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Elimination of Competing Hydrolysis and Coupling Side Reactions of A Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution Ronan M Kelly, Hans Leemhuis, Henriëtte J Rozeboom, Niels van Oosterwijk, Bauke W. Dijkstra, Lubbert Dijkhuizen To cite this version: Ronan M Kelly, Hans Leemhuis, Henriëtte J Rozeboom, Niels van Oosterwijk, Bauke W. Dijkstra, et al.. Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucan- otransferase by directed evolution. Biochemical Journal, Portland Press, 2008, 413 (3), pp.517-525. 10.1042/BJ20080353. hal-00478977 HAL Id: hal-00478977 https://hal.archives-ouvertes.fr/hal-00478977 Submitted on 30 Apr 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical Journal Immediate Publication. Published on 21 Apr 2008 as manuscript BJ20080353 Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution Ronan M. Kelly1, Hans Leemhuis1, Henriëtte J. Rozeboom2, Niels van Oosterwijk2, Bauke W. Dijkstra2 and Lubbert Dijkhuizen1* 1Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands, and Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. 2Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Running head: Removing enzyme side reactions. * Corresponding author. Tel: 31-50-3632150; Fax: 31-50-3632154; Email: [email protected] 1 Abbreviations: CGTase, cyclodextrin glucanotransferase; epPCR, error-prone polymerase chain reaction; GH13, glycoside hydrolase family 13; MBS, maltose binding site; Tabium, Thermoanaerobacterium thermosulfurigenes strain EM1; pNPG7, 4-nitrophenyl-Į-D- maltoheptaoside-4-6-O-ethylidene. Thermoanaerobacterium thermosulfurigenes To reduce the competing hydrolysis cyclodextrin glucanotransferase primarily reaction, while maintaining the cyclization catalyzes the formation of cyclic Į-(1,4)- activity, we applied directed evolution, linked oligosaccharides (cyclodextrins) from introducing random mutations throughout THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080353 starch. This enzyme also possesses the cgt gene by error-prone PCR. Mutations unusually high hydrolytic activity as a side in two residues, Ser-77 and Trp-239, on the reaction, thought to be due to partial outer region of the active site, lowered the retention of ancestral enzyme function. This hydrolytic activity up to 15-fold with side reaction is undesirable since it produces retention of cyclization activity. In contrast, short saccharides that are responsible for the mutations within the active site could not breakdown of the cyclodextrins formed, thus lower hydrolytic rates, indicating an limitingStage the yield of cyclodextrins 2(a) produced. POST-PRINTevolutionary optimized role for cyclodextrin 1 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 21 Apr 2008 as manuscript BJ20080353 formation by residues within this region. maintaining primary catalytic function. The crystal structure of the most effective Subsequent functional analysis of various mutant, S77P, showed no alterations to the glucanotransferases from the superfamily of peptide backbone. However, subtle glycoside hydrolases also suggests a gradual conformational changes to the side chains of evolutionary progression of these enzymes active site residues had occurred, which may from a common ‘intermediate-like’ ancestor explain the increased cyclization/hydrolysis toward specific transglycosylation activity. ratio. This indicates that secondary effects of mutations located on the outer regions of the Keywords: protein evolution; amylase; catalytic site are required to lower the rates CGTase, protein stability; side reactions; of competing side reactions, whilst saccharide Introduction diversification from a common enzyme Enzymes are essentially a product of ancestor. All family members either evolution. Continuous evolution of enzymes hydrolyze or transglycosylate Į-glucosidic results in intermediates partially retaining linkages to produce Į-anomeric mono- and the initial ancestral function while catalysing oligo-saccharides; catalysis proceeds via a new function [1]. Over several evolutionary double displacement mechanism with initial phases, aided by selective pressure, new formation of a covalent glycosyl enzyme function dominates while ancestral intermediate [7]. It is therefore the type of function decreases to a mere side reaction acceptor substrate utilised in the second half [2-4]. This evolutionary phenomenon is of the reaction which determines enzyme clearly evident among members of the reaction specificity, a water molecule in the case of Į-amylases and a hydroxyl group of glycoside hydrolase family 13 (GH13), THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080353 retaining a multitude of minor side reaction a sugar substrate for the glucanotransferases. specificities and activities [5]. Members of Despite highly specific primary activities of GH13 act on starch, glycogen and related GH13 enzymes, many glucanotransferase oligo- and polysaccharides, representing the enzymes also catalyze hydrolysis as a side largest family of glycoside hydrolases. Their reaction. Extensive analysis of cyclodextrin active site regions share high sequence glucanotransferases (CGTase) revealed that this side reaction has a considerable impact similarity embedded in a TIM (ȕ/Į)8 barrel structuralStage fold [6], indicating 2(a) evolutionary POST-PRINTon final product yields. CGTases primarily 2 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 21 Apr 2008 as manuscript BJ20080353 catalyze the glucanotransferase reaction of Ala-233 in Bacillus clarkii 7364 CGTase by cyclization, for the formation of circular Į- basic amino acids enhanced Ȗ-cyclodextrin (1,4)-linked oligosaccharides (cyclodextrins) forming activity over 4-fold in the neutral (Fig. 1) [8]. These enzymes also exhibit, to a pH range [13], while substitution of Tyr-54 lesser extent, Į-amylase-like activity, in the amylomaltase from Thermus hydrolysing starch into short linear aquaticus reduced hydrolytic activity but saccharides (Fig. 1). The build-up of short also enhanced cyclization activity saccharides from starch hydrolysis promotes significantly [14]. yet another CGTase side reaction, coupling To generate CGTase variants with a low (Fig. 1). In the coupling reaction hydrolytic activity, we applied directed cyclodextrin rings are cleaved and evolution, introducing random mutations transferred to (short) saccharide molecules to throughout the cgt gene by error-prone (ep) yield linear products [9]. While most PCR, followed by site-specific saturation CGTases generally exhibit low hydrolytic mutagenesis. Parallel screening for high rates, Thermoanaerobacterium thermosulf- cyclization and reduced hydrolytic activity urigenes strain EM1 (Tabium) CGTase on starch yielded three mutations, with no displays unusually high hydrolytic activity, direct interactions with substrate. The crystal but still low compared to Į-amylases [10]. structure of the most effective mutant, S77P, By determining the structural/molecular revealed subtle conformational changes of a factors responsible for the high hydrolytic network of amino acids at the active site rates of Tabium CGTase, we aimed at responsible for the strongly reduced side significantly reducing this side activity. reaction rates with retention of primary Reduced hydrolytic rates would effectively catalytic function. limit the breakdown of cyclodextrins via the Experimental Procedures coupling reaction, thus enhancing the enzyme’s applicability for industrial Bacterial strains, plasmids and protein THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20080353 applications [11]. Previous protein production – Escherichia coli strain engineering studies, aimed at improving the MC1061 was used for DNA manipulations cyclodextrin size specificity of CGTases, and library screening. CGTase proteins were have been of limited success. A double produced and purified using Bacillus subtilis mutant, Y89D/S146P, at the substrate strain DB104A carrying plasmid pCScgt-tt, binding sites of Bacillus circulans 251 as described [15]. Plasmid carrying strains CGTase, increased Į-cyclodextrin were grown on LB medium at 37q C in the productionStage two-fold [12]. 2(a)Replacement of POST-PRINTpresence of kanamycin (50 Pg/ml for E. coli 3 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society
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