Cell‐free production of integral membrane aspartic acid proteases reveals zinc‐dependent methyltransferase activity of theP seudomonas aeruginosaprepilin peptidase PilD
| Autor: | Emily T. Beebe, Carolina Sepúlveda, Katrina T. Forest, Chi Ho Chan, Michael A. Goren, Shin-ichi Makino, Khaled A. Aly, Brian G. Fox |
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| Rok vydání: | 2012 |
| Předmět: |
Models
Molecular Signal peptide Proteases Aspartic Acid Proteases S-adenosyl methionine Molecular Sequence Data Coenzymes Biology Methylation Microbiology In vitro translation Cell-free system 03 medical and health sciences Bacterial Proteins Endopeptidases Amino Acid Sequence Peptide sequence Original Research 030304 developmental biology chemistry.chemical_classification 0303 health sciences Cell-Free System type IV pili 030306 microbiology type II secretion Methyltransferases Zinc Enzyme Biochemistry chemistry Pilin Pseudomonas aeruginosa liposome biology.protein Fimbriae Proteins Protein Processing Post-Translational |
| Zdroj: | MicrobiologyOpen |
| ISSN: | 2045-8827 |
| DOI: | 10.1002/mbo3.51 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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