Cloning and Expression of Genes for Biodegrading Nodularin by Sphingopyxis sp. USTB-05.

Xu Xu,Liu Liu,Wang Wang,Yin Yin,Zhang Zhang,Ma Ma,Fan Fan,Yan Yan

doi:10.3390/toxins11100549

Xu Xu, Liu Liu + Show 6 more

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https://doi.org/10.3390/toxins11100549

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Abstract

Biodegradation is efficient for removing cyanobacterial toxins, such as microcystins (MCs) and nodularin (NOD). However, not all the microbial strains with the microcystin-biodegrading enzymes MlrA and MlrC could biodegrade NOD. Studies on genes and enzymes for biodegrading NOD can reveal the function and the biodegradation pathway of NOD. Based on successful cloning and expression of the USTB-05-A and USTB-05-C genes from Sphingopyxis sp. USTB-05, which are responsible for the biodegradation of MCs, the pathway for biodegrading NOD by these two enzymes was investigated in this study. The findings showed that the enzyme USTB-05-A converted cyclic NOD (m/z 825.4516) into its linear type as the first product by hydrolyzing the arginine and Adda peptide bond, and that USTB-05-C cut off the Adda and glutamic acid peptide bond of linearized NOD (m/z 843.4616) and produced dimeric Adda (m/z 663.4377) as the second product. Further, based on the homology modeling of enzyme USTB-05-A, site-directed mutants of USTB-05-A were constructed and seven crucial sites for enzyme USTB-05-A activity were found. A complete enzymatic mechanism for NOD biodegradation by USTB-05-A in the first step was proposed: glutamic acid 172 and histidine 205 activate a water molecule facilitating a nucleophilic attack on the arginine and Adda peptide bond of NOD; tryptophan 176 and tryptophan 201 contact the carboxylate side chain of glutamic acid 172 and accelerate the reaction rates; and histidine 260 and asparagine 264 function as an oxyanion hole to stabilize the transition states.

Highlights

Cyanobacteria are widespread in freshwater, brackish water, estuarine [1], and marine environments [2] naturally
We found that an initial concentration of 42.3 mg L−1 of MC-RR was completely eliminated within 36 h by Sphingopyxis sp
For treatment AC, the cell-free extract (CE) containing crude USTB-05-A protein was added to phosphate buffered saline (PBS) containing NOD at 0 h, and the CE containing crude USTB-05-C protein was added to the above solution at 12 h

Summary

Introduction

Cyanobacteria are widespread in freshwater, brackish water, estuarine [1], and marine environments [2] naturally. Cyanobacteria cause serious environmental hazards by growing excessively and producing various kinds of cyanobacterial toxins, especially microcystins (MCs) and nodularin (NOD) [3,4,5]. With water eutrophication and global warming, cyanobacterial blooms occur increasingly all over the world [4], including New Zealand [6], Australia [1], South Africa [7], the Baltic Sea [8] and the Mediterranean region [9]. More than 100 analogues of MCs and about nine analogues of NODs [10,11,12] have been described in the literature. Nodularia spumigena was Toxins 2019, 11, 549; doi:10.3390/toxins11100549 www.mdpi.com/journal/toxins

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