Abstract
The thermostable endo-processive xyloglucanase MtXgh74 from Myceliophthora thermophila was used to study the influence of aromatic amino acids in the catalytic cleft on the mode of action and the ability of enzyme to reduce xyloglucan viscosity. The enzyme derivative Mut I with mutations W64A/W67A in the “negative” subsites of the catalytic cleft resulted in a 5.5-fold increase of the Km value. Mut I produced oligosaccharides of various lengths in addition to xyloglucan building blocks. The W320A/W321A substitutions in the “positive” subsites of the mutated enzyme Mut II catalytic cleft increased the Km value 54-fold and resulted in an endo-dissociative mode of action. The ability of Mut II to reduce the viscosity of xyloglucan at 50 °C was much better than that of other MtXgh74 variants. Besides, Mut II efficiently reduced viscosity of a natural substrate, the pulp of xyloglucan-containing tamarind seed flour. The Km, Vmax, and kcat values and viscosity reduction ability of the enzyme derivative Mut III (W320A/W321A/G446Y) returned to levels close to that of MtXgh74. The pattern of xyloglucan hydrolysis by Mut III was typical for endo-processive xyloglucanases. The thermostability of Mut I and Mut II at 60 °C decreased significantly compared to the wild type, whereas the thermostability of Mut III at 60 °C restored almost to the MtXgh74-wt value. All mutants lost the ability to cleave the backbone of xyloglucan building blocks which was a characteristic of MtXgh74. Instead they acquired a low branch removing activity. Molecular dynamics simulations revealed the role of mutated amino acids in the complex action mechanism of GH74 enzymes.
Key Points
• Endo-processive mode of action of the xyloglucanase MtXgh74 was altered by rational design.
• The endo-dissociative mutant Mut II (W320A/W321A) efficiently reduced XyG viscosity.
• The substitutions W320A/W321A/G446Y in Mut III recovered the endo-processive mode.
• Mut II can be used to reduce the viscosity of biomass slurries containing tamarind seed flour.
Similar content being viewed by others
Data availability
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
References
Alahuhta M, Adney WS, Himmel ME, Lunin VV (2013) Structure of Acidothermus cellulolyticus family 74 glycoside hydrolase at 1.82 Å resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun 69(PT 12):1335–1338. https://doi.org/10.1107/S1744309113030005
Arnal G, Stogios PJ, Asohan J, Skarina T, Savchenko A, Brumer H (2018) Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase. Biochem J 475(24):3963–3978. https://doi.org/10.1042/BCJ20180763
Arnal G, Stogios PJ, Asohan J, Attia MA, Skarina T, Viborg AH, Henrissat B, Savchenko A, Brumer H (2019) Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74. J Biol Chem 294(36):13233–13247. https://doi.org/10.1074/jbc.RA119.009861
Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27(3):343–350. https://doi.org/10.1093/bioinformatics/btq662
Berezina OV, Herlet J, Rykov SV, Kornberger P, Zavyalov A, Kozlov D, Sakhibgaraeva L, Krestyanova I, Schwarz WH, Zverlov VV, Liebl W, Yarotsky SV (2017) Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein. Appl Microbiol Biotechnol 101(14):5653–5666. https://doi.org/10.1007/s00253-017-8297-2
Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42(W1):W252–W258. https://doi.org/10.1093/nar/gku340
Bienert S, Waterhouse A, de Beer TAP, Tauriello G, Studer G, Bordoli L, Schwede T (2017) The SWISS-MODEL repository-new features and functionality. Nucleic Acids Res 45(Database issue):D313–D319. https://doi.org/10.1093/nar/gkw1132
Bussi G, Donadio D, Parrinello M (2007) Canonical sampling through velocity rescaling. J Chem Phys 126(1):014101. https://doi.org/10.1063/1.2408420
Darden T, York D, Pedersen L (1993) Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. J Chem Phys 98(12):10089–10092. https://doi.org/10.1063/1.464397
Deng JH, Luo J, Mao YL, Lai S, Gong YN, Zhong DC, Lu TB (2020) π-π stacking interactions: non-negligible forces for stabilizing porous supramolecular frameworks. Sci Adv 6(2):eaax9976. https://doi.org/10.1126/sciadv.aax9976
Desmet T, Cantaert T, Gualfetti P, Nerinckx W, Gross L, Mitchinson C, Piens K (2007) An investigation of the substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina. FEBS J 274(2):356–363. https://doi.org/10.1111/j.1742-4658.2006.05582.x
Feng T, Yan KP, Mikkelsen MD, Meyer AS, Schols HA, Westereng B, Mikkelsen JD (2014) Characterisation of a novel endo-xyloglucanase (XcXGHA) from Xanthomonas that accommodates a xylosyl-substituted glucose at subsite -1. Appl Microbiol Biotechnol 98(23):9667–9679. https://doi.org/10.1007/s00253-014-5825-1
Fry SC, York WS, Albersheim P, Darvill A, Hayashi T, Joseleau JP, Kato Y, Lorences EP, Maclachlan GA, McNeil M, Mort AJ, Reid JSG, Seitz HU, Selvendran RR, Voragen AGJ, White AR (1993) An unambiguous nomenclature for xyloglucan-derived oligosaccharides. Physiol Plant 89(1):1–3. https://doi.org/10.1111/j.1399-3054.1993.tb01778.x
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316:695–696. https://doi.org/10.1042/bj3160695
Heimbach JT, Egawa H, Marone PA, Bauter MR, Kennepohl E (2013) Tamarind seed polysaccharide: a 28-day dietary study in Sprague-Dawley rats. Int J Toxicol 32(3):198–208. https://doi.org/10.1177/1091581813484069
Huang J, MacKerell AD (2013) CHARMM36 all-atom additive protein force field: validation based on comparison to NMR data. J Comput Chem 34(25):2135–2145. https://doi.org/10.1002/jcc.23354
Huang R, Chen H, Zhou W, Ma C, Zhang Y-HP (2018) Engineering a thermostable highly active glucose 6-phosphate dehydrogenase and its application to hydrogen production in vitro. Appl Microbiol Biotechnol 102:3203–3215. https://doi.org/10.1007/s00253-018-8798-7
Ichinose H, Araki Y, Michikawa M, Harazono K, Yaoi K, Karita S, Kaneko S (2012) Characterization of an endo-processive-type xyloglucanase having a β-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis. Appl Environ Microbiol 78(22):7939–7945
Kozlov DG, Yagudin TA (2008) Antibody fragments may be incorrectly processed in the yeast Pichia pastoris. Biotechnol Lett 30:1661–1663
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Le Bars R, Bianchi MW, Lefebvre C (2019) Three-dimensional surface rendering of ESCRT proteins microscopy data using UCSF Chimera software. Methods Mol Biol 1998:149–161. https://doi.org/10.1007/978-1-4939-9492-2_11
Mark P, Nilsson L (2001) Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. J Phys Chem A 105(43):9954–9960. https://doi.org/10.1021/jp003020w
Martinez-Fleites C, Guerreiro CI, Baumann MJ, Taylor EJ, Prates JA, Ferreira LM, Fontes CM, Brumer H, Davies GJ (2006) Crystal structures of Clostridium thermocellum xyloglucanase, XGH74A, reveal the structural basis for xyloglucan recognition and degradation. J Biol Chem 281(34):24922–24933. https://doi.org/10.1074/jbc.M603583200
Matsuzawa T, Saito Y, Yaoi K (2014) Key amino acid residues for the endo-processive activity of GH74 xyloglucanase. FEBS Lett 588(9):1731–1738. https://doi.org/10.1016/j.febslet.2014.03.023
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428
Morton JF (1987) Tamarind. In: Morton JF, Fruits of the warm climates. Wipf and Stock Publishers, Miami, pp 115–121
Munusamy K, Somani RS, Bajaj HC (2011) Tamarind seeds carbon: preparation and methane uptake. BioRes 6(1)537–551. https://doi.org/10.15376/BIORES.6.1.537-551
Nishinari K, Takemasa M, Zhang H, Takahashi R (2007) Storage plant polysaccharides: xyloglucans, galactomannans, glucomannans. In: Kamerling H (ed) Comprehensive glycoscience. From chemistry to systems biology. Reference module in chemistry, molecular sciences and chemical engineering, vol 2. Elsevier Science, pp 613–652. https://doi.org/10.1016/B978-044451967-2/00146-X
Parrinello M, Rahman A (1981) Polymorphic transitions in single crystals: a new molecular dynamics method. J Appl Phys 52:7182–7190. https://doi.org/10.1063/1.328693
Payne CM, Baban J, Horn SJ, Backe PH, Arvai AS, Dalhus B, Bjørås M, Eijsink VGH, Sørlie M, Beckham GT, Vaaje-Kolstad G (2012) Hallmarks of processivity in glycoside hydrolases from crystallographic and computational studies of the Serratia marcescens chitinases. J Biol Chem 287(43):36322–36330. https://doi.org/10.1074/jbc.M112.402149
Pronk S, Páll S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Michael R, Shirts MR, Smith JC, Kasson PM, van der Spoel D, Hess B, Lindahl E (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics. 29(7):845–854. https://doi.org/10.1093/bioinformatics/btt055
Rykov SV, Kornberger P, Herlet J, Tsurin NV, Zorov IN, Zverlov VV, Liebl W, Schwarz WH, Yarotsky SV, Berezina OV (2019) Novel endo-(1,4)-β-glucanase Bgh12A and xyloglucanase Xgh12B from Aspergillus cervinus belong to GH12 subgroup I and II, respectively. Appl Microbiol Biotechnol 103(18):7553–7566. https://doi.org/10.1007/s00253-019-10006-x
Schultink A, Liu L, Zhu L, Pauly M (2014) Structural diversity and function of xyloglucan sidechain substituents. Plants 3(4):526–542. https://doi.org/10.3390/plants3040526
Seeliger D, de Groot BL (2010) Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24:417–422. https://doi.org/10.1007/s10822-010-9352-6
Stierand K, Maass PC, Rarey M (2006) Molecular complexes at a glance: automated generation of two-dimensional complex diagrams. Bioinformatics 22(14):1710–1716. https://doi.org/10.1093/bioinformatics/btl150
Studer G, Rempfer C, Waterhouse AM, Gumienny R, Haas J, Schwede T (2020) QMEANDisCo-distance constraints applied on model quality estimation. Bioinformatics 36(6):1765–1771. https://doi.org/10.1093/bioinformatics/btz828
Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455–461. https://doi.org/10.1002/jcc.21334
Uzodinma EO, Osagiede EG, Chikwendu JN (2020) Effect of different processing methods on chemical and pasting properties of tamarind (Tamarindus indica L.) seed flours. Agro-Science 19(1):1–10. https://doi.org/10.4314/as.v19i1.1
Watanabe M, Matsuzawa T, Yaoi K (2018) Rational protein design for thermostabilization of glycoside hydrolases based on structural analysis. Appl Microbiol Biotechnol 102(20):8677–8684. https://doi.org/10.1007/s00253-018-9288-7
Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. Methods Enzymol 160:87–117. https://doi.org/10.1016/0076-6879(88)60109-1
Xiang Z (2006) Advances in homology protein structure modeling. Curr Protein Pepte 7(3):217–227. https://doi.org/10.2174/138920306777452312
Yaoi K, Mitsuishi Y (2002) Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase. J Biol Chem 277(50):48276–48281. https://doi.org/10.1074/jbc.M208443200
Yaoi K, Mitsuishi Y (2004) Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128. FEBS Lett 560(1-3):45–50. https://doi.org/10.1016/S0014-5793(04)00068-7
Yaoi K, Nakai T, Kameda Y, Hiyoshi A, Mitsuishi Y (2005) Cloning and characterization of two xyloglucanases from Paenibacillus sp. Strain KM21. Appl Environ Microbiol 71(12):7670–7678. https://doi.org/10.1128/AEM.71.12.7670-7678.2005
Yaoi K, Kondo H, Hiyoshi A, Noro N, Sugimoto H, Tsuda S, Miyazaki K (2009) The crystal structure of a xyloglucan-specific endo-beta-1,4-glucanase from Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate specificity. FEBS J 276(18):5094–5100. https://doi.org/10.1111/j.1742-4658.2009.07205.x
Zavyalov AV, Rykov SV, Lunina NA, Sushkova VI, Yarotsky SV, Berezina OV (2019) Plant polysaccharide xyloglucan and enzymes that hydrolyze it (review). Russ J Bioorg Chem 45(7):845–859. https://doi.org/10.1134/S1068162019070148
Zoete V, Cuendet MA, Grosdidier A, Michielin O (2011) SwissParam: a fast force field generation tool for small organic molecules. J Comput Chem 32(11):2359–2368. https://doi.org/10.1002/jcc.21816
Acknowledgments
We express our gratitude to Dr. Ilya Toropygin from the Institute of Biomedical Chemistry, Moscow, for performance of the mass-spectrometry experiments; to Pavel Volkov from NRC Kurchatov Institute–IREA, Moscow, for performance of the viscosity assay; to Dr. Nils Thieme from the TUM for assistance in the HPAEC-PAD experiments. Molecular dynamics simulations were performed using facilities of N.N. Semenov Federal Research Center for Chemical Physics RAS (state task АААА-А19-119012990175-9).
Funding
The work was supported by the Ministry of Science and Higher Education of the Russian Federation (Grant № 075-15-2019-1658).
Author information
Authors and Affiliations
Contributions
O.V.B., S.V.R., and S.V.Y. conceived and designed research. O.V.B., S.V.R., A.K.P., and M.B. conducted experiments. M.E.B. and A.V.S. performed MD simulations. O.V.B. and V.V.Z. analyzed data. O.V.B., V.V.Z., and W.H.S. wrote and edited the manuscript. All authors read and approved the manuscript.
Corresponding authors
Ethics declarations
The article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Oksana V. Berezina and Sergey V. Rykov are equal contributors.
Rights and permissions
About this article
Cite this article
Berezina, O.V., Rykov, S.V., Polyakova, A.K. et al. Strategic aromatic residues in the catalytic cleft of the xyloglucanase MtXgh74 modifying thermostability, mode of enzyme action, and viscosity reduction ability. Appl Microbiol Biotechnol 105, 1461–1476 (2021). https://doi.org/10.1007/s00253-021-11106-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-021-11106-3