Open AccessCell‐free production of integral membrane aspartic acid proteases reveals zinc‐dependent methyltransferase activity of the P seudomonas aeruginosa prepilin peptidase PilD
Author(s) -
Aly Khaled A.,
Beebe Emily T.,
Chan Chi H.,
Goren Michael A.,
Sepúlveda Carolina,
Makino Shinichi,
Fox Brian G.,
Forest Katrina T.
Publication year - 2013
Publication title -
microbiologyopen
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.881
H-Index - 36
ISSN - 2045-8827
DOI - 10.1002/mbo3.51
Subject(s) - pilin , rhomboid , biochemistry , biology , signal peptide , proteases , proteolysis , chemistry , enzyme , pilus , peptide sequence , virulence , gene
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 G ram‐negative P seudomonas aeruginosa PilD and the archaeal M ethanococcus 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.