title: Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD.
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.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Khaled A
authors.initials: KA
authors.lastname: Aly
authors.forename: Emily T
authors.initials: ET
authors.lastname: Beebe
authors.forename: Chi H
authors.initials: CH
authors.lastname: Chan
authors.forename: Michael A
authors.initials: MA
authors.lastname: Goren
authors.forename: Carolina
authors.initials: C
authors.lastname: Sepúlveda
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Makino
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
authors.forename: Katrina T
authors.initials: KT
authors.lastname: Forest
mesh_terms: D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000378:metabolism*; D002474:Cell-Free System; D003067:Coenzymes / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D036781:Fimbriae Proteins; D008780:Methyltransferases; D057055:Aspartic Acid Proteases; D010450:Endopeptidases; C067127:prepilin peptidase protein, Bacteria; D015032:Zinc