title: Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1.
abstract: The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD. Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.
authors.affiliation: Agrotechnological Research Institute, P. O. Box 17, 6700 AA Wageningen, The Netherlands. r.d.wind@ato.dlo.nl
authors.forename: R D
authors.initials: RD
authors.lastname: Wind
authors.forename: J C
authors.initials: JC
authors.lastname: Uitdehaag
authors.forename: R M
authors.initials: RM
authors.lastname: Buitelaar
authors.forename: B W
authors.initials: BW
authors.lastname: Dijkstra
authors.forename: L
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D001105:Archaea / Q000201:enzymology*; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability / Q000502:physiology; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D013213:Starch / Q000378:metabolism; D047391:alpha-Cyclodextrins
chemical_list: D019843:Archaeal Proteins; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D047391:alpha-Cyclodextrins; C016549:maltohexaose; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C032613:alpha-cyclodextrin