LSs of to in the enzyme to the of the residues belonging to the subsites +2, +3, and and for which is in residues of these subsites are in LSs of the Bacillus the site we that the LS a of the levanhexaose molecule the In the LSs of G. and in the and The G. LS a the LS a of which the of that the of this is for the elongation of levans in subtilis and which the elongation of in the of G. LS and the elongation of in the of the in these Owing to the structure of the a site in to the catalytic cavity was for the first in subtilis site the of a levanhexaose molecule by of its fructosyl a site from the catalytic site was identified in the catalytic pocket of in which was R. S. The structure of from a pocket for J. Sci. Scopus Google Scholar). is and Å the from the to the and in the complex The that the presence of the in the enzyme enzyme in the of in the In and have to such as the substrate the site, and S. and functional of in Biotechnol. 2012; PubMed Scopus Google Scholar). a of the levan elongation mechanism by the fructose acceptor and its from the the and the and of in the and as are with the the the of the site to the elongation of levan of as the site identified in from which as an to a the enzyme are in the site and In this the site a mechanism C. M. M. G. the molecular understanding of elongation by PubMed Scopus Google Scholar). In the site, five residues the levanhexaose molecule and of these residues are in enzymes from in the of the and and the functional of the which was in enzymes compared in this determined in LS to for the Km by the activity by and formation M. K. K. of important residues in Biochem. PubMed Scopus Google Scholar). is that the residues belonging to the site in the activity and polymerization in of the of and LSs showed a in the LSs from and G. that these LSs the of an oligosaccharide in this The of the LS this In is that is a the and in the subtilis and which a molecule in and of the is that the residues in this as of the acceptor are to this The of the structure in complex with a levan-type allowed us to for the first time, the of a levan and the central catalytic as as to identify the residues belonging to the subsites for this The interaction is by subsites that the the nonprocessive synthesis of levans. The of LSs that the in the identified acceptor subsites are for the of the and LSs the of and levans. Our results the of an additional the synthesis of levans that to

Topics & Concepts