Eijsink, Vincent

Madhuprakash, Jogi

Podile, AR

Rani, TS

Sørlie, Morten

English

Brage NMBU

Contents lists available at ScienceDirectBBA - Proteins and Proteomicsjournal homepage: www.elsevier.com/locate/bbapapThermodynamic insights into the role of aromatic residues inchitooligosaccharide binding to the transglycosylating chitinase-D fromSerratia proteamaculansJogi Madhuprakasha,b,⁎, T. Swaroopa Ranib, Appa Rao Podileb, Vincent G.H. Eijsinka,Morten Sørliea,⁎a Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432 Ås, NorwaybDepartment of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad, IndiaA R T I C L E I N F OKeywords:ChitinaseThermodynamicsSubstrate bindingTransglycosylationHydrolysisChitin is crystalline polysaccharide made of β-1–4 linked N-acetylglucosamine (GlcNAc) units. In α-chitin, the most abundant form ofchitin, individual chains interact through three-dimensional hydrogenbond arrangements and stacking of the hydrophobic faces to form acrystal [1]. This makes chitin recalcitrant and difficult for enzymes todegrade. In Nature, degradation is catalyzed by family 18 chitinasesthat hydrolyze glycosidic linkages, primarily producing chitobiose [2].Central for the ability of chitinases to degrade chitin is a series of sur-face exposed aromatic amino acids in the active site cleft that bystrongly interacting with a single polysaccharide chain, promoting itsdissociation from the crystal and guiding into the active site [3]. Theseinteractions mediated by aromatic amino acids are also beneficial forthe catalysis of transglycosylation [4,5]. SpChiD from Serratia protea-maculans is an interesting chitinase due to its ability to catalyze bothhydrolysis and transglycosylation [6]. The active-site cleft of SpChiDcontains five exposed aromatic amino acids stretching from subsites−1to +5 (Fig. 1.). In this work, we have used isothermal titration ca-lorimetry (ITC) and a series of engineered SpChiD variants to assess thecontribution of each individual aromatic amino acid to the binding freeenergy of a single chitin chain, (GlcNAc)6.Initially, (GlcNAc)6 binding was investigated with the wild typeSpChiD where the catalytic acid Glu153 was mutated to an alanine-re-sidue to avoid hydrolysis [7], yielding a Kd of 0.35 ± 0.09 μM(ΔGr° = −8.9 ± 0.1 kcal/mol, ΔHr° = 3.2 ± 0.1 kcal/mol, and–TΔSr° = −12.1 ± 0.1 kcal/mol) at t = 25 °C (Fig. 1). Exchange ofTrp395 (−1 subsite) by alanine yielded a Kd of 16 ± 3 μM(ΔGr° = −6.6 ± 0.4 kcal/mol, ΔHr° = −3.5 ± 0.2 kcal/mol, and–TΔSr° = −3.1 ± 0.4 kcal/mol). Exchange of Trp114 (+1 subsite)yielded a Kd of 147 ± 85 μM (ΔGr° = −5.2 ± 0.3 kcal/mol,ΔHr° = 5.7 ± 0.7 kcal/mol, and –TΔSr° = −10.9 ± 0.8 kcal/mol).Exchange of Tyr226 (+2 subsite) yielded a Kd of 2.7 ± 0.4 μM(ΔGr° = −7.6 ± 0.1 kcal/mol, ΔHr° = 3.9 ± 0.2 kcal/mol, and–TΔSr° = −11.5 ± 0.2 kcal/mol). Exchange of Trp160 (+3 subsite)yielded a Kd of 37 ± 14 μM (ΔGr° = −6.0 ± 0.2 kcal/mol,ΔHr° = 7.4 ± 0.7 kcal/mol, and –TΔSr° = −13.4 ± 0.7 kcal/mol).Finally, exchange of Trp290 (+5 subsite) yielded a Kd of 0.7 ± 0.2 μM(ΔGr° = −8.4 ± 0.2 kcal/mol, ΔHr° = 5.3 ± 0.4 kcal/mol, and–TΔSr° = −13.7 ± 0.4 kcal/mol).Interestingly, the distribution between enthalpic and entropic con-tributions to the binding free energy to SpChiD-W395A is dramaticallydifferent compared to the other variants. The average ΔHr° and –TΔSr°are 5.4 ± 0.6 and − 12.6 ± 0.6 kcal/mol, respectively, for the othervariants, while binding of (GlcNAc)6 to SpChiD-W395A is accompaniedby a (favorable) ΔHr° of −3.5 kcal/mol and a –TΔSr° of −3.1 kcal/mol,suggesting a very different binding mode. Structures of family 18chitinases (Fig. 1; [9]) show that Trp395 is crucial for the energeticallydemanding conformational change of the GlcNAc moiety binding to the−1 subsite change from the 4C1 chair conformation to the 1,4B boathttps://doi.org/10.1016/j.bbapap.2020.140414Received 30 January 2020; Received in revised form 17 March 2020; Accepted 19 March 2020⁎ Corresponding authors at: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432 Ås,Norway.E-mail addresses: madhuprakash25@gmail.com (J. Madhuprakash), morten.sorlie@nmbu.no (M. Sørlie).BBA - Proteins and Proteomics 1868 (2020) 140414Available online 26 March 20201570-9639/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).Tconfirmation that is necessary to form the Michaelis complex [10]. It isthus conceivable that the W395A mutation reduces the enthalpic pen-alty of substrate binding.All other mutations also resulted in reduced binding affinity. Thelargest reduction in binding free energy (3.6 kcal/mol) was observedfor the mutations in subsite +1 (W114A) (Fig. 1). Mutation of Trp160 inthe +3 subsite also had a large effect, whereas the effect of mutatingthe tyrosine in the +2 subsite was less profound. The relatively smallcontribution of the tyrosine is also apparent from previous studies,which showed that replacement of this Tyr or of an analogous Phe-residue ChiA from S. marcescens by tryptophan leads to improvedbinding and, consequently, increased transglycosylation [4,5]. Still,removal of the Phe residue in ChiA (exchange with an Ala-residue)reduced transglycosylation [4]. Similar observation has been made for aclass V chitinase from cycad, Cycas revoluta where a Phe mutation toAla in positive subsites reduced transglycosylation activity [11].Finally, the lowest reduction in binding free energy (0.4 kcal/mol)among the mutants was observed for SpChiD-W290A (subsite +5).Interaction with this subsite would require the binding of (GlcNAc)6from subsites −1 to +5 in SpChiD. This may indeed happen sinceprevious studies have shown that SpChiD can catalyze hydrolysis whenonly the −1 subsite is occupied. It has been shown that (GlcNAc)4productively binds to −1 to +3 (next to binding to–2 to +2) [6] andthat SpChiD can cleave (GlcNAc)2, which is a unique feature for family18 chitinases [12]. Still, in reactions with (GlcNAc)6 there is probablylow occupancy for the +5 subsite, which may explain the limited effectof the W290A mutation.Combined, our results demonstrate that all five investigated re-sidues provide significant binding free energy to the carbohydratesubstrate. The tryptophan residues seem essential in providing neces-sary binding free energy for the formation of a stable Michaelis com-plex. These results are in accordance with results from previous studies[3–5] showing that tryptophan-residues provide more binding freeenergy to GlcNAc-residues than phenylalanine- and tyrosine-residues.This is also a feature observed in other GH systems i.e. xylosaccharidebinding to a family 10 xylanase where a Tyr-residue contributed less toFig. 1. A) Crystal structure of SpChiD (pdb 4nzc [8]) with (GlcNAc)5 taken from pdb entry 1e6n [9]. B) A close-up view of aromatic amino acids in the active site ofSpChiD highlighting key interactions with the substrate. C) The thermodynamic parameters of (GlcNAc)6 binding to individual SpChiD mutants and their con-centrations in the isothermal titration calorimetry experiments.J. Madhuprakash, et al. BBA - Proteins and Proteomics 1868 (2020) 1404142the binding free energy as well as the change in heat capacity than aTrp-residue [13].Generation and expression of mutant enzymes and their purificationhas been thoroughly described previously [6]. Table S1 shows details ofprimers and templates used for generation of SpChiD variants. Execu-tion of ITC experiments and the analysis of calorimetric data has beenthoroughly described previously [14,15]. ITC experiments were per-formed with a VP-ITC system from Microcal, Inc. (Northampton, MA).Solutions of (GlcNAc)6 were placed in the syringe whereas the reactioncell of the calorimeter contained solutions purified SpChiD variants;details are provided in Fig. 1. Titrations were performed at 25 °C in20 mM potassium phosphate, pH 6.0. Next to the mutations used todenote the various enzyme variants, all variants carried a mutation ofthe catalytic acid (E153A), which abolishes hydrolytic activity, thusenabling ITC measurements. Errors in Ka and ΔHr° were obtained asstandard deviations from four individual experiments. Kd, ΔGr°, ΔSr°,and -TΔSr° were calculated from Eq. 1, and errors in these parameterswere obtained from propagation of error.° = ° − ° = − =G H T S RT K RT KΔ Δ Δ ln lnr r r a d (1)Credit author statementJM designed and executed experiments, performed data analysis,wrote original draft.TSR designed experiments, edited original draft.ARP designed experiments, edited original draft.VGHE designed experiments, edited original draft.MS designed and executed experiments, performed data analysis,wrote original draft.Declaration of Competing InterestThe authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.Appendix A. Supplementary dataSupplementary data to this article can be found online at https://doi.org/10.1016/j.bbapap.2020.140414.References[1] P. Sikorski, R. Hori, M. 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Thermodynamic insights into the role of aromatic residues in chitooligosaccharide binding to the transglycosylating chitinase-D from Serratia proteamaculans

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Thermodynamic insights into the role of aromatic residues in chitooligosaccharide binding to the transglycosylating chitinase-D from Serratia proteamaculans

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