The Burkholderia pseudomallei serine protease MprA is autoproteolytically activated to produce a highly stable enzyme

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Abstract

Burkholderia pseudomallei, a tropical pathogen and the causative agent of melioidosis, is known to secrete a serine metalloprotease (MprA) into the internal milieu of the infectious host. This protein has been shown to cause extensive damage to mammalian physiological proteins and its role in the pathogenesis of melioidosis is still under investigation. Previously, we have reported on the epitope mapping of B. pseudomallei protease that revealed a consensus peptide sequence of serine-methionine-alanine (SMA). The serine within this motif is involved in the serine protease catalytic triad and the SMA domain of the B. pseudomallei serine protease could be a major immunodominant domain. We undertook to further characterize the mprA gene and protein to gain deeper insight into the role and mechanism of this protein. Preliminary analysis showed that the crude lysate of expressed recombinant protease was able to hydrolyze gelatin, azocasein and skimmed milk. Further biochemical characterization demonstrated that the expressed protein maintained good proteolytic activity over a wide pH range of 5-11, is stable from 4 °C up to 68 °C and partially digested immunoglobulins A and G, transferrin and myosin. The proteolytic activity was strongly inhibited by phenylmethylsulfonyl fluoride. From our in vitro experimental evidence, we propose a proenzyme processing mechanism similar to that of subtilisin to produce the mature active protease.

Original languageEnglish
Pages (from-to)370-377
Number of pages8
JournalEnzyme and Microbial Technology
Volume40
Issue number2
DOIs
Publication statusPublished - 4 Jan 2007

Fingerprint

Burkholderia pseudomallei
Metalloproteases
Serine Proteases
Enzymes
Serine
Proteins
Melioidosis
Peptide Hydrolases
Alanine
Methionine
Phenylmethylsulfonyl Fluoride
Epitope Mapping
Epitopes
Subtilisin
Immunodominant Epitopes
Enzyme Precursors
Consensus Sequence
Pathogens
Gelatin
Myosins

Keywords

  • Auto-proteolysis
  • Burkholderia pseudomallei
  • Serine protease

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

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title = "The Burkholderia pseudomallei serine protease MprA is autoproteolytically activated to produce a highly stable enzyme",
abstract = "Burkholderia pseudomallei, a tropical pathogen and the causative agent of melioidosis, is known to secrete a serine metalloprotease (MprA) into the internal milieu of the infectious host. This protein has been shown to cause extensive damage to mammalian physiological proteins and its role in the pathogenesis of melioidosis is still under investigation. Previously, we have reported on the epitope mapping of B. pseudomallei protease that revealed a consensus peptide sequence of serine-methionine-alanine (SMA). The serine within this motif is involved in the serine protease catalytic triad and the SMA domain of the B. pseudomallei serine protease could be a major immunodominant domain. We undertook to further characterize the mprA gene and protein to gain deeper insight into the role and mechanism of this protein. Preliminary analysis showed that the crude lysate of expressed recombinant protease was able to hydrolyze gelatin, azocasein and skimmed milk. Further biochemical characterization demonstrated that the expressed protein maintained good proteolytic activity over a wide pH range of 5-11, is stable from 4 °C up to 68 °C and partially digested immunoglobulins A and G, transferrin and myosin. The proteolytic activity was strongly inhibited by phenylmethylsulfonyl fluoride. From our in vitro experimental evidence, we propose a proenzyme processing mechanism similar to that of subtilisin to produce the mature active protease.",
keywords = "Auto-proteolysis, Burkholderia pseudomallei, Serine protease",
author = "Chin, {Chui Yoke} and Roohaida Othman and Sheila Nathan",
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T1 - The Burkholderia pseudomallei serine protease MprA is autoproteolytically activated to produce a highly stable enzyme

AU - Chin, Chui Yoke

AU - Othman, Roohaida

AU - Nathan, Sheila

PY - 2007/1/4

Y1 - 2007/1/4

N2 - Burkholderia pseudomallei, a tropical pathogen and the causative agent of melioidosis, is known to secrete a serine metalloprotease (MprA) into the internal milieu of the infectious host. This protein has been shown to cause extensive damage to mammalian physiological proteins and its role in the pathogenesis of melioidosis is still under investigation. Previously, we have reported on the epitope mapping of B. pseudomallei protease that revealed a consensus peptide sequence of serine-methionine-alanine (SMA). The serine within this motif is involved in the serine protease catalytic triad and the SMA domain of the B. pseudomallei serine protease could be a major immunodominant domain. We undertook to further characterize the mprA gene and protein to gain deeper insight into the role and mechanism of this protein. Preliminary analysis showed that the crude lysate of expressed recombinant protease was able to hydrolyze gelatin, azocasein and skimmed milk. Further biochemical characterization demonstrated that the expressed protein maintained good proteolytic activity over a wide pH range of 5-11, is stable from 4 °C up to 68 °C and partially digested immunoglobulins A and G, transferrin and myosin. The proteolytic activity was strongly inhibited by phenylmethylsulfonyl fluoride. From our in vitro experimental evidence, we propose a proenzyme processing mechanism similar to that of subtilisin to produce the mature active protease.

AB - Burkholderia pseudomallei, a tropical pathogen and the causative agent of melioidosis, is known to secrete a serine metalloprotease (MprA) into the internal milieu of the infectious host. This protein has been shown to cause extensive damage to mammalian physiological proteins and its role in the pathogenesis of melioidosis is still under investigation. Previously, we have reported on the epitope mapping of B. pseudomallei protease that revealed a consensus peptide sequence of serine-methionine-alanine (SMA). The serine within this motif is involved in the serine protease catalytic triad and the SMA domain of the B. pseudomallei serine protease could be a major immunodominant domain. We undertook to further characterize the mprA gene and protein to gain deeper insight into the role and mechanism of this protein. Preliminary analysis showed that the crude lysate of expressed recombinant protease was able to hydrolyze gelatin, azocasein and skimmed milk. Further biochemical characterization demonstrated that the expressed protein maintained good proteolytic activity over a wide pH range of 5-11, is stable from 4 °C up to 68 °C and partially digested immunoglobulins A and G, transferrin and myosin. The proteolytic activity was strongly inhibited by phenylmethylsulfonyl fluoride. From our in vitro experimental evidence, we propose a proenzyme processing mechanism similar to that of subtilisin to produce the mature active protease.

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