| Both Pseudomonas syringae and P. aeruginosa overproduce the exopolysaccharide alginate, which contributes to pathogenicity during infection of their hosts. Alginate lyase, encoded by algL, is an enzyme known to degrade alginate and is produced by a variety of organisms including P. syringae and P. aeruginosa. A role for alginate lyase in the biosynthesis of alginate has been hypothesized but not proven.; In this study, algL from P. syringae pv. syringae was cloned, characterized and analyzed for structure and function. algL encoded a protein of 378 amino acids with a 28 amino acid signal sequence. It was overproduced in Escherichia coli as an ∼40 kDa protein that was recognized by antiserum produced against AlgL from P. aeruginosa. The biochemical properties of a crude extract of AlgL were determined, including optimal pH, optimal temperature, cation requirement, substrate specificity, Km and Kcat . AlgL was purified in one-step using a C-terminal hexahistidine tag. During purification, a functional, truncated version of AlgL, AlgL ′, was identified and its size was determined by mass spectrometry. Three C-terminal truncations were generated in an effort to understand the origin of AlgL′.; Site-directed mutagenesis of algL from P. syringae and P. aeruginosa was conducted to identify residues involved in catalysis. The results indicate that histidine and tryptophan residues are important for alginate lyase activity in both organisms. A putative three-dimensional structure of AlgL from P. syringae was constructed using Swiss PDBViewer and showed that the mutations were not detrimental to the structure of the enzyme. A histidine residue that was mutated to glutamine was chosen for further study because of results indicating the importance of this residue in catalysis. Results from limited proteolysis showed that the histidine residue plays a role in binding to the alginate substrate.; In this study, the alginate lyase from P. syringae pv. syringae was identified and characterized. Data from molecular modeling, site-directed mutagenesis and limited proteolysis provided important structural information about the enzyme, which may aid in resolving its role in alginate biosynthesis. |
