FEBS Letters 583 (2009) 1964–1968

journal homepage: www.FEBSLetters.org

Structural determinants of specificity of sucrose

Stéphanie Ravaud a,1, Xavier Robert a, Hildegard Watzlawick b, Richard Haser a, Ralf Mattes b, Nushin Aghajari a,* a Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, UMR 5086-CNRS/Université de Lyon, IFR128 ‘‘BioSciences Gerland – Lyon Sud”, 7 Passage du Vercors, F-69367 Lyon cedex 07, France b Universität Stuttgart, Institut für Industrielle Genetik, Allmandring 31, D-70569 Stuttgart, Germany article info abstract

Article history: The healthy sweetener isomaltulose is industrially produced from the conversion of sucrose by the Received 21 April 2009 sucrose SmuA from Protaminobacter rubrum. Crystal structures of SmuA in native and Accepted 2 May 2009 deoxynojirimycin complexed forms completed with modeling studies unravel the characteristics Available online 8 May 2009 of the isomaltulose synthases catalytic pocket and their binding mode. Comparison with the synthase MutB highlights the role of Arg298 and Arg306 residues and sur- Edited by Hans Eklund face charges in controlling product specificity of sucrose isomerases (isomaltulose versus trehalu- lose). The results provide a rationale for the specific design of optimized . Keywords: Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Sucrose isomerase Glycoside Aromatic clamp Crystal structure Deoxynojirimycin Molecular modeling

1. Introduction ature, pH and reaction conditions [5]. Crystal structures of two SI’s with distinct product specificity and physico-chemical parameters The market for alternative sweeteners holds considerable poten- (Supplementary Table 1) were determined: PalI an isomaltulose tial with the rising health concerns on diet, obesity and cardiovascu- synthase from Klebsiella sp. strain LX3 [6] and MutB a trehalulose lar diseases. Among the complex array of new compounds, synthase from Pseudomonas mesoacidophila MX-45 [8–10]. They palatinose is the only low and slow insulinaemic and glycemic car- display a catalytic core and an active site architecture characteristic bohydrate that provides prolonged energy supply. Further, its of glycoside hydrolase family 13 enzymes. Substrate recognition ‘tooth-friendliness’ and a sensory profile very similar to that of su- and processing as well as the reaction mechanism and specificity crose, opens up interesting opportunities for food and drink usage were earlier investigated for MutB pointing out the important role [1]. Present on the Japanese market since 1985, palatinose was re- of an aromatic clamp (Phe256/Phe280) and of the residues interacting cently approved for use in the US, the EU, Australia and New Zealand. with the substrate [9]. Such a complete characterization lacks for The current large-scale industrial production of palatinose (iso- isomaltulose synthases. Thus, besides the identification by se- maltulose, a-D-glucosylpyranosyl-1,6-D-fructofuranose) is mainly quence and biochemical analysis of the so-called isomerization mo- carried out using immobilized cells of Protaminobacter rubrum tif RLDRD located in a loop region [11], structural features ruling which is recognized as being safe for food ingredients and which out the product specificity of SI’s remain elusive. Identification of expresses the sucrose isomerase (SI; EC 5.4.99.11) SmuA [1]. the parameters determining the product ratio is crucial for optimiz- SI’s, identified in several microorganisms [2–7], catalyze the ing the efficiencies. SmuA from P. rubrum produces 85% isomerization of sucrose into variable amounts of isomaltulose isomaltulose, a minor quantity of trehalulose, monosaccharides and trehalulose (a-D-glucosylpyranosyl-1,1-D-fructofuranose) with and eventually isomaltose. Controlling and reducing the amounts the formation of glucose and fructose as by-products. The product of side reaction products within the isomaltulose production cur- ratio depends mainly on the bacterial strain but also on the temper- rently remain a considerable industrial problem. We therefore solved the crystal structures of SmuA in its native state and in complex with the competitive inhibitor deoxynojirimy- * Corresponding author. Fax: +33 (0)472722616. E-mail address: [email protected] (N. Aghajari). cin at 1.95 and 1.7 Å resolution, respectively. Compared to the cor- 1 Present address: Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075 CEA- responding MutB/deoxynojirimicyn structure [9], this first complex CNRS-Université Joseph Fourier, F-38027 Grenoble cedex 1, France. structure provides insights into the substrate binding mode in

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.05.002 S. Ravaud et al. / FEBS Letters 583 (2009) 1964–1968 1965

Table 1 Summary of refinement statistics.

Native SmuA SmuA/deoxynojirimycin

a b Rfactor /Rfree (%) 17.9/22.4 17.1/19.7 Content of the asymmetric unit Residues 558c 558c Water molecules 699 696 Ligandd – Deoxynojirimycin Rms deviation Bonds lengths (Å) 0.013 0.010 Bond angles (°) 1.60 1.60 Ramachandran plot analysis (%) Favored 88.2 88.6 Allowed 11.6 11.4 Disallowed 0.2 0.0 P jjF ðhÞjkjF ðhÞjj a R ¼ h Pobs calc with k, a scaling factor. jF ðhÞj h obs b Rfree was calculated as the Rfactor but from a test set constituted by 10% (native SmuA) or 5% (SmuA/deoxynojirimycin) of the total number of reflections randomly selected. c No electron density was observed for the first 15 residues of the mature protein. d In addition, eight ethylene glycol molecules and one citrate anion were located in both structures. isomaltulose synthases. With the structures of PalI and MutB, these data show general characteristics of the SI’s as well as typical and specific structural features linked to the product specificity.

2. Materials and methods

2.1. Crystallization, data collection and processing

Cloning, expression, purification, crystallization, collection and processing of native data were described [12]. The SmuA/deoxy- nojirimycin complex was obtained by co-crystallization in the crystallization buffer used for the native enzyme. Micro-crystals of native SmuA were used for microseeding in 24 h pre-equili- brated sitting drops containing 10 mM deoxynojirimycin. Diffrac- tion data (Supplementary Table 2) were processed with XDS [13].

2.2. Structure determination

The structure of SmuA was solved by molecular replacement (AMoRe) [14] using PalI as search model [6]. The SmuA/deoxynoj- irimycin structure was subsequently solved by direct phasing using the refined native structure. Models were refined using CNS [15], alternating with manual building (TURBO-FRODO) [16] and validated (PROCHECK and WHATCHECK) [17,18]. Refinement statistics are listed in Table 1. Figures were prepared with PyMOL [19] and GRASP [20]. Atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 3GBD and 3GBE).

3. Results and discussion

3.1. Overall structure Fig. 1. Structure of the SmuA/deoxynojirimycin complex. (A) Ribbon presentation of SmuA with the subdomain couloured in red, the N-terminal catalytic domain in green and the C-terminal domain in yellow. Catalytic residues are highlighted in The SmuA crystal structure shows the typical topology of GH13 pink and the deoxynojirimycin inhibitor bound in subsite 1 is shown as a stick enzyme structures with a three domain organization as described representation. (B) Close-up on the active site. 2Fobs Fcalc electron density map for the deoxynojirimycin is contoured at 1r. for PalI and MutB [6,9] (Fig. 1A). The central (b/a)8-barrel domain at the N-terminus comprises residues 16–119 and 190–493, the loop rich subdomain encompasses residues 120–190 and residues 3.2. Catalytic pocket interactions network 494–573 form two antiparallel b-sheets at the C-terminus. The overall structure of SmuA is highly similar to those of PalI and The azasugar derivative deoxynojirimycin, a known inhibitor of MutB with rmsd’s of 0.63 and 0.69 Å, respectively, between the carbohydrate processing enzymes [21], showed a strong inhibitory native structures in agreement with the sequence identities of effect on the isomaltulose synthase activity (Ki of 10 lM). As for 76.3% with PalI and 69.6% with MutB (Supplementary Fig. 1). MutB, deoxynojirimycin was used to probe the active site of SmuA 1966 S. Ravaud et al. / FEBS Letters 583 (2009) 1964–1968 and to mimic the oxocarbenium ion transition state [9]. In a 1.7 Å and/or further stabilization of the fructose ring for isomaltulose resolution structure one molecule of deoxynojirimycin was synthases. unequivocally located and oriented in the active site subsite 1 We generated a 3D molecular model (Supplementary data) and (Fig. 1A and B). As observed previously for MutB, native and inhib- analyzed the interaction network between SmuA and its substrate. itor complex structures of SmuA are nearly identical (rmsd of All MutB E254Q/sucrose interactions [9] are retrieved in the model 0.27 Å), indicating that protein–carbohydrate interactions do not (Supplementary Fig. 2A). Though, no direct or indirect interactions influence the overall structure. Upon deoxynojirimycin binding, between Arg306 and the sucrose molecule are seen, the model indi- rearrangements however occurred within the active site with cates that Arg306 certainly may adopt several conformations and Phe270 and Phe294 undergoing a concerted movement of 1–1.5 Å that such an interaction is plausible in term of distance and at the Cb levels. Glu268 (proton donor), Arg298 (part of isomeriza- geometry. tion motif, see below) and Arg429 turned slightly away from the inhibitor position. 3.3. Aromatic clamp residues MutB/deoxynojirimycin and SmuA/deoxynojirimycin structures are highly similar (rmsd of 0.64 Å). A backbone shift of 2.5 Å is Structural studies of MutB revealed an aromatic clamp, Phe256/ observed at the Na80–Na800 level. Located at the entrance of the Phe280 (MutB numbering) of significant importance in substrate rec- catalytic pocket, this region was previously highlighted as being ognition and reaction specificity [9], which is conserved in SmuA mobile in MutB [9] and this movement may have a direct impact (Phe270/Phe294). Comparing structures and models of SmuA indi- on the pocket volume. The orientation and the position of the cates that the aromatic clamp always moves in a concerted manner inhibitor ring and its interaction with the protein are nearly iden- and adopts several conformations as in the MutB structures (Fig. 2B). tical in the MutB and SmuA complexes (Fig. 2A, Supplementary These observations stress the importance of the clamp for all Table 3). The binding is mediated by 13 direct hydrogen bonds sucrose isomerases. The clamp is obviously not a direct determi- and by stacking to Tyr78. The nucleophile Asp214 and the catalytic nant of the isomaltulose/trehalulose product ratio since it is con- Asp342 perform strong interactions with respectively N-5 and O-2 served in both isomaltulose and trehalulose synthases, but is a of the ligand, while the proton donor Glu268 interacts only via a key player in the catalytic mechanism controlling/modulating the water molecule. The putative catalytic water, Wat1021, is also entrance/exit of the substrate/product. It might determine in direct conserved. line with the kinetics parameters of the enzyme, the time that the Besides a different orientation of the Glu268 side-chain, the most substrate stays in the pocket and favors or penalizes the tautomer- striking difference between the two complexes concerns Arg306 ization event required for trehalulose formation. which is located next to the entrance of the pocket delimiting sub- site +1. In MutB the Arg306 (Arg291 according to MutB numbering) side-chain adopts an orientation similar to that in native SmuA. In 3.4. Isomerization motif SmuA/deoxynojirimycin one additional conformation is seen in the electron density. The side chain of this second rotamer is oriented Earlier, 298RLDRD302 was defined as the isomerization motif [11]. in the opposite direction, pointing towards the active site (distance Mutations of the four charged residues were shown to influence the of 1.8 Å between Cf atoms of the two rotamers). Superimposition and product specificity of PalI and SmuA [11,22].It of SmuA/deoxynojirimycin onto the MutB inactive mutant E254Q/ was proposed, based on modeling studies, that this motif and espe- sucrose revealed that in SmuA this orientation would allow an cially the two arginines may be involved in fructose binding [22]. additional interaction with O-40 of the sucrose fructosyl moiety. Despite sequence differences, the motifs in MutB (284RYDRA288), Hence, this movement might be a prerequisite for recognition SmuA (298RLDRD302) and PalI (325RLDRD329) structures superim-

Fig. 2. Structural comparison of sucrose isomerase active sites. (A) Superimposition of SmuA/deoxynojirimycin and MutB/deoxynojirimycin active sites. Structures are shown in green and orange, respectively. Catalytic residues (SmuA, pale pink; MutB, pink), residues interacting with the inhibitor (SmuA, pale blue; MutB, dark blue), the aromatic clamp Phe residues (SmuA, pale grey; MutB, dark grey) and the isomerization motifs RLDRD/RYDRA (SmuA, purple; MutB, cyan; in addition with that of PalI, yellow) are highlighted. Deoxynojirimycin, perfectly superimposed in SmuA and MutB complexes, is presented in yellow ball-and-sticks. Residues are labelled according to SmuA sequence numbering. (B) Close-up on the aromatic clamp. Catalytic residues of native MutB are coloured in black and Phe residues (in double conformation) in pink, and counterparts in MutB/deoxynojirimycin (in ball-and-stick), native SmuA, SmuA/deoxynojirimycin, SmuA/sucrose and SmuA/isomaltulose models and PalI are shown in cyan, pale pink, red, yellow, blue and green, respectively. Residues are labelled according to SmuA sequence numbering. S. Ravaud et al. / FEBS Letters 583 (2009) 1964–1968 1967

Fig. 3. Molecular surfaces at the entrance of the catalytic pockets coloured as a function of the electrostatic potentials (red, negative and blue, positive charges): (A) MutB, (B) SmuA and (C) PalI. posed well especially at the Arg levels which are conserved in all excluded (mutations of the RLDRD motif lead to a strong decrease sucrose isomerases (Fig. 2A and Supplementary Fig. 3). of Vmax [11,22]), the surface charges at the catalytic pocket level The structure of MutB-E254Q/sucrose and the present study appeared as key parameters in line with the influence of the pH clearly show that the non-conserved L299 and D302 point away from on the catalytic reaction [5]. the active site and that the two Arg’s are not directly involved in the interaction with the fructosyl moiety of sucrose, neither with Acknowledgements the glucosyl moiety. Interestingly, the molecular model of SmuA/ isomaltulose (Supplementary Fig. 2B) suggests a significant rear- We acknowledge access to beamlines FIP BM30A and ID14-eh3 rangement at the +1 sub-site after isomerization and predicts a di- at the European Synchrotron Radiation facility (ESRF, Grenoble, rect interaction between the fructosyl moiety of isomaltulose and France) and the excellent support by the beamline scientists. Arg298, thus suggesting a possible role in the last step of the cata- lytic reaction rather than in substrate recognition. Appendix A. Supplementary data A deletion one residue downstream the 284RYDRA288 motif in MutB resulted in a rather large difference in the backbone and side Supplementary data associated with this article can be found, in chains conformations of residues 303–306 (SmuA numbering) be- the online version, at doi:10.1016/j.febslet.2009.05.002. tween isomaltulose synthases and trehalulose synthases which partially disturb the salt bridge network in the catalytic pocket References (Supplementary Table 4,R306–E401). The highly efficient and iso- maltulose specific Pantoea dispersa enzyme possesses a slightly dif- [1] Lina, B.A., Jonker, D. and Kozianowski, G. (2002) Isomaltulose (palatinose): a ferent motif RLDRY preceded by a proline. The relative flexibility of review of biological and toxicological studies. Food. Chem. Toxicol. 40, 1375– the motif might therefore be involved in the efficiency and in spec- 1381. [2] Cheetham, P.S. (1984) The extraction and mechanism of a novel isomaltulose- ificity variations between the different enzymes. synthesizing enzyme from Erwinia rhapontici. Biochem. J. 220, 213–220. [3] Nagai, Y., Sugitani, T. and Tsuyuki, K. (1994) Characterization of alpha- 3.5. Charge distribution glucosyltransferase from Pseudomonas mesoacidophila MX-45. Biosci. Biotechnol. Biochem. 58, 1789–1793. [4] Mattes, R., Klein, K., Schiweck, H., Kunz, M. and Munir, M. (1998) US Patent Analysis of the charge distribution at the surface of SmuA, PalI 5786140. and MutB showed significant differences at the active site entrance [5] Veronese, T. and Perlot, P. (1998) Proposition for the biochemical mechanism (Fig. 3). MutB is more negatively charged than the two isomaltu- occurring in the sucrose isomerase active site. FEBS Lett. 441, 348–352. [6] Zhang, D., Li, N., Lok, S.M., Zhang, L.H. and Swaminathan, K. (2003) lose synthases and some positive patches differ between the en- Isomaltulose synthase (PalI) of Klebsiella sp. LX3. Crystal structure and zymes. A number of arginines located in the vicinity of the active implication of mechanism. J. Biol. Chem. 278, 35428–35434. site are either involved in hydrogen bond interactions with the [7] Wu, L. and Birch, R.G. (2005) Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase substrate, the reaction intermediates and the product or in salt gene. Appl. Environ. Microbiol. 71, 1581–1590. bridges of the catalytic pocket. Slight perturbations in the charge [8] Ravaud, S., Watzlawick, H., Mattes, R., Haser, R. and Aghajari, N. (2005) balance may therefore influence the interaction network and the Towards the three-dimensional structure of a sucrose isomerase from Pseudomonas mesoacidophila MX-45. Biologia Cell. Mol. Biol. Sec. 60, 99–105. pocket geometry. Consequently, stabilization of the fructosyl [9] Ravaud, S., Robert, X., Watzlawick, H., Haser, R., Mattes, R. and Aghajari, N. moiety after the hydrolytic step and the product ratio could be (2007) Trehalulose synthase native and carbohydrate complexed structures affected, explaining the results described [11,22]. Increased stabil- provide insights into sucrose isomerization. J. Biol. Chem. 282, 28126–28136. [10] Ravaud, S., Robert, X., Watzlawick, H., Laurent, S., Haser, R., Mattes, R. and ization of this sugar moiety would prevent tautomerization and Aghajari, N. (2008) Insights into sucrose isomerisation from crystal structures favour formation of isomaltulose. of the Pseudomonas mesoacidophila MX45 sucrose isomerase, MutB. Biocatal. We have solved two 3D-structures of the P. rubrum sucrose Biotransfor. 26, 111–119. [11] Zhang, D., Li, N., Swaminathan, K. and Zhang, L.H. (2003) A motif rich in isomerase, SmuA, which is the industrially used enzyme due to charged residues determines product specificity in isomaltulose synthase. its high product specificity and to the fact that P. rubrum is FEBS Lett. 534, 151–155. accepted as safe for food ingredients. Taken together, our analyses [12] Ravaud, S., Watzlawick, H., Haser, R., Mattes, R. and Aghajari, N. (2006) and comparative studies with existing structures show that the Overexpression, purification, crystallization and preliminary diffraction studies of the Protaminobacter rubrum sucrose isomerase SmuA. Acta Cryst. sucrose isomerases product specificity determinants are subtle. F62, 74–76. No difference was observed at the subsite 1 between isomaltu- [13] Kabsch, W. (1993) Automatic processing of rotation diffraction data from lose and trehalulose synthases. At the +1 subsite, Arg298 and crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 306 795–800. Arg which are not directly involved in substrate recognition [14] Navaza, J. (2001) Implementation of molecular replacement in AMoRe. Acta may contribute to further stabilization of the fructose ring for iso- Cryst. D57, 1367–1372. maltulose synthases. They could therefore be envisioned as targets [15] Brünger, A.T. et al. (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Cryst. D54, 905–921. for the design of rationally modified enzymes. Although a link [16] Roussel, A. and Cambillau, C. (1992) TURBO-FRODO, Biographics AFMB, between the Vmax values and the product specificity can not be Marseille, France. 1968 S. Ravaud et al. / FEBS Letters 583 (2009) 1964–1968

[17] Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993) [21] Yu, J., Choi, S.Y., Moon, K.D., Chung, H.H., Youn, H.J., Jeong, S., Park, H. and PROCHECK: a program to check the stereochemical quality of protein Schultz, P.G. (1998) A glycosidase antibody elicited against a chair-like structures. J. Appl. Crystallogr. 26, 283–291. transition state analog by in vitro immunization. Proc. Natl. Acad. Sci. USA 95, [18] Hooft, R.W., Vriend, G., Sander, C. and Abola, E.E. (1996) Errors in protein 2880–2884. structures. Nature 381, 272. [22] Lee, H.C., Kim, J.H., Kim, S.Y. and Lee, J.K. (2008) Isomaltose production by [19] DeLano, W.L. (2002) PyMOL, DeLano Scientific, San Carlos, CA. modification of the fructose- on the basis of the predicted [20] Nicholls, A., Sharp, K.A. and Honig, B. (1991) Protein folding and association: structure of sucrose isomerase from Protaminobacter rubrum. Appl. Environ. insights from the interfacial and thermodynamic properties of hydrocarbons. Microbiol. 74, 5183–5194. Proteins 11, 281–296.