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. 2024 Apr 22:15:1389235.
doi: 10.3389/fmicb.2024.1389235. eCollection 2024.

Enzymatic mechanism of MlrB for catalyzing linearized microcystins by Sphingopyxis sp. USTB-05

Junhui Teng  1 , Qianqian Xu  1 , Haiyang Zhang  1 , Ruipeng Yu  2 , Chao Liu  1 , Meijie Song  1 , Xiaoyu Cao  1 , Xinyue Du  1 , Suxuan Tao  1 , Hai Yan  1
Affiliations

Enzymatic mechanism of MlrB for catalyzing linearized microcystins by Sphingopyxis sp. USTB-05

Junhui Teng et al. Front Microbiol. .

Abstract

Microcystins (MCs) are the most widespread cyanobacterial toxins in eutrophic water body. As high toxic intermediate metabolites, linearized MCs are further catalyzed by linearized microcystinase (MlrB) of Sphingopyxis sp. USTB-05. Here MlrB structure was studied by comprizing with a model representative of the penicillin-recognizing enzyme family via homology modeling. The key active sites of MlrB were predicted by molecular docking, and further verified by site-directed mutagenesis. A comprehensive enzymatic mechanism for linearized MCs biodegradation by MlrB was proposed: S77 transferred a proton to H307 to promote a nucleophilic attack on the peptide bond (Ala-Leu in MC-LR or Ala-Arg in MC-RR) of linearized MCs to form the amide intermediate. Then water was involved to break the peptide bond and produced the tetrapeptide as product. Meanwhile, four amino acid residues (K80, Y171, N173 and D245) acted synergistically to stabilize the substrate and intermediate transition states. This study firstly revealed the enzymatic mechanism of MlrB for biodegrading linearized MCs with both computer simulation and experimental verification.

Keywords: MlrB; active site; biodegradation; linearized microcystins; mechanism.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Ramachandran Plot to assess models of MlrB. (A) The model constructed by phyre2. (B) The model constructed by Alphafold (accessions: M4NG93).
Figure 2
Figure 2
Multiple sequence alignment of the template enzyme DAP (PDB ID: 1ei5; chain A) and the MlrB (AGG86526.1 from Sphingopyxis sp. USTB-05).
Figure 3
Figure 3
Linearized MC-LR and MC-RR molecules docked to cartoon depiction of MlrB. (A) Linearized MC-LR molecules docked to MlrB. (B) Linearized MC-RR molecules docked to MlrB. The linearized MC-LR and MC-RR are colored blue and green respectively, while the receptor proteins are all colored gray, with the positions of the six active sites shown as stick form. Colored cyan is the receptor-ligand interaction force, while the site where the receptor hydrolyses the ligand is denoted by a red dotted line. All active sites of MlrB are surrounded by the structural center, and the spatial arrangement of the six active sites is apparent.
Figure 4
Figure 4
Purification of recombinant MlrB and its site-directed mutants. Lane 1 represents the MlrB enzyme without the mutation (MlrB). Lane 2 represents the MlrB enzyme with a mutation at position 77, substituting serine with alanine (MlrB S77A). Lane 3 represents the MlrB enzyme with a mutation at position 80, substituting lysine with alanine (MlrB K80A). Lane 4 contains the MlrB enzyme responsible for converting tyrosine at position 171 into alanine (MlrB Y171A). Lane 5 contains the MlrB enzyme responsible for converting asparagine at position 173 into alanine (MlrB N173A). Lane 6 represents the MlrB enzyme that changes aspartic acid to alanine at position 245 (MlrB D245A). Lane 7 corresponds to the MlrB enzyme that alters histidine to alanine at position 307 (MlrB H307A).
Figure 5
Figure 5
Biodegradation kinetics of linearized MC-LR and MC-RR by MlrB and mutants. (A) Linearized MC-LR (B) Linearized MC-RR.
Figure 6
Figure 6
Catalytic mechanism of MlrB enzyme. Acylation reactions (① A→B and ② B→D), deacylation reactions (③–⑤ D→C→E→F). The MlrB enzyme is represented in green, while the yellow and purple colors represent the linearized MCs. The path of electron transfer and nucleophilic attack is indicated by red arrows.
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References

    1. Bompard-Gilles C., Remaut H., Villeret V., Prangé T., Fanuel L., Delmarcelle M., et al. . (2000). Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the “penicillin-recognizing enzyme” family. Structure 8, 971–980. doi: 10.1016/S0969-2126(00)00188-X, PMID: - DOI - PubMed
    1. Bouaïcha N., Miles C. O., Beach D. G., Labidi Z., Djabri A., Benayache N. Y., et al. . (2019). Structural diversity, Characterization and Toxicology of Microcystins. Toxins 11:714. doi: 10.3390/toxins11120714 - DOI - PMC - PubMed
    1. Bourne D. G., Jones G. J., Blakeley R. L., Jones A., Negri A. P., Riddles P. (1996). Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin Lr. Appl. Environ. Microbiol. 62, 4086–4094. doi: 10.1128/aem.62.11.4086-4094.1996, PMID: - DOI - PMC - PubMed
    1. Bourne D. G., Riddles P., Jones G. J., Smith W., Blakeley R. L. (2001). Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin Lr. Environ. Toxicol. 16, 523–534. doi: 10.1002/tox.10013, PMID: - DOI - PubMed
    1. Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. doi: 10.1016/0003-2697(76)90527-3, PMID: - DOI - PubMed