Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase

Krishan Kumar, Márcia A. S. Correia, Virgínia M. R. Pires, Arun Dhillon, Kedar Sharma, Vikky Rajulapati, Carlos M. G. A. Fontes, Ana Luísa Carvalho, Arun Goyal

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

Original languageEnglish
Pages (from-to)890-901
Number of pages12
JournalInternational Journal of Biological Macromolecules
Volume117
DOIs
Publication statusPublished - 1 Oct 2018

Fingerprint

Cellulosomes
Clostridium thermocellum
Clostridium
Glucans
Glycoside Hydrolases
Ions
Degradation
Freezing
Oligosaccharides
Mutagenesis
Gene encoding
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Ethylenediaminetetraacetic acid
Molecular mass
Structural integrity
Site-Directed Mutagenesis
Catalysis
Edetic Acid
Polymerization
Melting point

Keywords

  • Clostridium thermocellum
  • Laminarin
  • Thin layer chromatography
  • X-ray crystallography
  • β-1,3-Glucanase

Cite this

@article{4d54e0fb4c654a439b2b2fbcdceee4d2,
title = "Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase",
abstract = "The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 {\AA} resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.",
keywords = "Clostridium thermocellum, Laminarin, Thin layer chromatography, X-ray crystallography, β-1,3-Glucanase",
author = "Krishan Kumar and Correia, {M{\'a}rcia A. S.} and Pires, {Virg{\'i}nia M. R.} and Arun Dhillon and Kedar Sharma and Vikky Rajulapati and Fontes, {Carlos M. G. A.} and Carvalho, {Ana Lu{\'i}sa} and Arun Goyal",
note = "Sem PDF conforme despacho. info:eu-repo/grantAgreement/FCT/COMPETE/132972/PT# info:eu-repo/grantAgreement/FCT/5876/147258/PT# This research work was carried out under bilateral joint DST (Ministry of Science and Technology, India) project (No. DST/INT/Portugal/P-14/2013) in collaboration with FCT Lisbon, Portugal (grant references PTDC/BIA-MIC/5947/2014, PTDC/BBB-BEP/0869/2014, IF/01621/2013/CP1183/CT0002, PEst-C/EQB/LA0006/2013 and UID/Multi/04378/2013 to Research Unit UCIBIO also co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728)) and in part supported by funding from Indian Institute of Technology Guwahati (IIT), Guwahati, Assam, India. The authors are also grateful to Professor Maria Joao Roma, for usage of the Macromolecular Crystallography Lab facilities (FCT-NOVA, Caparica), to Central Instrument Facility, IIT Guwahati for providing the MALDI-TOF facility and to the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and the Diamond Light Source (DLS, Oxfordshire, UK) for access to synchrotron facilities.",
year = "2018",
month = "10",
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doi = "10.1016/j.ijbiomac.2018.06.003",
language = "English",
volume = "117",
pages = "890--901",
journal = "International Journal of Biological Macromolecules",
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publisher = "Elsevier Science B.V., Inc",

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Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase. / Kumar, Krishan; Correia, Márcia A. S.; Pires, Virgínia M. R.; Dhillon, Arun; Sharma, Kedar; Rajulapati, Vikky; Fontes, Carlos M. G. A.; Carvalho, Ana Luísa; Goyal, Arun.

In: International Journal of Biological Macromolecules, Vol. 117, 01.10.2018, p. 890-901.

Research output: Contribution to journalArticle

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T1 - Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase

AU - Kumar, Krishan

AU - Correia, Márcia A. S.

AU - Pires, Virgínia M. R.

AU - Dhillon, Arun

AU - Sharma, Kedar

AU - Rajulapati, Vikky

AU - Fontes, Carlos M. G. A.

AU - Carvalho, Ana Luísa

AU - Goyal, Arun

N1 - Sem PDF conforme despacho. info:eu-repo/grantAgreement/FCT/COMPETE/132972/PT# info:eu-repo/grantAgreement/FCT/5876/147258/PT# This research work was carried out under bilateral joint DST (Ministry of Science and Technology, India) project (No. DST/INT/Portugal/P-14/2013) in collaboration with FCT Lisbon, Portugal (grant references PTDC/BIA-MIC/5947/2014, PTDC/BBB-BEP/0869/2014, IF/01621/2013/CP1183/CT0002, PEst-C/EQB/LA0006/2013 and UID/Multi/04378/2013 to Research Unit UCIBIO also co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728)) and in part supported by funding from Indian Institute of Technology Guwahati (IIT), Guwahati, Assam, India. The authors are also grateful to Professor Maria Joao Roma, for usage of the Macromolecular Crystallography Lab facilities (FCT-NOVA, Caparica), to Central Instrument Facility, IIT Guwahati for providing the MALDI-TOF facility and to the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and the Diamond Light Source (DLS, Oxfordshire, UK) for access to synchrotron facilities.

PY - 2018/10/1

Y1 - 2018/10/1

N2 - The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

AB - The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

KW - Clostridium thermocellum

KW - Laminarin

KW - Thin layer chromatography

KW - X-ray crystallography

KW - β-1,3-Glucanase

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EP - 901

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