The sequence of a subtilisin-type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability.
The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 degrees C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80-130 degrees C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named "aerolysin") was determined. Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases. Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha-helices. Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.
Völkl
P
P
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
Markiewicz
P
P
Stetter
K O
KO
Miller
J H
JH
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D052899:Pore Forming Cytotoxic Proteins; D015183:Restriction Mapping; D016415:Sequence Alignment; D017421:Sequence Analysis
D001427:Bacterial Toxins; D018076:DNA, Complementary; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin