ABSTRACT
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 α-1 proteinase inhibitor, α2-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.