1DTU

BACILLUS CIRCULANS STRAIN 251 CYCLODEXTRIN GLYCOSYLTRANSFERASE: A MUTANT Y89D/S146P COMPLEXED TO AN HEXASACCHARIDE INHIBITOR

Summary for 1DTU

• Entry DOI: 10.2210/pdb1dtu/pdb
• Related: 1A47 1CDG 2CXG 2DIJ
• Related PRD ID: PRD_900001 PRD_900009
• Descriptor: PROTEIN (CYCLODEXTRIN GLYCOSYLTRANSFERASE), alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose, alpha-D-quinovopyranose-(1-4)-alpha-D-glucopyranose, ... (8 entities in total)
• Functional Keywords: alpha-amylase, acarbose, inhibitor complex, family 13 glycosyl hydrolase, mutant, product specificity, cyclodextrin, transferase
• Biological source: Bacillus circulans
• Total number of polymer chains: 1
• Total formula weight: 77282.96

Authors

Uitdehaag, J.C.M.,Kalk, K.H.,Dijkstra, B.W. (deposition date: 2000-01-13, release date: 2000-03-06, Last modification date: 2024-10-30)

Primary citation

van der Veen, B.A.,Uitdehaag, J.C.,Penninga, D.,van Alebeek, G.J.,Smith, L.M.,Dijkstra, B.W.,Dijkhuizen, L.
Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production.
J.Mol.Biol., 296:1027-1038, 2000

PubMed 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.

PubMed: 10686101
DOI: 10.1006/jmbi.2000.3528

Experimental method

X-RAY DIFFRACTION (2.4 Å)