THE REFINED CRYSTAL-STRUCTURE OF BACILLUS-CEREUS OLIGO-1,6-GLUCOSIDASE AT 2.0-ANGSTROM RESOLUTION - STRUCTURAL CHARACTERIZATION OF PROLINE-SUBSTITUTION SITES FOR PROTEIN THERMOSTABILIZATION

The crystal structure of oligo-1,6-glucosidase (dextrin 6-alpha-glucan ohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refine d to 2.0 Angstrom resolution with an R-factor of 19.6% for 43,328 refl ections. The final model contains 4646 protein atoms and 221 ordered w ater molecules with respective root-mean-square deviations of 0.015 An gstrom for bond lengths and of 3.166 degrees for bond angles from the ideal values. The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558). The N-termin al domain takes a (beta/alpha)(8)-barrel structure with additions of a n alpha-helix (N alpha 6') between the sixth strand N beta 6 and the s ixth helix N alpha 6, an alpha-helix (N alpha 7') between the seventh strand N beta 7 and the seventh helix N alpha 7 and three alpha-helice s (N alpha 8', N alpha 8 '' and N alpha 8''') between the eighth stran d N beta 8 and the eighth helix N alpha 8. The subdomain is composed o f an alpha-helix, a three-stranded antiparallel beta-sheet, and long i ntervening loops. The C-terminal domain has a beta-barrel structure of eight antiparallel beta-strands folded in double Greek key motifs, wh ich is distorted in the sixth strand C beta 6. Three catalytic residue s, Asp199, Glu255 and Asp329, are located at the bottom of a deep clef t formed by the subdomain and a cluster of the two additional alpha-he lices N alpha 8' and N alpha 8 '' in the (beta/alpha)(8)-barrel. The r efined structure reveals the locations of 21 proline-substitution site s that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases wit h different thermostability. These sites lie in loops, beta-turns and alpha-helices, in order of frequency, except for Cys515 in the fourth beta-strand C beta 4 of the C-terminal domain. The residues in beta-tu rns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu-487) are a ll found at their second positions, and those in alpha-helices (Asn109 , Glu175, Thr261 and Ile-403) are present at their N1 positions of the first helical turns. Those residues in both secondary structures adop t phi and phi values favorable for proline substitution. Residues prec eding the 21 sites are mostly conserved upon proline occurrence at the se 21 sites in more thermostable Bacillus oligo-1,6-glucosidases. Thes e structural features with respect to the 21 sites indicate that the s ites in beta-turns and alpha-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops. This well supports the previous finding that the replacement a t the appropriate positions in beta-turns or alpha-helices is the most effective for protein thermostabilization by proline substitution. (C ) 1997 Academic Press Limited.