Purification and characterization of LasD: a second staphylolytic proteinase produced by Pseudomonas aeruginosa

Summary We have previously described studies of a 22 kDa active fragment of the LasA proteinase. In follow‐up studies of LasA, we have discovered the separate existence of a 23 kDa proteinase which shares many of the enzymatic properties of LasA, including the ability to lyse heat‐killed staphylococoi. However, this apparent serine proteinase, which we designate LasD, is distinct from the 22 kDa active LasA protein for the following reasons: (i) the N‐terminal sequence of LasD shares no homology with LasA or the LasA precursor sequence; (ii) Pseudomonas aeruginosa LasA mutant strains AD1825 and FRD2128 do not produce LasA yet produce LasD; and (iii) specific antibodies to each proteinase do not show any cross‐reactivity. LasD appears to be produced as a 30 kDa protein, which is possibly cleaved to produce a 23 kDa active fragment. The purified LasD fragment (23 kDa) shows strong staphylolytic activity only at higher pH conditions, while LasA exhibits staphylolytic activity over a broad pH range, in addition to their ability to cleave at internal diglycine sites, both the LasD and LasA endoproteinases efficiently cleave β‐casein.