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

Khaled A. Aly, Emily T. Beebe, Chi H. Chan, Michael A. Goren, Carolina Sepúlveda, Shin Ichi Makino, Brian G. Fox, Katrina T. Forest

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)94-104
Number of pages11
JournalMicrobiologyOpen
Volume2
Issue number1
DOIs
StatePublished - Feb 2013

Keywords

  • In vitro translation
  • Liposome
  • S-adenosyl methionine
  • Type II secretion
  • Type IV pili

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