Crystal structure of a thermophilic fungal cyanase and its implications on the catalytic mechanism for bioremediation

Bibhuti Ranjan,Suren Singh,Liang Tong,Kugenthiren Permaul,Philip H Choi,Santhosh Pillai

doi:10.1038/s41598-020-79489-3

Bibhuti Ranjan, Suren Singh + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-79489-3

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Abstract

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved.

Highlights

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source
The role of Tl-Cyn in the bioremediation of cyanurated-waste is k nown[25,26,27], the molecular mechanism by which Tl-Cyn carries out decomposition of cyanate is unclear
The structure of the inhibitor-bound Tl-Cyn has elucidated the molecular mechanism of enzyme catalysis, in which the inhibitor interacts with the catalytic residues of the Tl-Cyn and explains the binding affinity between them (Fig. 3 a,b)

Summary

Introduction

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. The cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. Thermomyces lanuginosus, a thermophilic fungus, has been known to produce the highest amount of xylanase and it produces several other hydrolytic enzymes[22] It has a ubiquitin degradation pathway which plays an essential role in responses to various stress, such as nutrient limitation, heat shock, and heavy metal exposure[23]. We have successfully over-expressed Tl-Cyn and evaluated its potential in cyanate detoxification[25,26,27] This cyanase (Tl-Cyn) showed ~ 250-fold higher catalytic activity compared to other cyanases[25], suggesting that it could be used for large-scale applications. We found that the Y14A mutant has higher catalytic activity compared to the wild-type, due primarily to an increase in kcat

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