Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase

Abstract

The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasBi) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species.These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase.

Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion

“…When the codon in lasB encoding His-223, an active-site residue, is changed to encode Asp-223 (lasBl) or Tyr-223 (lasB2), overexpression of these mutant alleles in E. coli results in both loss of enzymatic activity and accumulation of the unprocessed 51-kDa proelastase (16). These results suggest that the rapid processing of proelastase to mature elastase is autocatalytic.…”

“…We previously described the lasBI allele which contains a single base pair mutation that changed the codon for His-223 (a substrate-binding residue) to encode Asp-223, a mutation which adversely affects proteolytic activity when expressed in E. coli (16). By a selectable cassette strategy, DNA containing the lasBI allele was cloned next to DNA containing Tn501-6 in the gene replacement vector pEMRZ3 (see Materials and Methods).…”

“…1). A 2.6-kb EcoRI-PstI fragment containing the lasBi allele was cut from pKSM5 (16) and cloned into pBluescript KS-(pBlue/lasBl). The PstI-TnSOl cassette was cloned in correct orientation into the single PstI site in pBlue/lasB1 (pBlue/lasBl/Tn6), from which a 2.9-kb EcoRI-KpnI fragment containing lasBi was cloned into the gene replacement vector pEMRZ3 (pZ3/lasBl).…”

“…We have recently shown that overexpression of lasB in Escherichia coli results in the intracellular accumulation of processed and enzymatically active 33-kDa elastase; however, little 51-kDa proelastase is seen (16). When the codon in lasB encoding His-223, an active-site residue, is changed to encode Asp-223 (lasBl) or Tyr-223 (lasB2), overexpression of these mutant alleles in E. coli results in both loss of enzymatic activity and accumulation of the unprocessed 51-kDa proelastase (16).…”

An extracellular zinc metalloprotease gene of Burkholderia cepacia

“…The deduced amino acid sequences of the B. cepacia and B. pseudomallei extracellular zinc metalloproteases have a preproenzyme structure similar to members of the thermolysin-like metalloprotease family (Hase & Finkelstein, 1991;Kessler & Safrin, 1988;McIver et al, 1991). The preproenzyme structure dictates secretion through the general secretory pathway (Pugsley, 1993).…”