PDBsum entry 1kcl

Transferase

PDB id: 1kcl

Contents

Protein chain

686 a.a. *

Ligands

GLC-GLC-GLC-BGC

BGC-GLC-GLC

GLC-GLC ×2

GLC

MPD

Metals

_CA ×2

Waters ×1023

* Residue conservation analysis

PDB id:

1kcl

Name:

Transferase

Title:

Bacillus ciruclans strain 251 cyclodextrin glycosyl transferase mutant g179l

Structure:

Cyclodextrin glycosyltransferase. Chain: a. Synonym: cyclomaltodextrin glucanotransferase. Engineered: yes. Mutation: yes

Source:

Bacillus circulans. Organism_taxid: 1397. Strain: 251. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.94Å R-factor: 0.171 R-free: 0.190

Authors:

H.J.Rozeboom,J.C.M.Uitdehaag,B.W.Dijkstra

Key ref:

H.Leemhuis et al. (2002). The remote substrate binding subsite -6 in cyclodextrin-glycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism. J Biol Chem, 277, 1113-1119. PubMed id: 11696539 DOI: 10.1074/jbc.M106667200

Date:

09-Nov-01 Release date: 16-Jan-02

Protein chain

P43379  (CDGT2_NIACI) -  Cyclomaltodextrin glucanotransferase from Niallia circulans

Seq: 713 a.a.

Struc: 686 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.4.1.19 - cyclomaltodextrin glucanotransferase.
• Reaction: Degrades starch to cyclodextrins by formation of a 1,4-alpha-D- glucosidic bond.

DOI no: 10.1074/jbc.M106667200

J Biol Chem 277:1113-1119 (2002)

PubMed id: 11696539

The remote substrate binding subsite -6 in cyclodextrin-glycosyltransferase controls the transferase activity of the enzyme via an induced-fit mechanism.

H.Leemhuis, J.C.Uitdehaag, H.J.Rozeboom, B.W.Dijkstra, L.Dijkhuizen.

ABSTRACT

Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of alpha-, beta-, and gamma-cyclodextrins (cyclic alpha-(1,4)-linked oligosaccharides of 6, 7, or 8 glucose residues, respectively) from starch. Nine substrate binding subsites were observed in an x-ray structure of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite -6 is conserved in CGTases, suggesting its importance for the reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G, N193L, and G179L/G180L). All subsite -6 mutants had decreased k(cat) values for beta-cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis. Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions. The results demonstrate that (i) subsite -6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii) Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently change cyclization and coupling activities, and (iv) substrate interactions at subsite -6 activate the enzyme in catalysis via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit mechanism in the alpha-amylase family.

Selected figure(s)

Figure 1.
Fig. 1. A, schematic overview of the interactions between the B. circulans strain 251 CGTase and a maltononaose substrate. For clarity not all interactions at subsites +1, 1, and 2 are shown (21). B, maltononaose (black) conformation in the active site of CGTase. The arrow indicates the cleavage site. B was made using Swiss-PDB-Viewer (41).

Figure 2.
Fig. 2. Production of cyclodextrins (g/liter) from 10% (w/v) pregelatinized starch by the action of (mutant) B. circulans strain 251 CGTases. circle , , and indicate -, -, and -cyclodextrin, respectively. WT, wild-type.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 1113-1119) copyright 2002.