PDBsum entry 3afg

Hydrolase

PDB id: 3afg

Contents

Protein chains

505 a.a. *

Metals

_CA ×4

Waters ×677

* Residue conservation analysis

PDB id:

3afg

Name:

Hydrolase

Title:

Crystal structure of pron-tk-sp from thermococcus kodakaraensis

Structure:

Subtilisin-like serine protease. Chain: a, b. Fragment: unp residues 24-562. Engineered: yes. Mutation: yes

Source:

Thermococcus kodakarensis. Organism_taxid: 311400. Gene: tk1689. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.184 R-free: 0.240

Authors:

T.Foophow,S.Tanaka,C.Angkawidjaja,Y.Koga,K.Takano,S.Kanaya

Key ref:

T.Foophow et al. (2010). Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability. J Mol Biol, 400, 865-877. PubMed id: 20595040

Date:

01-Mar-10 Release date: 04-Aug-10

Protein chains

Q5JIZ5  (TKSP_THEKO) -  Subtilisin-like serine protease from Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)

Seq: 663 a.a.

Struc: 505 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.21.62 - subtilisin.
• Reaction: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.

J Mol Biol 400:865-877 (2010)

PubMed id: 20595040

Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability.

T.Foophow, S.Tanaka, C.Angkawidjaja, Y.Koga, K.Takano, S.Kanaya.

ABSTRACT

Tk-SP is a hyperthermostable subtilisin-like serine protease from Thermococcus kodakaraensis and is autoprocessed from its precursor (Pro-Tk-SP) with N- and C-propeptides. The crystal structure of the active-site mutant of Pro-Tk-SP lacking C-propeptide, ProN-Tk-S359A, was determined at 2.0 A resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and beta-jelly roll domains. Two Ca(2+) ions bind to the beta-jelly roll domain. The overall structure of ProN-Tk-S359A without the beta-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca(2+) ions. To analyze the role of the beta-jelly roll domain of Tk-SP, we constructed a series of the active-site mutants of Tk-SP with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibited protease activities in gel assay, indicating that the beta-jelly roll domain is not required for folding or activity. However, the T(m) value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl(2) was lower than that of Tk-S359A by 29.4 degrees C. The T(m) value of Tk-S359A was decreased by 29.5 degrees C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca(2+)-bound form. Tk-SP highly resembles subtilisin-like serine proteases from Pyrococcus furiosus, Thermococcus gammatolerans, and Thermococcus onnurineus in size and amino acid sequence. We propose that attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment.