3FH4

Crystal Structure of Recombinant Vibrio proteolyticus aminopeptidase

Summary for 3FH4

• Entry DOI: 10.2210/pdb3fh4/pdb
• Related: 2IQ6
• Descriptor: Bacterial leucyl aminopeptidase, ZINC ION, 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL, ... (5 entities in total)
• Functional Keywords: recombinant vibrio proteolyticus aminopeptidase, aminopeptidase, hydrolase, metal-binding, protease, secreted, zinc, zymogen
• Biological source: Vibrio proteolyticus
• Cellular location: Secreted: Q01693
• Total number of polymer chains: 1
• Total formula weight: 32537.09

Authors

Yong, W.,Kim, J.-J.P.,Hartley, M.,Bennett, B. (deposition date: 2008-12-08, release date: 2009-11-17, Last modification date: 2024-11-20)

Primary citation

Hartley, M.,Bennett, B.
Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol
Protein Expr.Purif., 66:91-101, 2009

PubMed 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.

PubMed: 19233285
DOI: 10.1016/j.pep.2009.02.011

Experimental method

X-RAY DIFFRACTION (1.95 Å)