Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil(253 visite) Pennacchio A, Sannino V, Sorrentino G, Rossi M, Raia CA, Esposito L
*** IBB - CNR *** Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, Naples, 80131, Italy Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, Naples, 80134, Italy
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 A degrees C and a 30-min half-inactivation temperature of similar to 88 A degrees 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 alpha-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 resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.
222 Records (216 escludendo Abstract e Conferenze). Impact factor totale: 871.178 (850.832 escludendo Abstract e Conferenze). Impact factor a 5 anni totale: 880.993 (859.966 escludendo Abstract e Conferenze).