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

摘要: Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD the archaeal Methanococcus voltae FlaK were synthesized presence unilamellar liposomes a cell-free translation system. Cosynthesis with its full-length substrate, PilA, or FlaB2, led to complete cleavage substrate signal peptides. Scaled-up synthesis PilD, followed by solubilization dodecyl-β-d-maltoside chromatography, pure enzyme that retained both known biochemical activities: PilA peptide S-adenosyl methionine-dependent methylation mature pilin. X-ray fluorescence scans show first time is zinc-binding protein. Zinc required N-terminal pilin, but not cleavage. Taken together, our work identifies P. prepilin peptidase as zinc-dependent N-methyltransferase provides new platform large-scale other integral basic microbial virulence.