Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease.
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 His(6) 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 His(6)-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 H(465) with G(465) or A(465), E(377) with A(377) or D(377), or H(380) with P(380) or A(380). Mutagenesis of H(465), E(377), or H(380) resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H(380) 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 alpha-1 proteinase inhibitor, alpha(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.
Kooi
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Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary Health Sciences Centre, Calgary, Alberta, Canada T2N 4N1.
Corbett
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Sokol
P A
PA
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016956:Burkholderia cepacia / Q000201:enzymology*; D002614:Chelating Agents / Q000494:pharmacology; D002863:Chromogenic Compounds / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D009930:Organic Chemicals; D010618:Phenanthrolines / Q000494:pharmacology; D017354:Point Mutation; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc; D000511:alpha-Macroglobulins / Q000378:metabolism
D001426:Bacterial Proteins; D002614:Chelating Agents; D002863:Chromogenic Compounds; D024141:Collagen Type IV; D005353:Fibronectins; D009930:Organic Chemicals; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; C043626:hide powder azure; D007371:Interferon-gamma; D004492:Edetic Acid; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; C419871:MprA protein, Burkholderia; C502465:ZmpA protein, Burkholderia cenocepacia; D015032:Zinc