University of Groningen X-ray Structure of Cyclodextrin Glycosyltransferase Complexed with Acarbose. Implications for the Catalytic Mechanism of Glycosidases

Crystals of cyclodextrin glycosyltransferase (CGTase) from Bacillus circuluns strain 25 1 were soaked in buffer solutions containing the pseudotetrasaccharide acarbose, a strong amylaseand CGTase inhibitor. The X-ray structure of the complex was elucidated at 2.5-A resolution with a final crystallographic R value of 15.8% for all data between 8.0 and 2.5 A. Acarbose is bound near the catalytic residues Asp229, Glu257, and Asp328. The carboxylic group of Glu257 is at hydrogen bonding distance from the glycosidic oxygen in the scissile bond between the B and C sugars (residue A is at the nonreducing end of the inhibitor). Asp328 makes hydrogen bonds with the 4-amino-4,6-dideoxyglucose (residue B), and Asp229 is in a close van der Waals contact with the C1 atom of this sugar. From this we conclude that in CGTase Glu257 acts as the proton donor and Asp229 serves as the general base or nucleophile, while Asp328 is involved in substrate binding and may be important for elevating the pKa of Glu257. On the basis of these results it appears that the absence of the C6-hydroxyl group in the B sugar is responsible for the inhibitory properties of acarbose on CGTase. This suggests that the C6-hydroxyl group of this sugar plays an essential role in the catalytic mechanism of CGTase. The binding mode of acarbose in CGTase differs from that observed in the complex of pancreatic a-amylase with acarbose where the catalytic Glu was found to be hydrogen bonded to the glycosidic nitrogen linking the A and B residues [Qian, M., Haser, R., Buisson, G., Duke, E., & Payan, F. (1994) Biochemistry 33, 6284-62941. Cyclodextrin glycosyltransferases (CGTases;' EC 2.4.1.19) form a class of proteins which convert starch into cyclodextrins and related a-( 1 4)-linked glucose polymers (French, 1957). Cyclodextrins (CDs) are cyclic compounds consisting of six, seven, or eight a-(1 4)-linked D-glucopyranose units: a-CD, P-CD, and y-CD, respectively. CGTases generally produce a mixture of these three types of cyclodextrins, and, according to their most preferred product, they are divided into a-, /3-, and y-CGTases. The p-CGTases are especially of interest since P-cyclodextrin is most widely applied (Schmid, 1989). However, the industrially used CGTases suffer from product inhibition and their product specificity is not very high. For instance, the P-CGTase from Bacillus circulans strain 251, which is studied in our laboratories, produces a mixture of a-, p-, and y-cyclodextrins in a ratio of 13:64:23 (Penninga et al., 1995). Separation of the different cyclodextrins is expensive and makes This work was financially supported by the Nederlandse Programma Commissie voor Biotechnologie (PCB) of the Ministry of Economic Affairs and the Groningen Biomolecular Sciences and Biotechnology Institute (GBB). * Coordinates of the model of CGTase complexed with acarbose have been deposited with the Protein Data Bank, Brookhaven National Laboratory (Bemstein et al., 1977) (entry 1CXG). * To whom correspondence should be addressed. Fax: (++31) 50 63 48 00. E-mail: [email protected]. BIOSON Research Institute and Laboratory of Biophysical Chemistry. l 1 Department of Microbiology. @ Abstract published in Advance ACS Abstracts, February 1, 1995. I Abbreviations: CGTase, cyclodextrin glycosyltransferase; CD, cyclodextrin; MPD, 2-methyl-2.4-pentanediol; Hepes, N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid; rms, root-mean-square. 0006-2960/95/0434-2234$09 .OO/O 0 use of organic solvents. Therefore, one of our main goals is to increase the product specificity of the enzyme by means of protein engineering. Rational design of such mutants requires not only knowledge of the three-dimensional structure of the protein but also insight in the substrate binding mode and the detailed mechanism of the transglycosylation reaction. We have already elucidated the nucleotide sequence and the three-dimensional structure at 2.0-A resolution of the CGTase from B. circulans strain 251 (Lawson et al., 1994). The folding of this 75-kDa enzyme is very similar to that of the CGTase from B. circulans strain 8 (Klein & Schulz, 1991), in accordance with the 75% identity in amino acid sequence between the two proteins. The enzyme consists of five domains, A-E. Domains A, B, and C are structurally homologous to the three domains of the a-amylases. The E domain has been implicated in starch binding (Svensson et al., 1989). The active site of CGTase is located at the N-terminal side of the (Pla)8-barrel of domain A. The catalytic residues are Asp229, Glu257, and Asp328, although their exact roles in the catalytic mechanism have not been firmly established. Site-directed mutagenesis of each of them resulted, however, in an inactive protein, indicating their importance for catalysis (Nakamura