1cgx
From Proteopedia
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SITE DIRECTED MUTATIONS OF THE ACTIVE SITE RESIDUE TYROSINE 195 OF CYCLODEXTRIN GLYXOSYLTRANSFERASE FROM BACILLUS CIRCULANS STRAIN 251 AFFECTING ACTIVITY AND PRODUCT SPECIFICITY
Overview
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)
About this Structure
1CGX is a Single protein structure of sequence from Bacillus circulans with MAL and CA as ligands. Active as Cyclomaltodextrin glucanotransferase, with EC number 2.4.1.19 Full crystallographic information is available from OCA.
Reference
Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity., Penninga D, Strokopytov B, Rozeboom HJ, Lawson CL, Dijkstra BW, Bergsma J, Dijkhuizen L, Biochemistry. 1995 Mar 14;34(10):3368-76. PMID:7880832
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