Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD
Article first published online: 17 DEC 2012
© 2012 The Authors. MicrobiologyOpen published by Blackwell Publishing Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 2, Issue 1, pages 94–104, February 2013
How to Cite
MicrobiologyOpen 2013; 2(1): 94–104
- Issue published online: 13 FEB 2013
- Article first published online: 17 DEC 2012
- Manuscript Accepted: 5 NOV 2012
- Manuscript Revised: 30 OCT 2012
- Manuscript Received: 11 SEP 2012
- USPHS. Grant Numbers: GM59721, 1U54 GM094584, R37-GM40313
- In vitro translation;
- S-adenosyl methionine;
- type II secretion;
- type IV pili
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.