PDBsum entry 3fh4

Hydrolase

PDB id: 3fh4

Contents

Protein chain

291 a.a. *

Ligands

TRS

Metals

_NA ×2

_ZN ×2

Waters ×150

* Residue conservation analysis

PDB id:

3fh4

Name:

Hydrolase

Title:

Crystal structure of recombinant vibrio proteolyticus aminopeptidase

Structure:

Bacterial leucyl aminopeptidase. Chain: a. Engineered: yes

Source:

Vibrio proteolyticus. Organism_taxid: 671. Strain: ifo13287. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.95Å R-factor: 0.193 R-free: 0.228

Authors:

W.Yong,J.-J.P.Kim,M.Hartley,B.Bennett

Key ref:

M.Hartley et al. (2009). Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol. Protein Expr Purif, 66, 91. PubMed id: 19233285 DOI: 10.1016/j.pep.2009.02.011

Date:

08-Dec-08 Release date: 17-Nov-09

Protein chain

Q01693  (AMPX_VIBPR) -  Bacterial leucyl aminopeptidase from Vibrio proteolyticus

Seq: 504 a.a.

Struc: 291 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.11.10 - bacterial leucyl aminopeptidase.
• Reaction: Release of an N-terminal amino acid, preferentially leucine, but not glutamic or aspartic acids.
• Cofactor: Zn(2+)

DOI no: 10.1016/j.pep.2009.02.011

Protein Expr Purif 66:91 (2009)

PubMed id: 19233285

Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol.

M.Hartley, W.Yong, B.Bennett.

ABSTRACT

Metalloaminopeptidases (mAPs) are enzymes that are involved in HIV infectivity, tumor growth and metastasis, angiogenesis, and bacterial infection. Investigation of structure-function relationships in mAPs is a prerequisite to rational design of anti-mAP chemotherapeutics. The most intensively studied member of the biomedically important dinuclear mAPs is the prototypical secreted Vibrio proteolyticus di-zinc aminopeptidase (VpAP). The wild-type enzyme is readily purified from the supernatant of cultures of V. proteolyticus, but recombinant variants require expression in Escherichia coli. A greatly improved system for the purification of recombinant VpAP is described. A VpAP-(His)(6) polypeptide, containing an N-terminal propeptide, and a C-terminal (His)(6) adduct, was purified by metal ion affinity chromatography from the supernatant of cultures of E. coli. This single step replaced the sequence of (NH(4))(2)SO(4) fractionation, and anion-exchange and hydrophobic interaction chromatographic separations of earlier methods. Traditionally, recombinant VpAP proenzyme has been treated with proteinase K and with heat (70 degrees C), to remove the N- and C-terminal regions, and yield the mature active enzyme. This method is unsuitable for VpAP variants that are unstable towards these treatments. In the new method, the hitherto noted, but not fully appreciated, ability of VpAP to autocatalyze the hydrolysis of the N-terminal propeptide and C-terminal regions was exploited; extensive dialysis of the highly purified VpAP-(His)(6) full-length polypeptide yielded the mature active protein without recourse to proteinase K or heat treatment. Purification of variants that have previously defied isolation as mature forms of the protein was thus carried out.