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

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.

Genomic Analysis of Sphingopyxis sp. USTB-05 for Biodegrading Cyanobacterial Hepatotoxins

“…USTB-05 contains USTB-05-A , USTB-05-B and USTB-05-C genes, which are highly homologous to mlr A , mlr B, and mlr C , respectively [ 28 ]. The first enzyme encoded by USTB-05-A stimulates the first and most critical step in the biodegrading of [D-Asp 1 ]NOD, MC-LR, MC-YR, and MC-RR, including the stimulation of cyclic hepatotoxins to linear hepatotoxins as the first product [ 27 , 29 , 30 , 31 , 32 ]. The second enzyme encoded by USTB-05-B converts linear [D-Asp 1 ]NOD, MC-LR, MC-YR, and MC-RR to tetrapeptide by cutting off the Ala-Arg, Ala-Tyr bonds [ 27 , 28 , 31 , 33 ].…”

“…The first enzyme encoded by USTB-05-A stimulates the first and most critical step in the biodegrading of [D-Asp 1 ]NOD, MC-LR, MC-YR, and MC-RR, including the stimulation of cyclic hepatotoxins to linear hepatotoxins as the first product [ 27 , 29 , 30 , 31 , 32 ]. The second enzyme encoded by USTB-05-B converts linear [D-Asp 1 ]NOD, MC-LR, MC-YR, and MC-RR to tetrapeptide by cutting off the Ala-Arg, Ala-Tyr bonds [ 27 , 28 , 31 , 33 ]. The third enzyme encoded by USTB-05-C cleaves the Adda-Glu peptide bond, transforming it to Adda-Glu-Mdha-Ala into Adda [ 33 ] ( Figure 4 ).…”

“…Furthermore, the functions of enzymes encoded by the USTB-05-A , USTB-05-B , and USTB-05-C genes were validated in E. coli via heterologous expression [ 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. The first enzyme encoded by USTB-05-A catalyzes the first step in the biodegrading of MC-YR, MC-LR, MC-RR and [D-Asp 1 ]NOD, hydrolyzes the cyclic hepatotoxins into linear hepatotoxins as the first product [ 28 , 29 , 30 , 31 , 32 ]. The second enzyme encoded by USTB-05-B can transform linear MC-YR, MC-LR and MC-RR to a tetrapeptide by breaking the Ala-Tyr, Ala-Arg bonds [ 27 , 28 , 33 ].…”

Microcystinase – a review of the natural occurrence, heterologous expression, and biotechnological application of MlrA

“…Nodularin is a monocyclic nonribosomal pentapeptide structurally similar to MCs, with an Arg-Adda peptide bond that is the specific site of MlrA peptidase activity. Intriguingly, MlrA homologs described later have demonstrated nodularin degradation (Imanishi et al, 2005;Xu et al, 2019a;Wu et al, 2019a). This may suggest variance in the specificity of MlrA homologs and/or may be indicative of assay protocols not optimized for the slower nodularin degradation rates later observed (Xu et al, 2020).…”

Purification and Activity of the Second Recombinant Enzyme for Biodegrading Linearized Microcystins by Sphingopyxis sp. USTB-05

“…USTB-05 in 2002 [ 31 ]. Three MCs degradation genes were cloned, and recombinant enzymes with continuous catalytic degradation activity of MCs were expressed [ 32 , 33 , 34 , 35 , 36 ]. We successfully purified MlrA and MlrC and simulated the 3D structures to study the active sites of both enzymes [ 36 , 37 ].…”