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.
Document Type
Article
Publication Date
6-2-2016
Digital Object Identifier (DOI)
https://doi.org/10.1371/journal.pone.0156197
Funding Information
This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (NRF-2014H1C1A1067030), and the National Science Foundation (NSF grant CHE-1111761).
Repository Citation
Jang, Chul Ho; Piao, Yu Lan; Huang, Xiaoqin; Yoon, Eun Jeong; Park, So Hee; Lee, Kyoung; Zhan, Chang-Guo; and Cho, Hoon, "Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation" (2016). Molecular Modeling and Biopharmaceutical Center Faculty Publications. 6.
https://uknowledge.uky.edu/mmbc_facpub/6
S1 Fig. Tracked geometrical root-mean square deviations (RMSD) and distances in the MD-simulated AlgL-alginate binding structure.
journal.pone.0156197.s002.TIF (2491 kB)
S2 Fig. Tracked distances in the MD-simulated AlgL-alginate binding structure.
journal.pone.0156197.s003.TIF (2555 kB)
S3 Fig. Tracked distances in the MD-simulated AlgL-alginate binding structure.
journal.pone.0156197.s004.TIF (2950 kB)
S4 Fig. As tracked along the MD trajectory, the shortest distances between the positively charged head group at the K319 side chain of AlgL and the carbonyl oxygen atoms (O10 and O11) from the M3 unit of the substrate, and the hydroxyl oxygen atom (O13) at the M2 unit of the substrate.
journal.pone.0156197.s005.TIF (2427 kB)
S5 Fig. Tracked distances in MD-simulated AlgL-alginate binding structure.
Notes/Citation Information
Published in PLOS ONE, v. 11, no. 6, e0156197, p. 1-18.
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