Affiliation:
1. Department of Material and Life Science, Graduate School of Engineering, Osaka University
2. PRESTO, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Abstract
ABSTRACT
Subtilisin from the hyperthermophilic archaeon
Thermococcus kodakaraensis
KOD1 is a member of the subtilisin family.
T. kodakaraensis
subtilisin in a proform (
T. kodakaraensis
pro-subtilisin), as well as its propeptide (
T. kodakaraensis
propeptide) and mature domain (
T. kodakaraensis
mat-subtilisin), were independently overproduced in
E. coli
, purified, and biochemically characterized.
T. kodakaraensis
pro-subtilisin was inactive in the absence of Ca
2+
but was activated upon autoprocessing and degradation of propeptide in the presence of Ca
2+
at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (
T. kodakaraensis
mat-subtilisin*) but forms an inactive complex with
T. kodakaraensis
mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of
T. kodakaraensis
pro-subtilisin was autoprocessed into
T. kodakaraensis
propeptide and
T. kodakaraensis
mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise,
T. kodakaraensis
mat-subtilisin was inactive in the absence of Ca
2+
but was activated upon incubation with Ca
2+
at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of
T. kodakaraensis
mat-subtilisin*, indicating that
T. kodakaraensis
mat-subtilisin does not require
T. kodakaraensis
propeptide for folding. However, only ∼5% of
T. kodakaraensis
mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments.
T. kodakaraensis
propeptide was shown to be a potent inhibitor of
T. kodakaraensis
mat-subtilisin* and noncompetitively inhibited its activity with a
K
i
of 25 ± 3.0 nM at 20°C.
T. kodakaraensis
propeptide may be required to prevent the degradation of the
T. kodakaraensis
mat-subtilisin molecules that are activated later by those that are activated earlier.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
44 articles.
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