Volume 12, Issue 9 p. 2478-2487
Full Paper

From the Amelioration of a NADP+-dependent Formate Dehydrogenase to the Discovery of a New Enzyme: Round Trip from Theory to Practice

Dr. Marina Simona Robescu

Dr. Marina Simona Robescu

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

These two authors contributed equally to this work.

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Rudy Rubini

Rudy Rubini

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

These two authors contributed equally to this work.

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Dr. Elisa Beneventi

Dr. Elisa Beneventi

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

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Dr. Michele Tavanti

Dr. Michele Tavanti

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

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Chiara Lonigro

Chiara Lonigro

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

Laboratoire de Biologie Physico-Chimique des Protéines Membranaires UMR7099, CNRS, IBPC, Université Paris Diderot, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005 Paris, France.

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Dr. Francesca Zito

Dr. Francesca Zito

Laboratoire de Biologie Physico-Chimique des Protéines Membranaires UMR7099, CNRS, IBPC, Université Paris Diderot, Sorbonne Paris Cité, 13 rue Pierre et Marie Curie, 75005 Paris, France.

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Prof. Francesco Filippini

Prof. Francesco Filippini

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

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Dr. Laura Cendron

Corresponding Author

Dr. Laura Cendron

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

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Prof. Elisabetta Bergantino

Corresponding Author

Prof. Elisabetta Bergantino

Synthetic Biology and Biotechnology Unit Department of Biology, University of Padova, via U. Bassi 58B/viale G. Colombo 3, I-35131 Padova, Italy

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First published: 05 February 2020
Citations: 11

Graphical Abstract

The race towards NADP+-specificity: a new FDH was identified and successfully engineered in order to switch its cofactor specificity.

Abstract

NADP+-dependent formate dehydrogenases (FDHs) are biotechnologically relevant enzymes for cofactors regeneration in industrial processes employing redox biocatalysts. Their effective applicability is however hampered by the low cofactor and substrate affinities of the few enzymes described so far. After different efforts to ameliorate the previously studied GraFDH from the acidobacterium Granulicella mallensis MP5ACTX8, an enzyme having double (NAD+ and NADP+) cofactor specificity, we started over our search with the advantage of hindsight. We identified and characterized GraFDH2, a novel highly active FDH, which proved to be a good NAD+-dependent catalyst. A rational engineering approach permitted to switch its cofactor specificity, producing an enzyme variant that displays a 10-fold activity improvement over the wild-type enzyme with NADP+. Such variant resulted to be one of the best performing enzyme among the NADP+-dependent FDHs reported so far in terms of catalytic performance.

Conflict of interest

The authors declare no conflict of interest.