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New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase, PKSIIINc in Neurospora crassa

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Abstract

Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIIINc, a functionally and structurally characterized type III PKS from the fungus, Neurospora crassa, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIIINc through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIIINc catalysis. Site-directed mutagenesis of Cys120 and Ser186 to Ser and Cys, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIIINc product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIIINc products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of Cys120 to bulky Phe side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIIINc protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.

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Acknowledgements

P.S. gratefully acknowledges Innovative Young Biotechnologist Award (IYBA) of Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India and South Asian University (SAU) Startup Research Grant, SAU, India for providing funds and support for this study. A.P. acknowledges CSIR for Senior Research Fellowship. We are thankful to Dr. Rajesh S. Gokhale (NII, India) for kindly providing the pET28c-PKSIIINc recombinant expression plasmid. We thank Sciex, Gurugram, India for Mass Spectrometry data collection.

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  1. Samir Giri

    Present address: Department of Ecology, School of Biology, University of Osnabrück, Osnabrück, 49076, Germany

  2. Amreesh Parvez and Samir Giri these authors contributed equally to this work.

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  1. Chemical Biology Group, Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, 110021, India

    Amreesh Parvez, Samir Giri, Renu Bisht & Priti Saxena

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Parvez, A., Giri, S., Bisht, R. et al. New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase, PKSIIINc in Neurospora crassa. Indian J Microbiol 58, 268–277 (2018). https://doi.org/10.1007/s12088-018-0738-9

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