Applied Microbiology and Biotechnology

, Volume 97, Issue 9, pp 3949–3964

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

  • Angela Pennacchio
  • Vincenzo Sannino
  • Giosuè Sorrentino
  • Mosè Rossi
  • Carlo A. Raia
  • Luciana Esposito
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-012-4273-z

Cite this article as:
Pennacchio, A., Sannino, V., Sorrentino, G. et al. Appl Microbiol Biotechnol (2013) 97: 3949. doi:10.1007/s00253-012-4273-z

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.

Keywords

Archaea Sulfolobus acidocaldarius Short-chain dehydrogenases/reductases Crystal structure Bioreduction Benzil 

Supplementary material

253_2012_4273_MOESM1_ESM.pdf (1.1 mb)
ESM 1(PDF 1078 kb)

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Angela Pennacchio
    • 1
  • Vincenzo Sannino
    • 1
  • Giosuè Sorrentino
    • 2
  • Mosè Rossi
    • 1
  • Carlo A. Raia
    • 1
  • Luciana Esposito
    • 2
  1. 1.Istituto di Biochimica delle ProteineCNRNaplesItaly
  2. 2.Istituto di Biostrutture e BioimmaginiCNRNaplesItaly