Molecular characterization of an aldo-keto reductase from Marivirga tractuosa that converts retinal to retinol

Abstract

A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32 unit ml(-1) for all-trans-retinal with a 72 kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde (k(cat) = 446 min(-1)) whereas the affinity and catalytic efficiency were the highest for all-trans-retinal (K-m = 48 mu M, k(cat)/K-m = 427 mM(-1) min(-1)) among the tested substrates. The optimal reaction conditions for the production of all-trans-retinol from all-trans-retinal by M. tractuosa AKR were pH 7.5, 30 C, 5% (v/v) methanol, 1% (w/v) hydroquinone, 10 mM NADPH, 1710 mgl(-1) all-trans-retinal, and 3 unit ml(-1) enzyme. Under these optimized conditions, the enzyme produced 1090 mg ml(-1) all-trans-retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818 mgl(-1) h(-1). AKR from M. tractuosa showed no activity for all-trans-retinol using NADP(+) as a cofactor, whereas human AKR exhibited activity. When the cofactor-binding residues (Alal 58, Lys212, and Gln270) of M. tractuosa AKR were changed to the corresponding residues of human AKR (Ser160, Pro212, and Glu272), the A158S and Q270E variants exhibited activity for all-trans-retinol. Thus, amino acids at positions 158 and 270 of M. tractuosa AKR are determinant residues of the activity for all-trans-retinol. Crown Copyright (C) 2013 Published by Elsevier B.V. All rights reserved.