A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

Abstract

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains. Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains. In recent decades, microbial polysaccharides have gained attention as promising biosourced polymers that can be regularly supplied and are less sensitive to market and climate fluctuations than plant polymers (1Freitas F. Alves V.D. Reis M.A. Advances in bacterial exopolysaccharides: from production to biotechnological applications.Trends Biotechnol. 2011; 29 (21561675): 388-39810.1016/j.tibtech.2011.03.008Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 2Moscovici M. Present and future medical applications of microbial exopolysaccharides.Front. Microbiol. 2015; 6 (26483763): 101210.3389/fmicb.2015.01012Crossref PubMed Scopus (121) Google Scholar). In addition, today’s progress in structure–function studies and engineering of polymerases allows us to better control their structures and by extension their physicochemical properties. In this field, the glucansucrases belonging to GH70 (family 70 of glycoside hydrolases) (3Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42 (24270786): D490-D49510.1093/nar/gkt1178Crossref PubMed Scopus (3267) Google Scholar) are very interesting enzymes. These α-transglucosylases catalyze the formation of high-molar-mass (HMM) and low-molar-mass (LMM) homopolysaccharides of d-glucose from sucrose, a low-cost and abundant substrate. The panel of polymers produced by glucansucrases varies dramatically in terms of size, type, and arrangement of α-glycosidic linkages and degree of branching, with all these features defining polymer structural properties and, consequently, the range of ongoing or potential applications. Among them, dextran, an α-1,6-linked glucan, was the first microbial polymer to be commercialized and used as plasma substitute (4Moulis C. André I. Remaud-Siméon M. GH13 amylosucrases and GH70 branching sucrases, atypical enzymes in their respective families.Cell. Mol. Life Sci. 2016; 73 (27141938): 2661-267910.1007/s00018-016-2244-8Crossref PubMed Scopus (29) Google Scholar). 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PubMed Scopus Google Scholar, properties of alternansucrase PubMed Scopus (48) Google Scholar) are by a of microbial glycoside as from sucrose which are to Scopus Google Scholar) or P. A. and properties of a J. PubMed Scopus Google Scholar) and are to which is of for applications as Recently, the resolution of ASR 3D structure to and molecular the structural for and specificity and for the that is by the of the of the to M. Moulis C. Monties N. Pizzut-Serin S. D. S. Cioci G. M. an of the structural determinants involved in the specificity of Catal. Scopus Google Scholar). residues in the of the site also found to be for alternan In the of and by alanine in and of polymer In addition, four sugar-binding pockets and to found in other GH70 family enzymes have identified in ASR domain V. of the domain V not polymer formation but it by M. Moulis C. Monties N. Pizzut-Serin S. D. S. Cioci G. M. an of the structural determinants involved in the specificity of Catal. Scopus Google Scholar). The control of polymer elongation by ASR is residues of both the catalytic site and domain V. To insight in this mechanism, we in the resolution of ASR To this ASR with different in terms of degree of and We with different of in domains A and V. with the the of the domain V in alternan formation and on the importance of a binding site found in domain A but from the catalytic which is by residues and and was named To the structural determinants for alternan with different in and structures The and a with a of that of and of and the with and we a in the domain A, in a site that we named surface-binding site and that in GH70 family enzymes the four the was also in the domain V at a to the sugar-binding previously proposed to be a sugar-binding M. Moulis C. Monties N. Pizzut-Serin S. D. S. Cioci G. M. an of the structural determinants involved in the specificity of Catal. Scopus Google Scholar). In was found in the as also previously to be a sugar-binding In all the the ASR in the with the of two in the and a very as the previously are between and in all the we to the A in which the is better The of be in from their We used the and for which we the resolution to the and that are in with the resolution structures of the branching from L. citreum NRRL in complex with and the of The is in with and with and and and also with the of and The mainly with the and with the and which that is a for the of as in branching and DSR-M In the the of the which is by and with and at resolution the other in and have also on the of the established mainly with because the of the the between and the three could be into the which a different than the that an is in with this as by an α-1,3-linked on the of the is not and the resolution of this complex not a of this it be that in all two are a in the and the domain V of is with of the of of are into and they to bind at the of the two pockets of In structures and of are with and of A and different residues are found in the and this we can propose that the sugar-binding pockets and are the is to be because of the of the with a at the of the was to be a of sugar-binding M. Cioci G. Vuillemin M. Monties N. Roblin P. Lippens G. Remaud-Siméon M. Moulis C. Investigations on the determinants responsible for low molar mass dextran formation by DSR-M dextransucrase.ACS Catal. 2017; 7: 7106-711910.1021/acscatal.7b02182Crossref Scopus (22) Google Scholar, Y. Y. G. L. Remaud-Siméon M. S. into the carbohydrate binding of an branching from glycoside hydrolase family Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). The structural of pockets and revealed but also in the seems to be with a between the and the of the that the for at in the To the of and the and by an The in a of the polymer from for the to and for the the and the double and and not significantly for these three mutants of the The from sucrose at with of enzyme and is The of was was in The was by with the was on in domain in in domain V was on in a To the of the enzyme with glucan, we of DSR-M truncated that sugar-binding pockets and M. Cioci G. Vuillemin M. Monties N. Roblin P. Lippens G. Remaud-Siméon M. Moulis C. Investigations on the determinants responsible for low molar mass dextran formation by DSR-M dextransucrase.ACS Catal. 2017; 7: 7106-711910.1021/acscatal.7b02182Crossref Scopus (22) Google and branching truncated Y. Pijning T. Y. L. S. G. P. S. Remaud-Siméon M. Dijkstra B.W. and structural characterization of branching from Biol. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar) that sugar-binding pockets and Y. Y. G. L. Remaud-Siméon M. S. into the carbohydrate binding of an branching from glycoside hydrolase family Biol. 2016; Full Text Full Text PDF PubMed Scopus Google in The two enzymes also sugar-binding pockets in which to the of the three enzymes is in the of dextran because enzyme sugar-binding pockets bind to a to the In ASR was by the of alternan in the Focusing on ASR sugar-binding both single and double and the binding of the enzyme with dextran and a of the sugar-binding in A is between the of and mutants in with the of with the not that the than the and is in with we the of in the The SBS-A1 site was for other GH70 enzymes. To the found in we first to the of the in the binding To was in the two with or in with The to in with because this the in a low and also allows the between and with the in the of the A. J. Res. 2013; PubMed Scopus Google Scholar, J. T. in 2015; PubMed Scopus Google Scholar). To this binding of that the is with the in with A was for both the and the that and all with their the and that this is of and we as an and to in the with the in with and could be in SBS-A1 for the and that have residues are found in with is in with and In addition, the and of with the of and with the of The of with and the with and and the with is not by with the and of with and of and are the of binding the four residues as for or of is not by we also crystal with and but could not complex in we that at two α-1,6-linked are for a in and the other in The and of the residues of the are in the as the three residues of and The and are The of with and and the is with and is a between and and also a with The is also with and The with residues not identified previously with to and the of the of with the of the and also be a with is that is in with and The this it a of the The five residues in with both and and Tyr717) by an alanine to their importance for enzyme specificity and polymer The not the enzyme or The was also with all mutants between and of with The product was for the and mutants In the was by the of and the importance of these residues for the of polymer decreased from for the enzyme to and for the and mutants and the double and The of polymer with the mutants is also a of the polymer as by the used to the of and alternan in the alternan the to with a of and low the polymer is and to could be to of branching other the of linkages was with the be to the and alternan structures in and that alternan from and L. citreum alternansucrase K. P. S. T. M. R. α-1,3-linked produced by Leuconostoc citreum alternansucrase with and PubMed Scopus Google Scholar) an size. analysis by of the sucrose with and the that the of with the is the of the the of the for the 6 mutants to the as for for the These from and to previously with a at the M. Moulis C. Monties N. Pizzut-Serin S. D. S. Cioci G. M. an of the structural determinants involved in the specificity of Catal. Scopus Google Scholar, S. and of Res. PubMed Scopus Google Scholar). To binding with alternan or we of which is a truncated of ASR of domain or in conditions with dextran or The was to the SBS-A1 site could for dextran and alternan in the of domain V. We not between the and in the of dextran or that the of of SBS-A1 to binding than that by or of sugar-binding pockets from domain V The was with of the residues in the sugar-binding of the domain and for and The alternan yield was from for the single to and for the and double The not significantly the and the was than for both mutants polymer formation with than with the and and the production is as and and In formation is with the which is in with a formation of polymer polymer formation is for the double mutants and in the and yield of polymer formation than that in We five crystal structures of alternansucrase in complex with different are the first with this a a unique specificity the GH70 glucansucrases by alternating and These structures us to oligosaccharide-binding sites in the of located in the domain and a site was identified in the domain A. We have their in the the and the to is to the that this enzyme the synthesis of both and The this was to identify structural that could the of ASR for or polymer synthesis in the have we all the we we have found and in domain V. domain a of with by for we have identified two sugar-binding and the structural traits to previously We have found for in of revealed that ASR dextran DSR-M M. Cioci G. Vuillemin M. Monties N. Roblin P. Lippens G. Remaud-Siméon M. Moulis C. Investigations on the determinants responsible for low molar mass dextran formation by DSR-M dextransucrase.ACS Catal. 2017; 7: 7106-711910.1021/acscatal.7b02182Crossref Scopus (22) Google Scholar). More we also found in and the between alternan and the domain V of the the binding of this In DSR-M and branching not by that structural the sugar-binding pockets could be different from enzyme to In the of is different in both DSR-M and with the of a and that could the in ASR in the complex with the seems to bind at the of the two domains a of glucansucrase can bind to a single polymer and this is very to in of the revealed the as DSR-M and branching a and a with the the residues with alanine in a but on the polymer yield that these pockets with the alternan and elongation by for chains. in the pockets less to alternan formation than of the domain V named of polymer M. Moulis C. Monties N. Pizzut-Serin S. D. S. Cioci G. M. an of the structural determinants involved in the specificity of Catal. Scopus Google Scholar) for and for the single or double mutants targeting the of the sugar-binding pockets and The of the of the pockets and not be to the in the double and to to an it be that of the domain V of of the also the of the other domains as by of with and this could have an on the production of that domain V a in the formation of targeting the of the to with the of the pockets in ASR domain V. SBS-A1 is the first surface-binding site for a GH70 family located in the catalytic and at a to the catalytic is in with the of surface-binding sites in GH13 and which to the as GH70 enzymes S. E. and of carbohydrate binding sites in glycoside Biotechnol. 2012; PubMed Scopus Google Scholar, D. C. C. S. J. M. M. B. of surface binding sites in carbohydrate enzymes with on glycoside hydrolase and a 2014; Scopus Google Scholar). the in and bind to SBS-A1 but and not not A of the complex revealed that in the proposed a α-1,6-linked could not be to the of because of a with the In the of an α-1,3-linked the to be and also the proposed binding of the in The between surface and the that the site is to with alternating and a of residues to be and Their by polymer yield from for to and for the that is an of SBS-A1 to alternan elongation. In with this of with the of domain V to a of the of polymer produced from to SBS-A1 be involved in the in to the catalytic site for to was for the GH13 of C. V. M. P. M. G. the molecular of elongation by Struct. Scopus Google a that to the than ASR and sucrose as to catalyze the formation of an the of to seems not at all by the in that which is from the on the a we propose the site as a of alternansucrase specificity. and are in all and characterization of an glucansucrase from Leuconostoc NRRL that an alternating Res. PubMed Scopus Google Scholar, K. P. S. T. M. R. α-1,3-linked produced by Leuconostoc citreum alternansucrase with and PubMed Scopus Google Scholar) and alternansucrase identified by L. citreum NRRL NRRL and P. key features in structures with the Acids Res. 2014; 42 PubMed Scopus Google Scholar) of GH70 is found in alternansucrase and is mainly by an of or a of in the other glucansucrases a is an between SBS-A1 site and the domain V of To this we double mutants the with of the in of the two pockets and The alternan yield from (ASRΔ2) to and decreased to and for the and double was also with the of alternan formation and provides of an between SBS-A1 and the sugar-binding in with the SBS-A1 could as a molecular and the of elongation as previously by Moulis C. G. D. E. G. P. Remaud-Siméon M. the of family 70 Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). a of in the of surface-binding sites because not both the SBS-A1 and the domain V to have a in but they also is in with the that and carbohydrate binding in carbohydrate enzymes D. B. binding sites in carbohydrate an of structural and of Scopus Google Scholar). To we five 3D structures of ASR in complex with different and in domain A or in domain V The of the domain as as the site SBS-A1 proposed as a of alternansucrase We have mutants for the formation of less than of alternansucrase for the formation of alternan to the domain V and the pockets to the for alternan and the enzyme a of the with a alternan and be a the resolution of crystal structures in complex with is to be very the of different as could be of could also be to the of the site to the domain V the site

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