PDBsum entry 3bgo

Hydrolase

PDB id: 3bgo

Contents

Protein chains

71 a.a. *

263 a.a. *

Ligands

AZI

Metals

_ZN ×4

Waters ×293

* Residue conservation analysis

PDB id:

3bgo

Name:

Hydrolase

Title:

Azide complex of engineered subtilisin subt_bacam

Structure:

Subtilisin bpn'. Chain: p. Fragment: prodomain. Synonym: subtilisin novo, subtilisin dfe, alkaline protease. Engineered: yes. Mutation: yes. Subtilisin bpn'. Chain: s. Fragment: enzyme domain.

Source:

Bacillus amyloliquefaciens. Organism_taxid: 1390. Gene: apr. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Resolution:

1.80Å R-factor: 0.208 R-free: 0.262

Authors:

D.T.Gallagher,P.N.Bryan

Key ref:

T.Gallagher et al. (2009). Structure of a switchable subtilisin complexed with a substrate and with the activator azide. Biochemistry, 48, 10389-10394. PubMed id: 19761257

Date:

27-Nov-07 Release date: 03-Jun-08

Protein chain

P00782  (SUBT_BACAM) -  Subtilisin BPN' from Bacillus amyloliquefaciens

Seq: 382 a.a.

Struc: 71 a.a.*

Protein chain

P00782  (SUBT_BACAM) -  Subtilisin BPN' from Bacillus amyloliquefaciens

Seq: 382 a.a.

Struc: 263 a.a.*

* PDB and UniProt seqs differ at 35 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains P, S: 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.

Biochemistry 48:10389-10394 (2009)

PubMed id: 19761257

Structure of a switchable subtilisin complexed with a substrate and with the activator azide.

T.Gallagher, B.Ruan, M.London, M.A.Bryan, P.N.Bryan.

ABSTRACT

An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme's activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme has applications as an azide or fluoride-triggered expression-purification tool. We report activity measurements showing that the enzyme is activated more than 3000-fold by azide and describe the 1.8 A resolution structure of an inactive form (by replacing the catalytic nucleophile Ser 221 with alanine) of the protease, in complex with azide and with a substrate that spans the active site. Both enzyme and substrate have been engineered to increase their stability and the affinity of their interaction. The substrate is based on a stabilized subtilisin prodomain, extended across the active site by the addition of four residues at its C-terminus. In the crystal structure, the substrate is well-ordered across the active site, and the azide anion is observed bound adjacent to Ala 32. The structures of the substrate complex in three different crystals (anion-free, fluoride-soaked, and azide-soaked) are compared. These structures provide extensive information for understanding subtilisin's substrate binding and catalytic mechanism, and for the development of biotechnology tools based on anion-activated proteolysis. The mechanism of anion-dependent proteolysis appears to be a slight modification of the accepted charge-relay mechanism for serine proteases.