Structural insight into the oxidation of sinapic acid by CotA laccase

Xie, Tian; Liu, Zhongchuan; Liu, Qian; Wang, Ganggang

doi:10.1016/j.jsb.2015.03.005

Cited by 28 publications

(19 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The crystal structure reveals that its side chain rotates 10°upon SA binding, sandwiching the substrate between itself and the T1 Cu site. 55 Finally, the SASA analysis shows a more buried interaction of the substrate at the T1 copper site in CA32F1, particularly in the case of SAH ( Fig. S5E and F †).…”

Section: Computational Analysis Of Substrate Binding and Oxidationmentioning

confidence: 86%

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Pardo

Santiago

Gentili

et al. 2016

Catal. Sci. Technol.

View full text Add to dashboard Cite

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a ligninrelated phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction was affected. Quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets.This work was funded by INDOX (KBBE-2013-7-613549) European project and NOESIS (BIO2014-56388-R) and CTQ2013-\ud 48287-R Spanish National Projects. I. P. and G. S. acknowledge the Spanish Research Council (CSIC) and MINECO for their respective predoctoral fellowships.Peer ReviewedPostprint (published version

show abstract

Section: Computational Analysis Of Substrate Binding and Oxidationmentioning

confidence: 86%

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Pardo

Santiago

Gentili

et al. 2016

Catal. Sci. Technol.

View full text Add to dashboard Cite

show abstract

“…Purification of recombinant CotA and the mutant proteins was performed as described previously (Xie et al, 2015). Crystals of the CotA proteins were obtained at 18 C by the vapour-diffusion method from a reservoir solution consisting of 30-42%(v/v) ethylene glycol, 100 mM sodium citrate pH 5.6 ( Table 1).…”

Section: Protein Crystallization and Data Collectionmentioning

confidence: 99%

“…As a component of the spore-coat outer layer from B. subtilis, CotA may also exhibit specific features (McKenney et al, 2013). The sequences of CotA from strains of Bacillus are highly conserved (Xie et al, 2015). Therefore, all of the CotA enzymes could have a similar structure.…”

Section: Figurementioning

confidence: 99%

Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

Liu

Xie

Zhong

et al. 2016

Acta Crystallogr F Struct Biol Commun

Self Cite

View full text Add to dashboard Cite

The CotA laccase from Bacillus subtilis is an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.

show abstract

“…Laccase activity was measured using spectrophotometry at 50°C with 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich Co. LLC., Shanghai, China) as a substrate [ 28 ]. The oxidation of ABTS was monitored at 420 nm (ε 420 = 36 mM -1 cm -1 ).…”

Section: Methodsmentioning

confidence: 99%

“…CotA of B . altitudinis SYBC hb4, which is similar to homologous proteins from other Bacillus species, also exhibited catalytic oxidation toward SA and SNP [ 26 – 28 ]. Laccases and peroxidases typically transform phenolic substrates into oligomers that show less toxicity or lower bioactivity to organisms [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

Hydrogen Peroxide-Resistant CotA and YjqC of Bacillus altitudinis Spores Are a Promising Biocatalyst for Catalyzing Reduction of Sinapic Acid and Sinapine in Rapeseed Meal

Zhang

Hao

et al. 2016

PLoS ONE

View full text Add to dashboard Cite

For the more efficient detoxification of phenolic compounds, a promising avenue would be to develop a multi-enzyme biocatalyst comprising peroxidase, laccase and other oxidases. However, the development of this multi-enzyme biocatalyst is limited by the vulnerability of fungal laccases and peroxidases to hydrogen peroxide (H2O2)-induced inactivation. Therefore, H2O2-resistant peroxidase and laccase should be exploited. In this study, H2O2-stable CotA and YjqC were isolated from the outer coat of Bacillus altitudinis SYBC hb4 spores. In addition to the thermal and alkali stability of catalytic activity, CotA also exhibited a much higher H2O2 tolerance than fungal laccases from Trametes versicolor and Trametes trogii. YjqC is a sporulation-related manganese (Mn) catalase with striking peroxidase activity for sinapic acid (SA) and sinapine (SNP). In contrast to the typical heme-containing peroxidases, the peroxidase activity of YjqC was also highly resistant to inhibition by H2O2 and heat. CotA could also catalyze the oxidation of SA and SNP. CotA had a much higher affinity for SA than B. subtilis CotA. CotA and YjqC rendered from B. altitudinis spores had promising laccase and peroxidase activities for SA and SNP. Specifically, the B. altitudinis spores could be regarded as a multi-enzyme biocatalyst composed of CotA and YjqC. The B. altitudinis spores were efficient for catalyzing the degradation of SA and SNP in rapeseed meal. Moreover, efficiency of the spore-catalyzed degradation of SA and SNP was greatly improved by the presence of 15 mM H2O2. This effect was largely attributed to synergistic biocatalysis of the H2O2-resistant CotA and YjqC toward SA and SNP.

show abstract

Structural insight into the oxidation of sinapic acid by CotA laccase

Cited by 28 publications

References 47 publications

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

Hydrogen Peroxide-Resistant CotA and YjqC of Bacillus altitudinis Spores Are a Promising Biocatalyst for Catalyzing Reduction of Sinapic Acid and Sinapine in Rapeseed Meal

Contact Info

Product

Resources

About