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Structural and Functional Studies on Three Alginate Lyases from Vibrio alginolyticus

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To date, several microorganisms that can metabolize alginate have been characterized. In these microorganisms, a class of enzymes called alginate lyases (Alys) catalyze the depolymerization of alginate into oligomers via an endolytic β-elimination reaction. But it is not clear how these lyases differ on their enzymological functions or their divisions of labor. We focused on three extracellular secretory alginate lyases in the Gram-negative marine bacterium V.alginolyticus (ATCC 17749). We found differences in lyase function and catalytic specificity, depending on substrates. These properties apparently derived from demonstrable changes to the protein sequences. We found shared structural domains in alginate lyases specific to the pM and pG substrates through the measurements on enzyme activities and kinetic parameters. Substitution of hydrophobic amino acids in the isoleucine site of domain QIH could have an enormous influence on the high-affinity to pM or pG. This isoleucine was reconfirmed to be indispensable for recognition of the pG or G-G bond. We identified key amino acids located in the catalytic center by means of protein site-directed mutagenesis, and found specific amino acid fragments with exclusive recognitional and catalytic activity effects for different substrates. We reconstructed a series of proteins through splicing, with the goal of producing an engineered protein with the ability to degrade alginate on a variety of substrates and with high enzyme activity as well as temperature stability. Our study provides the theoretical basis for future work on alginate oligosaccharide, transport mechanisms, an area of research with potentially large ramifications for the chemical, medical, textile, printing and agricultural industries.
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Keywords: Alginate lyase; ESI-MS; NMR; chimeric protein; site-directed mutagenesis; substrate specificity

Document Type: Research Article

Publication date: February 1, 2014

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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