Asp-229, Glu-257, and Asp-328 constitute the catalytic residues in cyclodextrin glycosyl transferase from Bacillus circulans strain 251. Via site-directed mutagenesis constructed D229N, E257Q, and D328N mutant proteins showed a 4,000 -60,000-fold reduction of cyclization activity. A D229N/E257Q double mutant showed a 700,000-fold reduction and was crystallized for use in soaking experiments with ␣-cyclodextrin. Crystal structures were determined of wild type CGTase soaked at elevated pH with

more »

... yclodextrin (resolution, 2.1 Å) and maltoheptaose (2.4 Å). In addition, structures at cryogenic temperature were solved of the unliganded enzyme (2.2 Å) and of the D229N/E257Q mutant after soaking with ␣-cyclodextrin (2.6 Å). In the crystals soaked in ␣-cyclodextrin and maltoheptaose, a maltotetraose molecule is observed to bind in the active site. Residue 229 is at hydrogen bonding distance from the C-6 hydroxyl group of the sugar, which after cleavage will contain the new reducing end. In the D229N/E257Q double mutant structure, two ␣-cyclodextrins are observed to replace two maltoses at the E-domain, thus providing structural information on product inhibition via binding to the enzyme's raw starch binding domain. Cyclodextrin glycosyltransferases (CGTases; E.C. 2.4.1.19) are monomeric bacterial enzymes that catalyze the conversion of starch into cyclic or linear ␣(134)-linked glucopyranosyl chains (1). In general, CGTases produce mixtures of cyclic compounds named cyclodextrins (CDs), 1 consisting of six, seven, or eight D-glucopyranose units, which are referred to as ␣-, ␤-, and ␥-cyclodextrins, respectively. Depending on the major type of cyclodextrin that is produced, CGTases are classified as ␣-, ␤-, or ␥-CGTases. The ␤-CGTase from Bacillus circulans strain 251 is one of the CGTases that is currently used for the industrial production of ␤-cyclodextrins. This process is, how-* This work was financially supported by the Nederlandse Programma Commissie voor Biotechnologie of the Ministry of Economic Affairs and the Groningen Biomolecular Sciences and Biotechnology Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 (codes 1cxe, 1cxf, 1cxh, and 1cxi) have been deposited in the Protein Data Bank, Brookhaven . 1 The abbreviations used are: CD, cyclodextrin; CGTase, cyclodextrin glycosyl transferase; G7, maltoheptaose; CAPS, 3-(cyclohexylamino)-1propanesulfonic acid; r.m.s., root mean square.