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Microbiologyopen. 2013 Feb;2(1):94-104. doi: 10.1002/mbo3.51. Epub 2012 Dec 17.

Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD.

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1
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.

Abstract

Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-β-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence.

PMID:
23255525
PMCID:
PMC3584216
DOI:
10.1002/mbo3.51
[Indexed for MEDLINE]
Free PMC Article
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