PDBsum entry 1cgx

Glycosyltransferase

PDB id: 1cgx

Contents

Protein chain

686 a.a. *

Ligands

GLC-GLC ×3

Metals

_CA ×2

Waters ×184

* Residue conservation analysis

PDB id:

1cgx

Name:

Glycosyltransferase

Title:

Site directed mutations of the active site residue tyrosine 195 of cyclodextrin glyxosyltransferase from bacillus circulans strain 251 affecting activity and product specificity

Structure:

Cyclomaltodextrin glucanotransferase. Chain: a. Engineered: yes

Source:

Bacillus circulans. Organism_taxid: 1397

Resolution:

2.50Å R-factor: 0.153

Authors:

B.V.Strokopytov,B.W.Dijkstra

Key ref:

D.Penninga et al. (1995). Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity. Biochemistry, 34, 3368-3376. PubMed id: 7880832 DOI: 10.1021/bi00010a028

Date:

05-Aug-94 Release date: 07-Feb-95

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: 10.1021/bi00010a028

Biochemistry 34:3368-3376 (1995)

PubMed id: 7880832

Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity.

D.Penninga, B.Strokopytov, H.J.Rozeboom, C.L.Lawson, B.W.Dijkstra, J.Bergsma, L.Dijkhuizen.

ABSTRACT

Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 angstrum resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)