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

Authors

  • Khaled A. Aly,

    1. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI
    Current affiliation:
    1. Microbiology Department, KAA University of Chicago, Chicago, IL
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  • Emily T. Beebe,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
    2. Transmembrane Protein Center, University of Wisconsin-Madison, Madison, WI
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  • Chi H. Chan,

    1. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI
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  • Michael A. Goren,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
    2. Transmembrane Protein Center, University of Wisconsin-Madison, Madison, WI
    Current affiliation:
    1. Department of Biochemistry, MAG Weill Cornell Medical College, New York, NY
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  • Carolina Sepúlveda,

    1. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI
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  • Shin-ichi Makino,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
    2. Transmembrane Protein Center, University of Wisconsin-Madison, Madison, WI
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  • Brian G. Fox,

    1. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
    2. Transmembrane Protein Center, University of Wisconsin-Madison, Madison, WI
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  • Katrina T. Forest

    Corresponding author
    1. Transmembrane Protein Center, University of Wisconsin-Madison, Madison, WI
    • Department of Bacteriology, University of Wisconsin-Madison, Madison, WI
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Correspondence

Katrina T. Forest, Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706. Tel: (608)-265-3566; Fax: (608)-262-9865; E-mail: forest@bact.wisc.edu

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.

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