Analysis of Surface Binding Sites (SBS) within GH62, GH13, and GH77

Abstract

Certain interactions between carbohydrate active enzymes and polysaccharides involve surface binding sites (SBS) situated on catalytic domains outside of the active site. We recently undertook to develop a toolbox for SBS identification and characterization. In affinity gel electrophoresis (AGE) SBS containing proteins are migrating slower in native polyacrylamide electrophoresis gels cast with polysaccharide versus without polysaccharide. Amylolytic enzymes from GH13 and GH77 and xylanases from GH10 and GH11 are the best studied GH families with respect to SBS, presenting about half of the reported SBSs. In GH13 SBSs have been seen in 17 subfamilies including SBSs with highly diverse functions in the same enzyme. Circumstantial evidence is provided for an SBS in the GH77 MalQ from Escherichia coli, the bacterial orthologue of Arabidopsis DPE2 involved in starch metabolism. Furthermore, Aspergillus nidulans α-L-arabinofuranosidase AnAbf62A-m2,3 of GH62 that has very high activity on wheat arabinoxylan (WAX) shows an unusually strongly retarded migration by WAX during AGE analysis. Using a recent GH62 crystal structure as template, Trp²³ and Tyr⁴⁴ in an AnAbf62A-m2,3 model are proposed to form an SBS situated about 30 Å from the catalytic site. Compared to wild-type, W23A/Y44A AnAbf62A-m2,3 retained 45% activity on WAX and was less retarded in AGE by WAX as well as by barley β-glucan and birchwood xylan, which are neither hydrolysed nor inhibiting activity towards WAX. The presence of a functional SBS agrees with W23A/Y44A AnAbf62A-m2,3 retaining only 3-25% activity for arabinoxylo-oligosaccharides (AXOS) of DP 3-5 possibly reflecting allosteric activation of wild-type through SBS occupation by AXOS.

Abbreviations

ABF, α-L-arabinofuranosidase; AGE, affinity gel electrophoresis; AMY1, barley α-amylase 1; AXOS, arabinoxylo-oligosaccharides; CBM, carbohydrate binding module; CLSM, confocal laser scanning microscopy; DP, degree of polymerization; DPE2, disproportionating enzyme 2; GH10, GH11, GH13, GH62, GH77, glycoside hydrolase families 10, 11, 13, 62, 77; ITC, isothermal titration calorimetry; PAGE, polyacrylamide gel electrophoresis; SBD, starch binding domain; SPR, surface plasmon resonance; WAX, wheat arabinoxylan.

INTRODUCTION

Interactions between carbohydrate-active enzymes and polysaccharide substrates or polysaccharides residing in complex biological matrices, such as plant cell walls are characterized by the interacting macromolecules being much larger than the enzyme and often of very diverse structural organization. As a step to facilitate intimate and appropriately persisting contact the enzymes have evolved a number of handles. Best known and by far the most widely occurring are carbohydrate binding modules (CBMs).¹⁾ CBM polysaccharide specificity can be the same as that of the enzyme catalytic domain or different from this, e.g., for a non-substrate polysaccharide component in plant biomass. Examples of the first case are starch binding domains (SBDs) attached to the starch-converting enzymes glucoamylase, cyclodextrin glucanotransferase or glucan water dikinase,²⁾ while an example of the second case can be a cellulose binding domain attached to a xylanase.^{3) 4)} In particular during the past decade, however, a growing number of studies reported about surface binding sites (SBSs), also called secondary binding sites, in carbohydrate-active enzyme that interact with polysaccharides.^{5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24)} SBSs are characteristically situated at a distance of the active site on the surface of the enzyme catalytic domain or of a domain intimately associated with the catalytic domain. SBSs are able to engage in specific binding providing for example a polysaccharide anchoring function comparable to that of CBMs. Recently, SBSs have been the topic of reviews and a book chapter.^{25) 26) 27)} It is noted that SBSs and CBMs appear also to play complimentary roles as some enzymes contain both these types of structures.²⁶⁾ Even though the majority of SBSs have been identified in glycoside hydrolases, they are also reported in glucan phosphatases, starch, and glycogen synthases as well as plant α-glucan phosphorylase PHS2 and from early on in glycogen phosphorylases.^{17) 18) 19) 20) 28) 29) 30) 31)}

FUNCTIONAL ROLES PLAYED BY SBSS

To summarize the type of roles played by SBSs in carbohydrate-active enzymes,^{25) 26) 27)} classically SBSs can target enzymes to their polysaccharide substrate, guide the substrate polysaccharide chain into the active site and probably be involved in disentangling of supramolecular structures by isolating separate constituting polysaccharide chains for access by the enzyme active site.^{8) 9) 10) 11) 12) 16) 17) 18) 19) 20) 23) 24) 32) 33)} These various mechanisms may apply to water-insoluble as well as water-soluble or solubilised (dissolved) polysaccharides.^{8) 16) 32) 34)} By effectively localizing the enzyme to a polysaccharide target and directing the polysaccharide chain to the active site, SBSs can facilitate processive action, also referred to as multiple attack, which stands for consecutive catalytic events carried out in a single enzyme substrate encounter, hence augmenting the efficacy of product formation which is important once an enzyme is functionally positioned on a large substrate.^{8) 35)} In fact a large number of polysaccharide converting enzymes are processive.³⁶⁾ Furthermore, the event of SBS occupation by carbohydrate ligands may influence on the properties of the active site, considered as an allosteric regulation. A small number of cases thus support SBSs as binding sites for regulators, typically the substrate and/or the product of the enzyme.^{15) 35) 37) 38) 39)} One may speculate about the driving force behind evolving sites with this regulatory behavior, but substrate or product triggered activation/deactivation can have clear benefits for carbohydrate-active enzymes. A related function is seen for an SBS able to control favorable degradation by barley α-amylase of the substrate amylopectin,³³⁾ hence involving SBS influence on the enzyme specificity. Additional roles described for SBSs include assisting with passing products on to neighboring carbohydrate binding modules and transporters as seen in uptake by the gut bacterium Bacteriodes thetaiotaomicron of maltooligosaccharides produced by its own α-amylase, SusG, attached to the cell membrane at the outer surface of the bacterium. These proteins are all encoded by the same gene locus (Sus, starch utilization locus).^{34) 40)} An SBS can also merely serve as an anchor keeping the enzyme to a specific microenvironment for example being attached to the cell walls, starch granules or the surface of microbes that benefit from the action of the enzyme.⁴¹⁾ Finally, SBSs binding of carbohydrates may improve the conformational or the biological stability of an enzyme, as practically applied in the use of pharmacological chaperones in enzyme replacement therapy, for example with human α-galactosidase, as well as potentially enzymes connected with glycogen storage disease.^{42) 43) 44)}

METHODS FOR IDENTIFICATION AND CHARACTERIZATION OF SBSS

By far the largest number of SBSs have been identified in crystal structures, which were solved either after co-crystallization or soaking of preformed crystals with substrates, products, inhibitors or substrate mimics.^{5) 6) 7) 9) 10) 11) 13) 18) 19) 21) 22) 23) 24) 25) 26) 27) 29) 34) 39) 45)} NMR has also been to identify and characterize an SBS in a xylanase.¹²⁾ To validate the possible importance of the sites with observed capacity to bind oligosaccharides, mutational analysis represents a straightforward approach that consists in site-directed mutagenesis of the recombinant protein followed by characterization of functional and stability properties of the variants. It has been sought to uncover characteristic structural elements of SBSs for the purpose of mining structural databases. However, no robust tool has yet been developed possibly reflecting a wide structural diversity of SBSs (Darrell Cockburn, unpublished work). It was also noticed that a larger number of enzymes interacting with different α-glucans can share architectural features of their SBSs, whereas several enzymes interacting with xylan apply distinctly different modes of binding (Casper Wilkins, unpublished work). Characteristic features of SBSs include adjacent carbohydrate residues stacking onto a couple of aromatic rings^{5) 6) 7) 9) 34) 45)} which may be from neighboring residues in the protein sequence. Other SBSs look like clamps or coin slots constructed by two aromatic rings and yet others also appear as clamps and undergo conspicuous conformational changes by grasping the sugar ligand as a “pair of tongs”.^{5) 7) 19) 23) 39) 41) 45) 46)}

Despite the hurdles encountered with developing useful prediction tools for identifying SBS candidates, insight available on the structural characteristics of SBSs has been exploited in attempts to introduce improvements of SBSs by protein engineering. A few relatively successful cases have been reported on such structure-guided SBS engineering as well as a case of in vitro evolution in different xylanases.^{14) 15) 47)} However, for rational introduction of novel functionally advantageous SBSs one shall probably consider in silico protein engineering procedures involving computational structural design.

Identification of novel SBSs by simply screening protein crystals with oligosaccharides (e.g., via soaking experiments) for occupation on the surface at a distance of the active site seems very attractive. However, such an approach is practical only for those working with crystallography groups and is likely to result in false positives and false negatives due to interactions of carbohydrates in protein crystals that are not having a functional foundation. Another rather simple way to test an enzyme for the presence of SBSs is by affinity gel electrophoresis (AGE) in which native PAGE in gels cast with soluble polysaccharide causes slower migration of an interacting protein versus its migration in a control without polysaccharide.^{12) 26) 27) 32)} (Wilkens et al., in preparation). We conducted screens using AGE for about 50 enzymes and 20 polysaccharides. An excerpt of our data is given in Fig. 1 (Darrell Cockburn and Casper Wilkens, unpublished work).

Fig. 1.

Affinity gel electrophoresis.

Screening of interaction of enzymes from different glycoside hydrolase families with soluble polysaccharides typically embedded at 0.1‒0.5% (wt/vol).

AGE can also be used to estimate the binding constant, K_d for the enzyme complex formation from a series of experiments giving migration distances measured relative to a reference protein and applying the equation (1/R_mi = (1/R_mo)・{1+(c/K_D)} in plots of 1/R_mi versus the polysaccharide concentration (c), where R_mi and R_mo are mobilities of the protein relative to reference protein in the presence and absence of polysaccharide.⁴⁸⁾

SBSs have also be characterized for oligosaccharide binding and specificity by aid of surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) analyses.^{8) 12) 14) 15) 20) 25) 26) 27) 32) 49) 50)} Combination of SPR and ITC as well as AGE with a mutational analysis is usually very powerful for elucidation and characterization of the roles of key functional residues in SBS. This strategy has been employed also to investigate individual SBSs in the same enzyme and discriminate interactions at SBSs from those occurring at the active site, which is generally an important shortcoming of screening for SBSs using AGE and SPR. Even though active site mutagenesis may counter this complication, it can still be problematic to efficiently eliminate binding along the entire length of the substrate binding area at the active site and only few suitable reactive carbohydrate derivatives or analogues of substrates or products are available for mechanism-based covalent modification at the catalytic site or have a level of affinity suitable for discriminating between the active site and the SBS(s) in question.^{25) 26) 27)} Finally direct observation of the binding onto insoluble substrates, e.g., starch granules can be pursued combining confocal laser scanning microscopy (CLSM) and mutational analysis of SBSs.³²⁾

SURFACE BINDING SITES IN GH13

The most thoroughly investigated SBSs in GH13 belong to barley α-amylase 1 (AMY1).^{5) 7) 8) 32) 33) 35) 37) 45) 49)} This very large GH13 family was first subdivided into 34 subfamilies⁵¹⁾ and today 17 of the more than 40 subfamilies in GH13 have been found to contain an SBS.^{26) 27)} In the past we published work on the discrimination between the two SBSs of AMY1 (SBS1 and SBS2). SBS1 has a prominent role in adsorption onto starch granules, while SBS2 was involved in interaction with single α-glucan chains and associated with the fast and high-affinity component in the kinetics of amylopectin hydrolysis.^{8) 33)} Recently, we thus extended characterization of SBS1 and SBS2 in AMY1 to include interaction with starch granules of different botanical origin and type (i.e., normal, waxy, high-amylose) as well as the retardation by amylose, amylopectin, pullulan, and glycogen in AGE and distinction of interactions of maltooligosaccharides at the active site.³²⁾ The binding to starch granules was determined using the Langmuir adsorption isotherm and the modified Langmuir adsorption isotherm:

B is the fraction of bound enzyme, [S] the concentration of starch granules, B_max the maximum bound fraction, K_d the dissociation constant, and K_s the substrate inhibition constant. While K_d for normal corn starch was 0.24 mg/mL, the affinity was 50% higher for waxy and only 60% for high-amylose corn starch granules compared to normal ones.³²⁾ When analyzing the SBS2 Y380A mutant, its affinity for all three types of starch granules was approximately 10% of that of wild-type, however, compared to wild-type AMY1 for the SBS1 mutant W278A/W279A the affinity was 50, 75, and 30 fold reduced for normal, waxy and high-amylose corn starch granules, respectively. This indicated both the higher affinity for the waxy, i.e., “all amylopectin” starch granules and the lower affinity for the high-amylose granules as well as the larger importance of SBS1 versus SBS2 in binding onto starch granules. Secondly it underscored the preference for amylopectin binding by SBS2 over SBS1. The binding data were complemented by CLSM analyses of the SBS1, SBS2 and double SBS1/SBS2 mutants, of which the latter failed to bind to the granule surface. However, at damaged areas of the granules, e.g., cracks interrupting surface integrity, even the double SBS1/SBS2 mutant showed intense binding to the cracks suggesting that in this case the active site was able to interact with these newly exposed distinctly different structural elements of the granule.³²⁾

When analyzing the same SBS mutants and wild-type AMY1 in AGE for interaction with amylopectin, glycogen, amylose, and pullulan in solution (Fig. 2) retardation of wild-type was largest by glycogen followed by pullulan, amylopectin and amylose, decreasing in this order. The SBSs mutants again migrated illustrating the greater role of SBS1 in interaction with the α-glucans. A much more prominent effect, however, resulted with the SBS1/SBS2 double mutant W279A/Y380A, indeed the two SBSs in AMY1 are acting in synergy. SPR analysis of the different SBS1, SBS2, and SBS1/SBS2 mutants in combination with the binding of β-cyclodextrin, that has 20 fold higher affinity for SBS2 than SBS1 in wild-type AMY1,⁸⁾ indicated that binding to maltoheptaose primarily occurred at SBS2 with K_d around 1.2 mM, while K_m for the active site was 1.1 mM, and binding to SBS1 was concluded to be much weaker than to SBS2 and the active site.³²⁾

Fig. 2.

Affinity gel electrophoresis of barley AMY1 wild-type and mutants in SBS1 and SBS2.

The relative mobility is given for various soluble α-glucans: amylopectin and glycogen (0.1%), amyloase and pullulan (0.05%). (modified from Ref. 32).

SURFACE BINDING SITES IN GH77

The disproportionating enzymes of GH77 belong to the same clan GH-H of glycoside hydrolases as members of GH13, also called the α-amylase family. The clan GH-H enzymes share similar substrates and protein structural and domain organization including catalytic residues and catalytic site geometry. An enigmatic GH77 enzyme DPE2 involved in starch metabolism in plants has a counterpart in MalQ from Escherichia coli that is able to complement an Arabidopsis mutant plant lacking the DPE2 protein via a dpe2 gene knock out.⁵⁰⁾ MalQ does not contain a starch binding domain (SBD), while DPE2 has two SBDs of the CBM20 family N-terminally to the catalytic domain. Both MalQ and DPE2 wild-type bind the small starch mimic β-cyclodextrin with a sub-millimolar K_d as measured by SPR. Since β-cyclodextrin is not expected to be able to bind at the active site, this raised the question as to whether MalQ has an SBS that serves the same role as the CBM20(s) in DPE2.⁵⁰⁾ However, comparing the binding of maltooligosaccharides of DP 3‒7 to DPE2, MalQ and a variant with the proximal DPE2 CBM20 fused to the N-terminus of MalQ, showed the characteristic almost three orders of magnitude larger affinity of MalQ compared to DPE2 being essentially preserved in the CBM20-MalQ protein chimera. The DPE2 has a preference for maltohexaose as opposed to MalQ and CBM20-MalQ both showing increasing affinity with decreasing DP of maltooligosaccharides. Thus in spite of MalQ being capable of complementing for the physiological loss of DPE2 in dpe2 knock out plants, the two enzymes display distinctly different substrate preference and range of affinity.⁵⁰⁾

Previously, the amylomaltase from Thermus aquaticus was found to have an SBS in the crystal structure.³⁹⁾ Mutational analysis of Tyr⁵⁴⁾ situated at the SBS suggested that a resulting loss of binding at this site was accompanied by favoured flexibility for substrate enzyme interactions at the active site compatible with the unimolecular cyclization reaction producing large cyclodextrins. By contrast allosteric regulation through occupation of the SBS was needed for the disproportionation, hydrolysis, and coupling (transglycosylation) reactions also catalysed by this enzyme and this was negatively influenced by mutation of the SBS.³⁸⁾

SURFACE BINDING SITES IN GH62

α-L-Arabinofuranosidases (ABFs) are found in five different GH families.¹⁾ Recently we cloned and produced recombinantly an annotated enzyme from Aspergillus nidulans,⁵²⁾ AnAbf62A-m2,3 belonging to the small GH62 family that only contains ABFs and found that the enzyme exhibited exceptionally high activity towards WAX. AnAbf62A-m2,3 was exceptionally strongly retarded in AGE by WAX (Fig. 1) (Wilkens et al., in preparation), suggesting the presence of extra binding sites, since the enzyme has no CBM as deduced from analysis of the sequence (Casper Wilkens, unpublished observation). Lack of any crystal structure from the GH62 family left us at that time with a working model obtained using several GH43 ABF structures as template as GH62 and GH43 constitute clan GH-F.¹⁾ We also demonstrated the inverting mechanism by the release of β-arabinose from arabinoxylo-oligosaccharides (AXOS; Fig. 3) monitored by using NMR in addition showing that 1,3-linked arabinose was released about three times faster from the backbone xylose residue versus 1,2-linked arabinose (Wilkens et al., in preparation). In 2014 five crystal structures were published of GH62 enzymes from various fungi and bacteria.^{53) 54) 55) 56)} In a model of AnAbf62A-m2,3 made using the crystal structure of SthAbf62A from Streptomyces thermoviolacius⁵⁵⁾ (PDB ID 4O8O) as template, our attention was now drawn to Trp²³ and Tyr⁴⁴ in AnAbf62A-m2,3 situated near each other and adjacent to the extended putative substrate binding cleft. The candidate SBS was located at a distance of about 30 Å from the catalytic site (Fig. 4), where the AXOS decorating arabinose residue is accommodated in a narrow pocket at subsite －1 and cleaved off from the backbone xylose residue situated at subsite +1. The double mutant W23A/Y44A was much less retarded in AGE by WAX, but still retained 45% activity of wild-type on this polysaccharide substrate. Notably, two other cell wall polysaccharides barley β-glucan and birchwood xylan retarded AnAbf62A-m2,3 in AGE even though these are neither substrates nor inhibit the hydrolysis of WAX. The retardation in AGE by these two polysaccharides was affected by the mutation similarly to the effect on retardation by WAX suggesting that their binding interactions occur at an SBS that accommodate these different polysaccharides (Wilkens et al., in preparation). Surprisingly, the W23A/Y44A mutant lost 65‒96% of the activity of wild-type towards AXOS substrates (Fig. 3). This larger reduction than found with WAX hinted at that AXOS binding, which was lost in the W23A/Y44A mutant, in wild-type elicits allosteric activation, reminiscent to roles played by oligosaccharide substrate SBS interactions observed for other enzymes.^{11) 33) 37) 38) 39)} It was not possible to predict based on structures and sequence comparisons if a similar functional site will be found in other GH62 enzymes. However, with regard to the two subgroups defined by phylogenetic analysis of GH62,⁵³⁾ Trp²³⁾ is conserved in 75% of all family members, but not strictly within subgroups 1 or 2, while Tyr⁴⁴ is only present in 8% of the GH62 members, all from subgroup 2.

Fig. 3.

Arabinoxylo-oligosaccharides (AXOS) preparations AX2(I), AX3 (I), and AX4 (I) of DP 3‒5 used for characterization of the activity of GH62 α-L-arabinofuranosidase AnAbg62A-m2,3 from Aspergillus nidulans wild-type and double mutant W23A/Y44A at the putative surface binding site (SBS) (see Fig. 4).

Fig. 4.

Close-up of the putative surface binding site containing Trp²³ and Tyr⁴⁴ of AnAbf62A-m2,3 from Aspergillus nidulans modelled using SthAbf62A from Streptomyces thermoviolacius⁵⁵⁾ (PDB ID 4O8O) as template.

The non-reducing end of xylopentaose from the complex with ScAbf62A (PDB ID 3WN2) from Streptomyces coelicolor⁵⁴⁾ is shown accommodated at main chain subsite +3NR. Arabinofuranose (in orange) is shown at subsite －1 from SthAbf62A.⁵⁵⁾

CONCLUSION AND PROSPECTS

Even though SBSs have been identified in rather few among the very large number of carbohydrate-active enzymes¹⁾ they do represent a functional feature confined to a well defined non-catalytic structural site. Cases are reported where SBSs have been proven to be essential for function and there is a need to establish a comprehensive database of SBSs and develop predictive search tools for structural databases. It is relevant to provide the scientific community with a knowledge base on the one hand delivering access to insights on SBSs and on the other generating a starting point for rational engineering to achieve desired properties by introduction of functional SBSs.

ACKNOWLEDGMENTS

We are grateful to our colleagues Andreas Blennow, Morten M. Nielsen, Pedro M. Coutinho, Jean-Guy Berrin, Claire Dumon, Matthew Gentry, Hans Erik Mølager Christensen, Martin Willemoës, Jonas Nielsen, and Harry Brumer for stimulating discussions. This work was supported by the Danish Natural Science Research Council, the Danish Strategic Research Council, the Carlsberg Foundation, a H.C. Ørsted postdoctoral fellowship from the Technical University of Denmark (to DC), and PhD fellowships from the Technical University of Denmark (to CW and SA).

REFERRENCES

• 1)  V. Lombard, H.G. Ramulu, E. Drula, P.M. Coutinho, and B. Henrissat: The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res., 42, D490-D495 (2014).
• 2)  C. Christiansen, M. Abou Hachem, Š. Janeček, A. Viksø-Nielsen, A. Blennow, and B. Svensson: The carbohydrate binding module family 20―diversity, structure and function. FEBS J., 276, 5006-5029 (2009).
• 3)  T. Ponyi, L. Szabó, T. Nagy, L. Orosz, P.J. Simpson, M.P. Williamson, and H.J. Gilbert: Trp22, Trp24, and Tyr8 play a pivotal role in the binding of the family 10 cellulose-binding module from Pseudomonas xylanase A to insoluble ligands. Biochemistry, 39, 985-991 (2000).
• 4)  G. Paës, J.-G. Berrin, and J. Beaugrand: G11 xylanases: Structure/function/properties relationships and applications. Biotechnol. Adv., 30, 564-592 (2012).
• 5)  A. Kadziola, M. Søgaard, B. Svensson, and R. Haser: Molecular structure of a barley α-amylase-inhibitor complex: implications for starch binding and catalysis. J. Mol. Biol., 278, 205-217 (1998).
• 6)  Z. Dauter, M. Dauter, A.M. Brzozowski, S. Christensen, T.V. Borchert, L. Beier, K.S. Wilson, and G.J. Davies: X-ray structure of Novamyl, the five-domain “maltogenic” α-amylase from Bacillus stearothermophilus: Maltose and acarbose complexes at 1.7 Å resolution. Biochemistry, 38, 8385-8392 (1999).
• 7)  X. Robert, R. Haser, T.E. Gottschalk, F. Ratajczak, H. Driguez, B. Svensson, and N. Aghajari: The structure of barley α-amylase isozyme 1 reveals a novel role of domain C in substrate recognition and binding: “a pair of sugar tongs”. Structure, 11, 973-984 (2003).
• 8)  M.M. Nielsen, S. Bozonnet, E.-S. Seo, J.A. Mótyán, J.M. Andersen, A. Dilokpimol, M. Abou Hachem, G. Gyémánt, H. Næsted, L. Kandra, B.W. Sigurskjold, and B. Svensson: Two secondary carbohydrate binding sites on the surface of barley α-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules. Biochemistry, 48, 7686-7697 (2009).
• 9)  C. Ragunath, S.G. Manuel, V. Venkataraman, H.B.R. Sait, C. Kasinathan, and N. Ramasubbu: Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary α-amylase in substrate hydrolysis and bacterial binding. J. Mol. Biol., 384, 1232-1248 (2008).
• 10)  S.B. Larson, J.S. Day, and A. McPherson: X-ray crystallographic analyses of pig pancreatic α-amylase with limit dextrin, oligosaccharide, and α-cyclodextrin. Biochemistry, 49, 3101-3115 (2010).
• 11)  L.K. Skov, O. Mirza, D. Sprogøe, I. Dar, M. Rémaud-Siméon, C. Albenne, P. Monsan, and M. Gajhede: Oligosaccharide and sucrose complexes of amylosucrase. J. Biol. Chem., 227, 47741-47747 (2002).
• 12)  M.L. Ludwiczek, M. Heller, T. Kantner, and L.P. McIntosh: A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase. J. Mol. Biol., 373, 337-354 (2007).
• 13)  E. Vandermarliere, T.M. Bourgois, S. Rombouts, S. Van Campenhout, G. Volckaert, S.V. Strelkov, J.A. Delcour, A. Rabijns, and C.M. Courtin: Crystallographic analysis shows substrate binding at the －3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-β-xylanases. Biochem. J., 410, 71-79 (2008).
• 14)  S. Cuyvers, E. Dornez, M. Abou Hachem, B. Svensson, M. Horhorn, J. Chory, J.A. Delcour, and C.M. Courtin: Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase. Anal. Biochem., 420, 90-92 (2011).
• 15)  S. Cuyvers, E. Dornez, M.N. Rezaei, A. Pollet, J.A. Delcour, and C.M. Courtin: Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases. FEBS J., 278, 1098-1111 (2011).
• 16)  S. Cuyvers, E. Dornez, J.A. Delcour, and C.M. Courtin: The secondary substrate binding site of the Pseudoalteromonas haloplanktis GH8 xylanase is relevant for activity on insoluble but not soluble substrates. Appl. Microbiol. Biotechnol., 92, 539-549 (2011).
• 17)  S. Baskaran, V.M. Chikwana, C.J. Contreras, K.D. Davis, W.A. Wilson, A.A. Depaoli-Roach, P.J. Roach, and T.D. Hurley: Multiple glycogen binding sites in eukaryotic glycogen synthase are required for high catalytic efficiency toward glycogen. J. Biol. Chem., 286, 33999-34006 (2011).
• 18)  D.A. Meekins, H.-F. Guo, S. Husodo, B.C. Paasch, T.M. Bridges, D. Santelia, O. Kötting, C.W. Vander Kooi, and M.S. Gentry: Structure of the Arabidopsis glucan phosphatase LIKE SEXFOUR2 reveals a unique mechanism for starch dephosphorylation. Plant Cell, 25, 2302-2314 (2013).
• 19)  J.A. Cuesta-Seijo, M.M. Nielsen, L. Marri, H. Tanaka, S.R. Beeren, and M.M. Palcic: Structure of starch synthase I from barley: insight into regulatory mechanisms of starch synthase activity. Acta Crystallogr. Sect. D. Biol. Crystallogr., 69, 1013-1025 (2013).
• 20)  C. Wilkens, J.A. Cuesta-Seijo, M.M. Palcic, and B. Svensson: Selectivity of the surface binding site (SBS) on barley starch synthase I. Biologia, 69, 1118-1121 (2014).
• 21)  L. Sim, S.R. Beeren, J. Findinier, D. Dauvillée, S. Ball, A. Henriksen, and M.M. Palcic: Crystal structure of the Chlamydomonas starch debranching enzyme isoamylase ISA1 reveals insights into the mechanism of branch trimming and complex assembly. J. Biol. Chem., 289, 22991-23003 (2014).
• 22)  M.S. Møller, M.S. Windahl, L. Sim, M. Bøjstrup, M. Abou Hachem, O. Hindsgaul, M. Palcic, B. Svensson, and A. Henriksen: Oligosaccharide and substrate binding in the starch debranching enzyme barley limit dextrinase. J. Mol. Biol., 427, 1263-1277 (2015).
• 23)  Z.M. Ye, H. Miyake, M. Tatsumi, S. Nishimura, and Y. Nitta: Two additional carbohydrate-binding sites in β-amylase from Bacillus cereus var. mycoides are involved in hydrolysis and raw starch-binding. J. Biochem. (Tokyo), 135, 355-363 (2004).
• 24)  J. Allouch, W. Helbert, B. Henrissat, and M. Czjzek: Parallel substrate binding sites in a β-agarase suggest a novel mode of action on double-helical agarose. Structure, 12, 623-632 (2004).
• 25)  S. Cuyvers, E. Dornez, J.A. Delcour, and C.M. Courtin: Occurrence and functional significance of secondary carbohydrate binding sites in glycoside hydrolases. Crit. Rev. Biotechnol., 32, 93-107 (2012).
• 26)  D. Cockburn and B. Svensson: Surface binding sites in carbohydrate active enzymes: an emerging picture of structural diversity. in SPR Carbohydrate Chemistry―Chemical and Biological Approaches, T.K. Lindhorst and A.P. Rauter, eds., Royal Society of Chemistry, Cambridge, pp. 204-221 (2013).
• 27)  D. Cockburn, C. Wilkens, S. Andersen, J.W. Nielsen, A.M. Smith, R.A. Field. M. Willemoës, M. Abou Hachem, and B. Svensson: Analysis of surface binding sites (SBS) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77―A mini-review. Biologia, 69, 705-712 (2014).
• 28)  G. Philip, G. Gringel, and D. Palm: Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site. Biochemistry, 21, 3043-3050 (1982).
• 29)  E.J. Goldsmith, R.J. Fletterick, and S.G. Withers: The three-dimensional structure of acarbose bound to glycogen phosphorylase. J. Biol. Chem., 262, 1449-1455 (1987).
• 30)  H. Mori, K. Tanizawa, and T. Fukui: Engineered plant phosphorylase showing extraordinarily high affinity for various α-glucan molecules. Protein Sci., 2, 1621-1629 (1993).
• 31)  E.C. O’Neill, A. Rashid, C.E.M. Stevenson, A.-C. Hetru, A.P. Gunning, M. Rejzek, S.A. Nepogodiev, S. Bornemann, D.M. Lawson, and R.A. Field: Sugar-coated sensor chip and nanoparticle surfaces for the in vitro enzymatic synthesis of starch-like materials. Chem. Sci., 5, 341-350 (2014).
• 32)  D. Cockburn, M.M. Nielsen, C. Christiansen, J.M. Andersen, J.B. Rannes, A. Blennow, and B. Svensson: Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation. Int. J. Biol. Macromol., 75, 338-345 (2015).
• 33)  J.W. Nielsen, B. Kramhøft, S. Bozonnet, M. Abou Hachem, S.L.S. Stipp, B. Svensson, and M. Willemoës: Degradation of the starch components amylopectin and amylose by barley α-amylase 1: Role of surface binding site 2. Arch. Biochem. Biophys., 528, 1-6 (2012).
• 34)  N.M. Koropatkin and T.J. Smith: SusG: A unique cell-membrane-associated α-amylase from a prominent human gut symbiont targets complex starch molecules. Structure, 18, 200-215 (2010).
• 35)  B. Kramhøft, K.S. Bak-Jensen, H. Mori, N. Juge, J. Nøhr, and B. Svensson: Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley α-amylase. Biochemistry, 44, 1824-1832 (2005).
• 36)  J.F. May, R.A. Splain, C. Brotschi, and L.L. Kiessling: A tethering mechanism for length control in a processive carbohydrate polymerization. Proc. Natl. Acad. Sci. USA, 106, 11851-11856 (2009).
• 37)  N. Oudjeriouat, Y. Moreau, M. Santimone, B. Svensson, G. Marchis-Mouren, and V. Desseaux: On the mechanism of barley α-amylase. Acarbose and cyclodextrin inhibition of barley amylase isozymes. Eur. J. Biochem., 270, 3871-3879 (2003).
• 38)  K. Fujii, H. Minagawa, Y. Terada, T. Takaha, T. Kuriki, J. Shimada, and H. Kaneko: Function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase. J. Biosci. Bioeng., 103, 167-173 (2007).
• 39)  I. Przylas, Y. Terada, K. Fujii, T. Takaha, W. Saenger, and N. Sträter: X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus. Implications for the synthesis of large cyclic glucans. Eur. J. Biochem., 267, 6903-6912 (2000).
• 40)  D.N. Bolam and N.M. Koropatkin: Glycan recognition by the Bacteroidetes Sus-like systems. Curr. Opin. Struct. Biol., 22, 563-569 (2012).
• 41)  W.M. Patrick, Y. Nakatani, S.M. Cutfield, M.I. Sharpe, R.J. Ramsay, and J.F. Cutfield: Carbohydrate binding sites in Candida albicans exo-β-1,3-glucanase and the role of the Phe-Phe ‘clamp’ at the active site entrance. FEBS J., 277, 4549-4561 (2010).
• 42)  A.I. Guce, N.E. Clark, E.N. Salgado, D.R. Ivanen, A.A. Kulminskaya, H. Brumer, and S.C. Garman: Catalytic mechanism of human α-galactosidase. J. Biol. Chem., 285, 3625-3632 (2010).
• 43)  N. Asano, S. Ishii, H. Kizu, K. Ikeda, K. Yasuda, A. Kato, O.R. Martin, and J.Q. Fan: In vitro inhibition and intracellular enhancement of lysosomal α-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. FEBS J., 267, 4179-4186 (2000).
• 44)  G. Parenti, C. Pignata, P. Vajro, and M. Salerno: New strategies for the treatment of lysosomal storage diseases (Review). Int. J. Mol. Med., 31, 11-20 (2013).
• 45)  X. Robert, R. Haser, H. Mori, B. Svensson, and N. Aghajari: Oligosaccharide binding to barley α-amylase 1. J. Biol. Chem., 280, 32968-32978 (2005).
• 46)  J. Sevcík, E. Hostinová, A. Solovicová, J. Gasperík, Z. Dauter, and K.S. Wilson: Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain. FEBS J., 273, 2161-2171 (2006).
• 47)  L. Song, B. Siguier, C. Dumon, S. Bozonnet, and M.J. O’Donohue: Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy. Biotechnol. Biofuels, 5, article 3 (2012).
• 48)  K. Takeo and S. Nakamura: Dissociation-constants of glucan phosphorylases of rabbit tissues studied by polyacrylamide-gel disk-electrophoresis. Arch. Biochem. Biophys., 153, 1-7 (1972).
• 49)  S. Bozonnet, M.T. Jensen, M.M. Nielsen, N. Aghajari, M.H. Jensen, B. Kramhøft, M. Willemoës, S. Tranier, R. Haser, and B. Svensson: The “pair of sugar tongs” site on the non-catalytic domain C of barley α-amylase participates in substrate binding and activity. FEBS J., 274, 5055-5067 (2007).
• 50)  C. Ruzanski, J. Smirnova, M. Rejzek, D. Cockburn, H.L. Pedersen, M. Pike, W.G.T. Willats, B. Svensson, M. Steup, A.M. Smith, and R.A. Field: A bacterial glucanotransferase can replace the complex maltose metabolism required for starch to sucrose conversion in leaves at night. J. Biol. Chem., 288, 28581-28598 (2013).
• 51)  M.R. Stam, E.G.J. Danchin, C. Rancurel, P.M. Coutinho, and B. Henrissat: Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of α-amylase-related proteins. Prot. Eng. Des. Sel., 19, 555-562 (2006).
• 52)  S. Bauer, P. Vasu, S. Persson, A.J. Mort, and C.R. Somerville: Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. Proc. Natl. Acad. Sci. USA, 103, 11417-11422 (2006).
• 53)  B. Siguier, M. Haon, V. Nahoum, M. Marcellin, O. Burlet-Schiltz, P.M. Coutinho, B. Henrissat, L. Mourey, M.J. O’Donohue, J.G. Berrin, S. Tranier, and C. Dumon (2014) First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies. J. Biol. Chem., 289, 5261-5273 (2014).
• 54)  T. Maehara, Z. Fujimoto, H. Ichinose, M. Michikawa, K. Harazono, and S. Kaneko (2014) Crystal structure and characterization of the glycoside hydrolase family 62 α-L-arabinofuranosidase from Streptomyces coelicolor. J. Biol. Chem., 289, 6962-6872 (2014).
• 55)  W. Wang, G. Mai-Gisondi, P.J. Stogios, A. Kaur, X. Xu, H. Cui, O. Turunen, A. Savchenko, and E.R. Master: Elucidation of the molecular basis for arabinoxylan-debranching activity of a thermostable family GH62 α-L-arabinofuranosidase from Streptomyces thermoviolaceus. Appl. Environ. Microbiol., 80, 5317-5329 (2014).
• 56)  A.P. Kaur, B.P. Nocek, X. Xu, M.J. Lowden, J.F. Leyva, P.J. Stogios, H. Cui, R. Di Leo, J. Powlowski, A. Tsang, and A. Savchenko: Functional and structural diversity in GH62 α-L-arabinofuranosidases from the thermophilic fungus Scytalidium thermophilum. Microb. Biotechnol., 8, 419-433 (2015).