• Open Access

Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome

Authors

  • José María Vieites,

    1. CSIC, Institute of Catalysis, 28049 Madrid, Spain.
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    • These authors contributed equally to the work.

  • Azam Ghazi,

    1. CSIC, Institute of Catalysis, 28049 Madrid, Spain.
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    • These authors contributed equally to the work.

  • Ana Beloqui,

    1. CSIC, Institute of Catalysis, 28049 Madrid, Spain.
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    • These authors contributed equally to the work.

  • Julio Polaina,

    1. CSIC, Instituto de Agroquímica y Tecnología de Alimentos, 46980 Valencia, Spain.
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  • José M. Andreu,

    1. CSIC, Centro de Investigaciones Biológicas, 28040 Madrid, Spain.
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  • Olga V. Golyshina,

    1. Environmental Microbiology Laboratory, HZI-Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
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  • Taras Y. Nechitaylo,

    1. Environmental Microbiology Laboratory, HZI-Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
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  • Agnes Waliczek,

    1. Environmental Microbiology Laboratory, HZI-Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
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  • Michail M. Yakimov,

    1. Istituto per l'Ambiente Marino Costiero, CNR, Messina 98122, Italy.
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  • Peter N. Golyshin,

    Corresponding author
    1. Environmental Microbiology Laboratory, HZI-Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
    2. School of Biological Sciences, Bangor University, Gwynedd LL57 2UW, UK.
    3. Centre for Integrated Research in the Rural Environment, Aberystwyth University-Bangor University Partnership (CIRRE), Ceredigion, SY23 3BF.
      **E-mail p.golyshin@bangor.ac.uk; Tel. (+44) 1248 38 3629; Fax (+44) 1248 38 2569.
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    • These two last authors contributed equally to the work.

  • Manuel Ferrer

    Corresponding author
    1. CSIC, Institute of Catalysis, 28049 Madrid, Spain.
      *E-mail mferrer@icp.csic.es; Tel. (+34) 91 585 4928; Fax (+34) 91 585 4760;
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    • These two last authors contributed equally to the work.


*E-mail mferrer@icp.csic.es; Tel. (+34) 91 585 4928; Fax (+34) 91 585 4760;

**E-mail p.golyshin@bangor.ac.uk; Tel. (+44) 1248 38 3629; Fax (+44) 1248 38 2569.

Summary

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(kcat/Km)]CE/[(kcat/Km)]FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys281, Asp282, Asn316 and Lys317) situated close to the catalytic core (Ser143/Asp273/His305) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in kcat/Km values) and enzymes with inverted specificity ((kcat/Km)CE/(kcat/Km)FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol−1), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol−1) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.

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