title: Zn2+-dependent autocatalytic activity of the Bordetella pertussis CyaA-hemolysin.
abstract: Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015-1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline‒an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015-1088. Moreover, Arg997‒one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity.
authors.affiliation: Division of Biochemistry and Biochemical Technology, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Veerada
authors.initials: V
authors.lastname: Raksanoh
authors.affiliation: Division of Biochemistry and Biochemical Technology, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. Electronic address: lalida_shank@yahoo.com.
authors.forename: Lalida
authors.initials: L
authors.lastname: Shank
authors.affiliation: Division of Biochemistry and Biochemical Technology, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Panchika
authors.initials: P
authors.lastname: Prangkio
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors.forename: Mattayaus
authors.initials: M
authors.lastname: Yentongchai
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors.forename: Somsri
authors.initials: S
authors.lastname: Sakdee
authors.affiliation: Division of Biology, Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand.
authors.forename: Chompounoot
authors.initials: C
authors.lastname: Imtong
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand; Laboratory of Molecular Biophysics and Chemical Biology, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D001886:Bordetella pertussis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D000072417:Protein Domains; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D010618:Phenanthrolines; D011498:Protein Precursors; D011994:Recombinant Proteins; D015032:Zinc; C025205:1,10-phenanthroline
keywords: Autocatalysis; Bordetella pertussis; CyaA-RTX; Hydrophobic pore-forming region; Proteolytic degradation; Zn(2+)-metalloproteases