PDBsum entry 1v0a

Hydrolase

PDB id: 1v0a

Contents

Protein chain

170 a.a. *

Ligands

SO4 ×2

Metals

_CA ×2

Waters ×182

* Residue conservation analysis

PDB id:

1v0a

Name:

Hydrolase

Title:

Family 11 carbohydrate-binding module of cellulosomal cellulase lic26a-cel5e of clostridium thermocellum

Structure:

Endoglucanase h. Chain: a. Synonym: cellulose binding protein a, egh, endo-1,4-beta-glucanase, cellulase h. Engineered: yes

Source:

Clostridium thermocellum. Organism_taxid: 1515. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.98Å R-factor: 0.195 R-free: 0.232

Authors:

A.L.Carvalho,M.J.Romao,A.Goyal,J.A.M.Prates,V.M.R.Pires, L.M.A.Ferreira,D.N.Bolam,H.J.Gilbert,C.M.G.A.Fontes

Key ref:

A.L.Carvalho et al. (2004). The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site. J Biol Chem, 279, 34785-34793. PubMed id: 15192099 DOI: 10.1074/jbc.M405867200

Date:

25-Mar-04 Release date: 12-Jan-05

Protein chain

P16218  (GUNH_CLOTH) -  Endoglucanase H from Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372)

Seq: 900 a.a.

Struc: 170 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.4 - cellulase.
• Reaction: Endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans.

DOI no: 10.1074/jbc.M405867200

J Biol Chem 279:34785-34793 (2004)

PubMed id: 15192099

The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site.

A.L.Carvalho, A.Goyal, J.A.Prates, D.N.Bolam, H.J.Gilbert, V.M.Pires, L.M.Ferreira, A.Planas, M.J.Romão, C.M.Fontes.

ABSTRACT

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.

Selected figure(s)

Figure 2.
FIG. 2. Ribbon representation of the three-dimensional structure of CtCBM11. a, Stereo representation of the overall structure of CtCBM11. The CtCBM11 structure consists of a distorted -jelly roll fold composed of two six-stranded antiparallel -sheets, which form a convex side ( -strands drawn in dark blue) and a concave side ( -strands drawn in light blue). The two calcium ions are indicated as green spheres. The concave side of CtCBM11 forms a cleft. Residues inside the putative binding cleft are shown as ball-and-stick models. Tyrosine residues are represented in red, arginines are in yellow, histidines are in pink, and aspartates are in orange. The picture was drawn with the program MOLMOL (42). b, stereo view of the CtCBM11 cleft, in the same orientation as in a, occupied by the C terminus residues of a symmetry-related molecule. The residues are labeled in red and represented in ball-and-stick. The 2mF[o] - DF[c] electron density map around the molecule is shown in dark blue and contoured at 1.1 . Residues inside the cleft (color code) are shown as ball-and-stick models and labeled blue. The calcium-binding site, Ca1, is represented as a green sphere. The picture was produced with the program TURBO-FRODO (27).

Figure 3.
FIG. 3. The Ca^2+-binding sites of CtCBM11. Stereo view of the Ca^2+ coordination in CtCBM11 superimposed in the 2mF[o] - DF[c] electron density map, contoured at 1.3 . The residues involved in calcium binding are represented as stick models and labeled black. The polypeptide chain atoms are represented in color code, and the Ca^2+ ions are shown as orange spheres. The pictures were produced with the program TURBO-FRODO (27).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 34785-34793) copyright 2004.

Figures were selected by an automated process.