title: Molecular characterization of N-terminal pro-sequence of keratinase Ker P from Pseudomonas aeruginosa: identification of region with chaperone activity.
abstract: In silico analysis of keratinase Ker P from Pseudomonas aeruginosa revealed that its full gene of 1,497 bp constituted of a 72-bp signal sequence along with a long 520 bp pro-sequence and 905 bp core region. Position specific multiple sequence alignment of Ker P protein with other distant proteases revealed high variability within their N-terminal regions while the core protein was considerably conserved. Ker P (F1) and its four N-terminal truncations (F2-F5) lacking 72, 177, 405, 507 bp, respectively, were cloned and constitutively expressed as extracellular protein in pEZZ-18 secretory vector with Escherichia coli HB101 as the expression host. Ker P F1, Ker P F2, Ker P F3 and Ker P F4 products were active whereas no keratinolytic activity was obtained in Ker P F5. Further analysis revealed that only 187 bp pro-sequence region is required for correct folding of the protein into its active conformation and, thus, has chaperone-like activity. Further, comparative biochemical characterization revealed that the full-length keratinase Ker P F1 was catalytically more efficient than the truncated forms. Among the truncated enzymes, keratinase Ker P F4 exhibited better thermostability than Ker P F2 with a t(1/2) of >1 h at 60 °C. It also had a higher V (max) and K (m) on casein as compared with Ker P F2. However, no significant variation was observed with respect to kinetics on synthetic substrates.
authors.affiliation: Department of Microbiology, University of Delhi, South Campus, New Delhi 110021, India.
authors.forename: Richa
authors.initials: R
authors.lastname: Sharma
authors.forename: N Apurva Ratan
authors.initials: NA
authors.lastname: Murty
authors.forename: Rani
authors.initials: R
authors.lastname: Gupta
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D007218:Industrial Microbiology / Q000379:methods*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D018832:Molecular Chaperones; D010447:Peptide Hydrolases; C035855:keratinase