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Journal of Bacteriology, July 2005, p. 4421-4429, Vol. 187, No. 13
0021-9193/05/$08.00+0     doi:10.1128/JB.187.13.4421-4429.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Analysis of the Burkholderia cenocepacia ZmpA Metalloprotease

C. Kooi, C. R. Corbett, and P. A. Sokol^*

Department of Microbiology & Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received 1 December 2004/ Accepted 29 March 2005

Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His₆ tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His₆-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H₄₆₅ with G₄₆₅ or A₄₆₅, E₃₇₇ with A₃₇₇ or D₃₇₇, or H₃₈₀ with P₃₈₀ or A₃₈₀. Mutagenesis of H₄₆₅, E₃₇₇, or H₃₈₀ resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H₃₈₀ was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil -1 proteinase inhibitor, ₂-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.

Present address: National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, MB, Canada R3E 3R2.

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