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Computational design of enhanced detoxification activity of a zearalenone lactonase from Clonostachys rosea in acidic medium.

ABSTRACT: Computational design of pH-activity profiles for enzymes is of great importance in industrial applications. In this research, a computational strategy was developed to engineer the pH-activity profile of a zearalenone lactonase (ZHD101) from Clonostachys rosea to promote its activity in acidic medium. The active site pK _a values of ZHD101 were computationally designed by introducing positively charged lysine mutations on the enzyme surface, and the experimental results showed that two variants, M2(D157K) and M9(E171K), increased the catalytic efficiencies of ZHD101 modestly under acidic conditions. Moreover, two variants, M8(D133K) and M9(E171K), were shown to increase the turnover numbers by 2.73 and 2.06-fold with respect to wild type, respectively, though their apparent Michaelis constants were concomitantly increased. These results imply that the active site pK _a value change might affect the pH-activity profile of the enzyme. Our computational strategy for pH-activity profile engineering considers protein stability; therefore, limited experimental validation is needed to discover beneficial mutations under shifted pH conditions.

SUBMITTER: Lin M

PROVIDER: S-EPMC9072336 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Computational design of enhanced detoxification activity of a zearalenone lactonase from Clonostachys rosea in acidic medium.

Lin Min M Tan Jian J Xu Zhaobin Z Huang Jin J Tian Ye Y Chen Bo B Wu Yandong Y Tong Yi Y Zhu Yushan Y

RSC advances 20191002 54

Computational design of pH-activity profiles for enzymes is of great importance in industrial applications. In this research, a computational strategy was developed to engineer the pH-activity profile of a zearalenone lactonase (ZHD101) from Clonostachys rosea to promote its activity in acidic medium. The active site pK a values of ZHD101 were computationally designed by introducing positively charged lysine mutations on the enzyme surface, and the experimental results s ...[more]

PMID: 35527979

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Project description:BackgroundClonostachys rosea strain IK726 is a mycoparasitic fungus capable of controlling mycotoxin-producing Fusarium species, including F. graminearum and F. culmorum, known to produce Zearalenone (ZEA) and Deoxynivalenol (DON). DON is a type B trichothecene known to interfere with protein synthesis in eukaryotes. ZEA is a estrogenic-mimicing mycotoxin that exhibits antifungal growth. C. rosea produces the enzyme zearalenone hydrolase (ZHD101), which degrades ZEA. However, the molecular basis of resistance to DON in C. rosea is not understood. We have exploited a genome-wide transcriptomic approach to identify genes induced by DON and ZEA in order to investigate the molecular basis of mycotoxin resistance C. rosea.ResultsWe generated DON- and ZEA-induced cDNA libraries based on suppression subtractive hybridization. A total of 443 and 446 sequenced clones (corresponding to 58 and 65 genes) from the DON- and ZEA-induced library, respectively, were analysed. DON-induced transcripts represented genes encoding metabolic enzymes such as cytochrome P450, cytochrome c oxidase and stress response proteins. In contrast, transcripts encoding the ZEA-detoxifying enzyme ZHD101 and those encoding a number of ATP-Binding Cassette (ABC) transporter transcripts were highly frequent in the ZEA-induced library. Subsequent bioinformatics analysis predicted that all transcripts with similarity to ABC transporters could be ascribed to only 2 ABC transporters genes, and phylogenetic analysis of the predicted ABC transporters suggested that they belong to group G (pleiotropic drug transporters) of the fungal ABC transporter gene family. This is the first report suggesting involvement of ABC transporters in ZEA tolerance. Expression patterns of a selected set of DON- and ZEA-induced genes were validated by the use of quantitative RT-PCR after exposure to the toxins. The qRT-PCR results obtained confirm the expression patterns suggested from the EST redundancy data.ConclusionThe present study identifies a number of transcripts encoding proteins that are potentially involved in conferring resistance to DON and ZEA in the mycoparasitic fungus C. rosea. Whilst metabolic readjustment is potentially the key to withstanding DON, the fungus produces ZHD101 to detoxify ZEA and ABC transporters to transport ZEA or its degradation products out from the fungal cell.

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Computational design of enhanced detoxification activity of a zearalenone lactonase from Clonostachys rosea in acidic medium.

Publications

Computational design of enhanced detoxification activity of a zearalenone lactonase from <i>Clonostachys rosea</i> in acidic medium.

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