PDBsum entry 2e02

Hydrolase

PDB id: 2e02

Contents

Protein chain

443 a.a. *

Waters ×122

* Residue conservation analysis

PDB id:

2e02

Name:

Hydrolase

Title:

Crystal structure of h369l mutant of yeast bleomycin hydrolase

Structure:

Cysteine proteinase 1. Chain: a. Synonym: y3, bleomycin hydrolase, blm hydrolase. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.20Å R-factor: 0.227 R-free: 0.260

Authors:

P.A.O'Farrell,L.Joshua-Tor

Key ref:

P.A.O'Farrell and L.Joshua-Tor (2007). Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure. Biochem J, 401, 421-428. PubMed id: 17007609

Date:

01-Oct-06 Release date: 14-Aug-07

Protein chain

Q01532  (BLH1_YEAST) -  Cysteine proteinase 1, mitochondrial from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 483 a.a.

Struc: 443 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.22.40 - bleomycin hydrolase.
• Reaction: Inactivates bleomycin B2 (a cytotoxic glycometallopeptide) by hydrolysis of a carboxyamide bond of b-aminoalanine, but also shows general aminopeptidase activity. The specificity varies somewhat with source, but amino acid arylamides of Met, Leu and Ala are preferred.

Biochem J 401:421-428 (2007)

PubMed id: 17007609

Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure.

P.A.O'Farrell, L.Joshua-Tor.

ABSTRACT

Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes.