Sequence regions of Bacilli metalloproteinases that can affect enzyme thermostability.
By a computer analysis of the five Bacilli metalloprotease sequences it was found that mesophilic Bacillus amyloliquefaciens and B. subtilis proteases had lost two Ca(2+)-binding sites due to the substitutions Asp----Ser 57, Asp----Thr 59, Asp----Pro 200, in comparison with the thermostable B. thermoproteolyticus thermolysin and B. stearothermophilus protease, which conserved three Ca(2+)-binding sites, and B. cereus protease with the intermediate thermostability, which had presumably lost only one site (Ile----Lys 197 substitution). The multiple substitutions within the sequence regions 91-101, 150-154, and 275-280 of the mesophilic enzymes also corresponded with the decrease in the proteinase thermostability value. On the known X-ray structure of thermolysin these sequence regions are spatially drawn together, being located near the central alpha-helix opposite the active site hole and providing the contact of the N- and C-terminal domains. It may be concluded that to increase the thermostability of the mesophilic Bacilli proteinases it is necessary to substitute the sequence regions 91-101, 150-154, 275-280 for the thermolysin ones and restore the Ca(2+)-binding sites of the enzymes.
Strongin
A
A
Division of Dermatology, Washington University Medical School, St. Louis, MO 63110.
Kostrov
S
S
Kaydalova
N
N
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D005075:Biological Evolution; D002118:Calcium / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D004795:Enzyme Stability; D008666:Metalloendopeptidases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity
D010450:Endopeptidases; D008666:Metalloendopeptidases; D002118:Calcium