Engineered Bacillus lentus subtilisins having altered flexibility.
The three-dimensional structures of engineered variants of Bacillus lentus subtilisin having increased enzymatic activity, K27R/N87S/V104Y/N123S/T274A (RSYSA) and N76D/N87S/S103A/V104I (DSAI), were determined by X-ray crystallography. In addition to identifying changes in atomic position we report a method that identifies protein segments having altered flexibility. The method utilizes a statistical analysis of variance to delineate main-chain temperature factors that represent significant departures from the overall variance between equivalent regions seen throughout the structure. This method reveals changes in main-chain mobility in both variants. Residues 125-127 have increased mobility in the RSYSA variant while residues 100-104 have decreased mobility in the DSAI variant. These segments are located at the substrate-binding site and changes in their mobility are believed to relate to the observed changes in proteolytic activity. The effect of altered crystal lattice contacts on segment flexibility becomes apparent when identical variants, determined in two crystal forms, are compared with the native enzyme.
Graycar
T
T
Genencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA.
Knapp
M
M
Ganshaw
G
G
Dauberman
J
J
Bott
R
R
D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D007527:Isoenzymes / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D012044:Regression Analysis; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics; D013696:Temperature
D007527:Isoenzymes; D012697:Serine Endopeptidases; D013381:Subtilisins; C032923:microbial serine proteinases