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Journal of Molecular Biology

Journal of Molecular Biology

IF: 4.7
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Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production.

Published:3 March 2000 DOI: 10.1006/jmbi.2000.3528 PMID: 10686101
B A van der Veen, J C Uitdehaag, D Penninga, G J van Alebeek, L M Smith, B W Dijkstra, L Dijkhuizen

Abstract

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.

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Related products
Procduct Name CAS Molecular Formula Supplier Price
β-Cyclodextrin 7585-39-9 C42H70O35 769 suppliers $9.00-$1810.00
Starch 9005-25-8 (C6H10O5)n 679 suppliers $13.50-$1900.00
γ-Cyclodextrin 17465-86-0 C48H80O40 518 suppliers $5.00-$14030.00
α-Cyclodextrin 10016-20-3 C36H60O30 505 suppliers $5.00-$3430.00
MALTOHEXAOSE 34620-77-4 C36H62O31 240 suppliers $44.00-$4800.00
maltononaose 6471-60-9 C54H92O46 51 suppliers $65.00-$2400.00
CYCLODEXTRIN 85220-53-7 C54H90O45 17 suppliers Inquiry

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