Journal of Biological Chemistry
Volume 271, Issue 51, 20 December 1996, Pages 32777-32784
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Protein Chemistry and Structure
The Raw Starch Binding Domain of Cyclodextrin Glycosyltransferase from Bacillus circulans Strain 251*

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The E-domain of cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) from Bacillus circulans strain 251 is a putative raw starch binding domain. Analysis of the maltose-dependent CGTase crystal structure revealed that each enzyme molecule contained three maltose molecules, situated at contact points between protein molecules. Two of these maltoses were bound to specific sites in the E-domain, the third maltose was bound at the C-domain.

To delineate the roles in raw starch binding and cyclization reaction kinetics of the two maltose binding sites in the E-domain, we replaced Trp-616 and Trp-662 of maltose binding site 1 and Tyr-633 of maltose binding site 2 by alanines using site-directed mutagenesis. Purified mutant CGTases were characterized with respect to raw starch binding and cyclization reaction kinetics on both soluble and raw starch. The results show that maltose binding site 1 is most important for raw starch binding, whereas maltose binding site 2 is involved in guiding linear starch chains into the active site. β-Cyclodextrin causes product inhibition by interfering with catalysis in the active site and the function of maltose binding site 2 in the E-domain.

CGTase mutants in the E-domain maltose binding site 1 could no longer be crystallized as maltose-dependent monomers. Instead, the W616A mutant CGTase protein was successfully crystallized as a carbohydrate-independent dimer; its structure has been refined to 2.2 Å resolution. The three-dimensional structure shows that, within the error limits, neither the absence of carbohydrates nor the W616A mutation caused significant further conformational changes. The modified starch binding and cyclization kinetic properties observed with the mutant CGTase proteins thus can be directly related to the amino acid replacements.

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This work was financially supported by the Netherlands Programma Commissie voor Biotechnologie (PCB) of the Netherlands Ministry of Economic Affairs and the Groningen Biomolecular Sciences and Biotechnology Institute (GBB) and by grants of the Netherlands Ministry of Economic Affairs, the Ministry of Education, Culture and Science, the Ministry of Agriculture, Nature Management and Fisheries, and the Netherlands Organization for Scientific Research (NWO) in the framework of the biotechnology programme of the Association of Biotechnology Research Schools in the Netherlands (ABON). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1TCM) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§

Contributed equally to this work.