Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation.

Abstract

Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect.

• Authors: Chul Ho Jang, Yu Lan Piao, Xiaoqin Huang, Eun Jeong Yoon, So Hee Park, Kyoung Lee, Chang-Guo Zhan, Hoon Cho
• Journal: PloS one (PLoS One) Vol. 11 Issue 6 Pg. e0156197 ( 2016) ISSN: 1932-6203 [Electronic] United States
• PMID: 27253324 (Publication Type: Journal Article)

Chemical References

• Alginates
• Anti-Bacterial Agents
• Hexuronic Acids
• Mutant Proteins
• Penicillanic Acid
• Glucuronic Acid
• Polysaccharide-Lyases
• poly(beta-D-mannuronate) lyase
• Tazobactam
• Piperacillin

Topics

• Alginates (chemistry, metabolism)
• Amino Acid Sequence (genetics)
• Anti-Bacterial Agents (chemistry, pharmacology)
• Azotobacter vinelandii (enzymology)
• Biofilms (drug effects)
• Catalysis
• Glucuronic Acid (chemistry, metabolism)
• Hexuronic Acids (chemistry, metabolism)
• Mutagenesis, Site-Directed
• Mutant Proteins (chemistry, genetics, pharmacology)
• Penicillanic Acid (analogs & derivatives, pharmacology)
• Piperacillin (pharmacology)
• Polysaccharide-Lyases (chemistry, genetics, pharmacology)
• Pseudomonas aeruginosa (drug effects)
• Tazobactam