文献类型：期刊文献

英文题名：Structural and Functional Studies on Three Alginate Lyases from Vibrio alginolyticus

作者：Deng, Shuping[1];Ye, Jiang[1];Xu, Qingqing[1];Zhang, Huizhan[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2014

卷号：21

期号：2

起止页码：179

外文期刊名：PROTEIN AND PEPTIDE LETTERS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000331170100011)】；

语种：英文

外文关键词：Alginate lyase; chimeric protein; ESI-MS; NMR; site-directed mutagenesis; substrate specificity

摘要：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 beta-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.