Solvent-stable Pseudomonas aeruginosa PseA protease gene: identification, molecular characterization, phylogenetic and bioinformatic analysis to study reasons for solvent stability.
We have previously isolated a solvent-stable protease from a novel solvent-tolerant strain of Pseudomonas aeruginosa (PseA). Here we report cloning and characterization of the gene coding for this solvent-tolerant protease. A homology search of the N-terminal amino acid sequence of the purified PseA protease revealed an exact match to a P. aeruginosa PST-01 protease gene, lasB. The c-DNA sequence of the PST-01 protease was used to design primers for the amplification of a 1,494-bp open reading frame encoding a 53.6-kDa, 498-amino-acid PseA LasB polypeptide. The deduced PseA LasB protein contained a 23-residue signal peptide (2.6 kDa) followed by a propeptide of 174 residues and a 33-kDa mature product of 301 residues. A phylogenetic analysis placed PseA lasB closest to the known zinc metalloproteases from P. aeruginosa. This gene was also found to contain a conserved HEXXH zinc-binding motif, characteristic of all zinc metallopeptidases. The 3D structure analysis of PseA protease revealed the presence of 7 alpha-helices (36% of the sequence). The molecule was found to have two disulfide bonds (between Cys-227 and Cys-255 and between Cys-467 and Cys-494) and had a number of hydrophobic clusters at the protein surface. These hydrophobic patches (21% of the sequence) and disulfide bonds may possibly be responsible for the solvent-stable nature of the enzyme.
Gupta
Anshu
A
Chemistry Department, Indian Institute of Technology, New Delhi, India.
Ray
Swatismita
S
Kapoor
Sanjay
S
Khare
S K
SK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012997:Solvents / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012997:Solvents; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa