PDBsum entry 1oss

Hydrolase

PDB id: 1oss

Contents

Protein chain

223 a.a. *

Ligands

SO4 ×2

BEN

Metals

_CA

Waters ×193

* Residue conservation analysis

PDB id:

1oss

Name:

Hydrolase

Title:

T190p streptomyces griseus trypsin in complex with benzamidine

Structure:

Trypsin. Chain: a. Synonym: sgt. Engineered: yes. Mutation: yes

Source:

Streptomyces griseus. Organism_taxid: 1911. Gene: sprt. Expressed in: bacillus subtilis. Expression_system_taxid: 1423.

Resolution:

1.93Å R-factor: 0.167 R-free: 0.212

Authors:

M.J.Page,S.L.Wong,J.Hewitt,N.C.Strynadka,R.T.Macgillivray

Key ref:

M.J.Page et al. (2003). Engineering the primary substrate specificity of Streptomyces griseus trypsin. Biochemistry, 42, 9060-9066. PubMed id: 12885239 DOI: 10.1021/bi0344230

Date:

20-Mar-03 Release date: 19-Aug-03

Protein chain

P00775  (TRYP_STRGR) -  Trypsin from Streptomyces griseus

Seq: 259 a.a.

Struc: 223 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.4.21.4 - trypsin.
• Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.

DOI no: 10.1021/bi0344230

Biochemistry 42:9060-9066 (2003)

PubMed id: 12885239

Engineering the primary substrate specificity of Streptomyces griseus trypsin.

M.J.Page, S.L.Wong, J.Hewitt, N.C.Strynadka, R.T.MacGillivray.

ABSTRACT

Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity. Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity. The recombinant and native proteases have nearly identical enzymatic properties and structures. Four SGT mutants with alterations in the S1 substrate binding pocket (T190A, T190P, T190S, and T190V) were also expressed. The T190P mutant demonstrated the largest shift to a preference for Arg versus Lys in the P1 site. This was shown by a minor reduction in catalytic activity toward an Arg-containing substrate (k(cat) reduction of 25%). The crystal structures of the recombinant SGT and the T190P mutant in a complex with the inhibitor benzamidine were obtained at high resolution (approximately 1.9 A). The increase in P1 specificity, achieved with minimal effect on the catalytic efficiency, demonstrates that the T190P mutant is an ideal candidate for the design of additional substrate specificity engineered into the S2 to S4 binding pockets.