title: 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.affiliation: Department of Otolaryngology, Chonnam National University Medical School, Gwangju, 501-757, Republic of Korea.
authors.forename: Chul Ho
authors.initials: CH
authors.lastname: Jang
authors.affiliation: Department of Polymer Science & Engineering, Chosun University, Gwangju, 501-759, South Korea.
authors.forename: Yu Lan
authors.initials: YL
authors.lastname: Piao
authors.affiliation: Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, Kentucky, 40536, United States of America.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Polymer Science & Engineering, Chosun University, Gwangju, 501-759, South Korea.
authors.forename: Eun Jeong
authors.initials: EJ
authors.lastname: Yoon
authors.affiliation: Department of Polymer Science & Engineering, Chosun University, Gwangju, 501-759, South Korea.
authors.forename: So Hee
authors.initials: SH
authors.lastname: Park
authors.affiliation: Department of Microbiology, Changwon National University, Changwon, Kyongnam, 641-773, Republic of Korea.
authors.forename: Kyoung
authors.initials: K
authors.lastname: Lee
authors.affiliation: Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, Kentucky, 40536, United States of America.
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
authors.affiliation: Department of Polymer Science & Engineering, Chosun University, Gwangju, 501-759, South Korea.
authors.forename: Hoon
authors.initials: H
authors.lastname: Cho
mesh_terms: D000464:Alginates / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D016948:Azotobacter vinelandii / Q000201:enzymology; D018441:Biofilms / Q000187:drug effects; D002384:Catalysis; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D010878:Piperacillin / Q000494:pharmacology; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects*; D000078142:Tazobactam
chemical_list: D000464:Alginates; D000900:Anti-Bacterial Agents; D006603:Hexuronic Acids; D050505:Mutant Proteins; D010397:Penicillanic Acid; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase; D000078142:Tazobactam; D010878:Piperacillin