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Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil

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Abstract

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ∼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2–NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.

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Acknowledgments

This work was funded by FIRB (Fondo per gli Investimenti della Ricerca di Base) RBNE034XSW and by the ASI project MoMa n. 1/014/06/0.

Atomic coordinates as well as structure factors have been deposited within the PDB under the accession code 4FN4.

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Authors and Affiliations

  1. Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, 80131, Naples, Italy

    Angela Pennacchio, Vincenzo Sannino, Mosè Rossi & Carlo A. Raia

  2. Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Naples, Italy

    Giosuè Sorrentino & Luciana Esposito

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  1. Angela Pennacchio
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  2. Vincenzo Sannino
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  3. Giosuè Sorrentino
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  4. Mosè Rossi
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  5. Carlo A. Raia
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  6. Luciana Esposito
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Correspondence to Angela Pennacchio or Luciana Esposito.

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Pennacchio, A., Sannino, V., Sorrentino, G. et al. Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil. Appl Microbiol Biotechnol 97, 3949–3964 (2013). https://doi.org/10.1007/s00253-012-4273-z

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