CRYSTAL-STRUCTURE AT 2.3 ANGSTROM RESOLUTION AND REVISED NUCLEOTIDE-SEQUENCE OF THE THERMOSTABLE CYCLODEXTRIN GLYCOSYLTRANSFERASE FROM THERMOANAEROBACTERIUM THERMOSULFURIGENES EM1

The crystal structure of the cyclodextrin glycosyltransferase (CGTase) from the thermophilic microorganism Thermoanaerobacterium thermosulfu rigenes EM1 has been elucidated at 2.3 Angstrom resolution. The final model consists of all 683 amino acid residues, two calcium ions and 34 3 water molecules, and has a crystallographic X-factor of 17.9% (R(fre e) 24.9%) with excellent stereochemistry. The overall fold of the enzy me is highly similar to that reported for mesophilic CGTases and diffe rences are observed only at surface loop regions. Closer inspection of these loop regions and comparison with other CGTase structures reveal s that especially loops 88-95, 335-339 and 534-539 possibly contribute with novel hydrogen bonds and apolar contacts to the stabilization of the enzyme. Other structural features that might confer thermostabili ty to the T. thermosulfurigenes EM1 CGTase are the introduction of fiv e new salt-bridges and three Gly to Ala/Pro substitutions. The abundan ce of Ser, Thr and Tyr residues near the active site and oligosacchari de binding sites might explain the increased thermostability of CGTase in the presence of starch, by allowing amylose chains to bind non-spe cifically to the protein. Additional stabilization of the A/E domain i nterface through apolar contacts involves residues Phe273 and Tyr187. No additional or improved calcium binding is observed in the structure , suggesting that the observed stabilization in the presence of calciu m ions is caused by the reduced exchange of calcium from the protein t o the solvent, rendering it less susceptible to unfolding. The 50% dec rease in cyclization activity of the T. thermosulfurigenes EM1 CGTase compared with that of B. circulans strain 251 appears to be caused by the changes in the conformation and amino acid composition of the 88-9 5 loop. In the T. thermosulfurigenes EM1 CGTase there is no residue ho mologous to Tyr89, which was observed to take part in stacking interac tions with bound substrate in the case of the B. circulans strain 251 CGTase. The lack of this interaction in the enzyme-substrate complex i s expected to destabilize bound substrates prior to cyclization. Appar ently, some catalytic functionality of CGTase has been sacrificed for the sake of structural stability by modifying loop regions near the ac tive site. (C) 1996 Academic Press Limited