High-resolution crystal structures of Caldicellulosiruptor strain Rt8B.4 carbohydrate-binding module CBM27-1 and its complex with mannohexaose

Yvette Roske, Anwar Sunna, Wolfgang Pfeil, Udo Heinemann

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Despite the large number of putative CBMs being identified by amino acid sequence alignments, only few representatives have been experimentally shown to have a carbohydrate-binding function. Caldicellulosiruptor strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase β-mannanase which contains two non-catalytic modules in tandem at its N terminus. These modules were recently shown to function primarily as β-mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. The N-terminal CBM27 (CsCBM27-1) of Man26 from Caldicellulosiruptor Rt8B.4 displays high-binding affinity towards mannohexaose with a Ka of 1×107M-1. Accordingly, the high-resolution crystal structures of CsCBM27-1 native and its mannohexaose complex were solved at 1.55Å and 1.06Å resolution, respectively. In the crystal, CsCBM27-1 shows the typical β-sandwich jellyroll fold observed in other CBMs with a single metal ion bound, which was identified as calcium. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. Based on binding affinity and thermal unfolding experiments this structural calcium is shown to play a role in the thermal stability of CsCBM27-1 at high temperatures. The higher binding affinity of CsCBM27-1 to mannooligosaccharides when compared to other members of CBM family 27 might be explained by the different orientation of the residues forming the "aromatic platform" and by differences in the length of loops. Finally, evidence is presented, on the basis of fold similarities and the retention of the position of conserved motifs and a calcium ion, for the consolidation of related CBM families into a superfamily of CBMs.

LanguageEnglish
Pages543-554
Number of pages12
JournalJournal of molecular biology
Volume340
Issue number3
DOIs
Publication statusPublished - 9 Jul 2004
Externally publishedYes

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Carbohydrates
Calcium
Hot Temperature
beta-Mannosidase
Ions
Mannans
Sequence Alignment
Glycoside Hydrolases
Plant Cells
Tryptophan
Cell Wall
Hydrogen
Amino Acid Sequence
Hydrolysis
Metals
Temperature
Enzymes

Cite this

@article{4d8a671e27dd400585ebe909d5ab03e8,
title = "High-resolution crystal structures of Caldicellulosiruptor strain Rt8B.4 carbohydrate-binding module CBM27-1 and its complex with mannohexaose",
abstract = "Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Despite the large number of putative CBMs being identified by amino acid sequence alignments, only few representatives have been experimentally shown to have a carbohydrate-binding function. Caldicellulosiruptor strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase β-mannanase which contains two non-catalytic modules in tandem at its N terminus. These modules were recently shown to function primarily as β-mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. The N-terminal CBM27 (CsCBM27-1) of Man26 from Caldicellulosiruptor Rt8B.4 displays high-binding affinity towards mannohexaose with a Ka of 1×107M-1. Accordingly, the high-resolution crystal structures of CsCBM27-1 native and its mannohexaose complex were solved at 1.55{\AA} and 1.06{\AA} resolution, respectively. In the crystal, CsCBM27-1 shows the typical β-sandwich jellyroll fold observed in other CBMs with a single metal ion bound, which was identified as calcium. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. Based on binding affinity and thermal unfolding experiments this structural calcium is shown to play a role in the thermal stability of CsCBM27-1 at high temperatures. The higher binding affinity of CsCBM27-1 to mannooligosaccharides when compared to other members of CBM family 27 might be explained by the different orientation of the residues forming the {"}aromatic platform{"} and by differences in the length of loops. Finally, evidence is presented, on the basis of fold similarities and the retention of the position of conserved motifs and a calcium ion, for the consolidation of related CBM families into a superfamily of CBMs.",
author = "Yvette Roske and Anwar Sunna and Wolfgang Pfeil and Udo Heinemann",
year = "2004",
month = "7",
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language = "English",
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High-resolution crystal structures of Caldicellulosiruptor strain Rt8B.4 carbohydrate-binding module CBM27-1 and its complex with mannohexaose. / Roske, Yvette; Sunna, Anwar; Pfeil, Wolfgang; Heinemann, Udo.

In: Journal of molecular biology, Vol. 340, No. 3, 09.07.2004, p. 543-554.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - High-resolution crystal structures of Caldicellulosiruptor strain Rt8B.4 carbohydrate-binding module CBM27-1 and its complex with mannohexaose

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AU - Heinemann, Udo

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AB - Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Despite the large number of putative CBMs being identified by amino acid sequence alignments, only few representatives have been experimentally shown to have a carbohydrate-binding function. Caldicellulosiruptor strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase β-mannanase which contains two non-catalytic modules in tandem at its N terminus. These modules were recently shown to function primarily as β-mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. The N-terminal CBM27 (CsCBM27-1) of Man26 from Caldicellulosiruptor Rt8B.4 displays high-binding affinity towards mannohexaose with a Ka of 1×107M-1. Accordingly, the high-resolution crystal structures of CsCBM27-1 native and its mannohexaose complex were solved at 1.55Å and 1.06Å resolution, respectively. In the crystal, CsCBM27-1 shows the typical β-sandwich jellyroll fold observed in other CBMs with a single metal ion bound, which was identified as calcium. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. Based on binding affinity and thermal unfolding experiments this structural calcium is shown to play a role in the thermal stability of CsCBM27-1 at high temperatures. The higher binding affinity of CsCBM27-1 to mannooligosaccharides when compared to other members of CBM family 27 might be explained by the different orientation of the residues forming the "aromatic platform" and by differences in the length of loops. Finally, evidence is presented, on the basis of fold similarities and the retention of the position of conserved motifs and a calcium ion, for the consolidation of related CBM families into a superfamily of CBMs.

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