1CGY

SITE DIRECTED MUTATIONS OF THE ACTIVE SITE RESIDUE TYROSINE 195 OF CYCLODEXTRIN GLYXOSYLTRANSFERASE FROM BACILLUS CIRCULANS STRAIN 251 AFFECTING ACTIVITY AND PRODUCT SPECIFICITY

1CGY の概要

• エントリーDOI: 10.2210/pdb1cgy/pdb
• 関連するBIRD辞書のPRD_ID: PRD_900001
• 分子名称: CYCLOMALTODEXTRIN GLUCANOTRANSFERASE, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose, CALCIUM ION, ... (4 entities in total)
• 機能のキーワード: glycosyltransferase
• 由来する生物種: Bacillus circulans
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 75705.56

構造登録者

Strokopytov, B.V.,Dijkstra, B.W. (登録日: 1994-08-05, 公開日: 1995-02-07, 最終更新日: 2024-10-09)

主引用文献

Penninga, D.,Strokopytov, B.,Rozeboom, H.J.,Lawson, C.L.,Dijkstra, B.W.,Bergsma, J.,Dijkhuizen, L.
Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity.
Biochemistry, 34:3368-3376, 1995

PubMed 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)

PubMed: 7880832
DOI: 10.1021/bi00010a028

実験手法

X-RAY DIFFRACTION (2.5 Å)