PDBsum entry 4jp8

Hydrolase

PDB id: 4jp8

Contents

Protein chain

395 a.a.

Metals

_CA ×6

Waters ×79

PDB id:

4jp8

Name:

Hydrolase

Title:

Crystal structure of pro-f17h/s324a

Structure:

Tk-subtilisin. Chain: a. Fragment: unp residues 25-422. Engineered: yes. Mutation: yes

Source:

Thermococcus kodakarensis. Organism_taxid: 69014. Strain: atcc baa-918 / jcm 12380 / kod1. Gene: tk1675. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.21Å R-factor: 0.232 R-free: 0.280

Authors:

K.Yuzaki,D.J.You,R.Uehara,Y.Koga,S.Kanaya

Key ref:

K.Yuzaki et al. (2013). Increase in activation rate of Pro-Tk-subtilisin by a single nonpolar-to-polar amino acid substitution at the hydrophobic core of the propeptide domain. Protein Sci, 22, 1711-1721. PubMed id: 24115021 DOI: 10.1002/pro.2371

Date:

19-Mar-13 Release date: 29-Jan-14

Protein chain

P58502  (TKSU_THEKO) -  Tk-subtilisin from Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)

Seq: 422 a.a.

Struc: 395 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.21.- - ?????

DOI no: 10.1002/pro.2371

Protein Sci 22:1711-1721 (2013)

PubMed id: 24115021

Increase in activation rate of Pro-Tk-subtilisin by a single nonpolar-to-polar amino acid substitution at the hydrophobic core of the propeptide domain.

K.Yuzaki, Y.Sanda, D.J.You, R.Uehara, Y.Koga, S.Kanaya.

ABSTRACT

Tk-subtilisin (Gly70-Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk-subtilisin is produced from its inactive precursor, Pro-Tk-subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk-propeptide. Tk-propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar-to-polar amino acid substitution at this core affects the activation rate of Pro-Tk-subtilisin, the Pro-Tk-subtilisin derivative with the Phe17 → His mutation (Pro-F17H), Tk-propeptide derivative with the same mutation (F17H-propeptide), and two active-site mutants of Pro-F17H (Pro-F17H/S324A and Pro-F17H/S324C) were constructed. The crystal structure of Pro-F17H/S324A was nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-subtilisin. The refolding rate of Pro-F17H/S324A and autoprocessing rate of Pro-F17H/S324C were also nearly identical to those of their parent proteins (Pro-S324A and Pro-S324C). However, the activation rate of Pro-F17H greatly increased when compared with that of Pro-Tk-subtilisin, such that Pro-F17H is efficiently activated even at 40°C. The far-UV circular dichroism spectrum of F17H-propeptide did not exhibit a broad trough at 205-230 nm, which is observed in the spectrum of Tk-propeptide. F17H-propeptide is more susceptible to chymotryptic degradation than Tk-propeptide. These results suggest that F17H-propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk-subtilisin. Thus, destabilization of the hydrophobic core of Tk-propeptide by a nonpolar-to-polar amino acid substitution is an effective way to increase the activation rate of Pro-Tk-subtilisin.