1dtu
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BACILLUS CIRCULANS STRAIN 251 CYCLODEXTRIN GLYCOSYLTRANSFERASE: A MUTANT Y89D/S146P COMPLEXED TO AN HEXASACCHARIDE INHIBITOR
OverviewOverview
Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular, bacterial enzymes that generate cyclodextrins from starch. All known, CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A, maltononaose inhibitor bound to the active site of the CGTase from, Bacillus circulans strain 251 revealed sugar binding subsites, distant, from the catalytic residues, which have been proposed to be involved in, the cyclodextrin size specificity of these enzymes. To probe the, importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we, have constructed three single and one double mutant, Y89G, Y89D, S146P and, Y89D/S146P, using site-directed mutagenesis. The mutations affected the, cyclization, coupling; disproportionation and hydrolyzing reactions of the, enzyme. The double mutant Y89D/S146P showed a twofold increase in the, production of alpha-cyclodextrin from starch. This mutant protein was, crystallized and its X-ray structure, in a complex with a maltohexaose, inhibitor, was determined at 2.4 A resolution. The bound maltohexaose, molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity., Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute, strongly to the size of cyclodextrin products formed and thus to CGTase, product specificity. Changes in sugar binding subsites -3 and -7 thus, result in mutant proteins with changed cyclodextrin production, specificity.
About this StructureAbout this Structure
1DTU is a Single protein structure of sequence from Bacillus circulans with CA and ADH as ligands. Active as Cyclomaltodextrin glucanotransferase, with EC number 2.4.1.19 Full crystallographic information is available from OCA.
ReferenceReference
Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production., van der Veen BA, Uitdehaag JC, Penninga D, van Alebeek GJ, Smith LM, Dijkstra BW, Dijkhuizen L, J Mol Biol. 2000 Mar 3;296(4):1027-38. PMID:10686101
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