Structural Insight into Substrate Differentiation of the Sugar-metabolizing Enzyme Galactitol Dehydrogenase from Rhodobacter sphaeroides D

Abstract

Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo-and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn 116 , Ser 144 , Tyr 159 , and Lys 163 . GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser 144 , Ser 146 , and Asn 151 are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.Dehydrogenases represent an important class of enzymes in biotechnological processes increasingly used in the chemical or pharmaceutical industry due to their enantio-and stereoselective oxidative and reductive catalytic properties (1-4). A dehydrogenase with a promising catalytic potential is galactitol dehydrogenase (galactitol:NAD ϩ 5-oxidoreductase (GatDH) 3 ), an enzyme originally isolated from a galactitol-utilizing mutant of the bacterium Rhodobacter sphaeroides (5). GatDH is a homotetrameric protein that requires Mg 2ϩ for maintenance of its quaternary structure and enzymatic activity. It catalyzes the dehydrogenation of a variety of polyvalent aliphatic alcohols and polyols to the corresponding ketones and ketoses, respectively, and in the reverse reaction it reduces prochiral ketones with high stereoselectivity yielding the corresponding S-configured secondary alcohols (5-7). Based on these catalytic capabilities, GatDH was used with cofactor recycling (8, 9) for several preparative conversions. Oxidation at C5 of galactitol gave the rare sugar L-tagatose in almost quantitative yields (6). L-Tagatose is a precursor for the synthesis of the glycosidase inhibitor 1-deoxygalactonojirimycin (10) and is a substrate of pyranose 2-oxidase yielding the interesting synthon 5-keto-psicose (11). Similarly, xylitol is oxidized at C4 to give L-xylulose (5). We also demonstrated the preparation of several (R)-1,2-diols by racemic resolution with GatDH as well as the synthesis of several S-configured aliphatic alcohols by reducing corresponding prochiral ketones (7). Furthermore, the suitability of GatDH for conversions with electrochemical cofactor regeneration was demons...

Structural characterization of the thermostableBradyrhizobium japonicumD-sorbitol dehydrogenase

“…The amino acids building the catalytic tetrad are conserved throughout the SDR family. A structural comparison of the active site of BjSDH with those of galactitol dehydrogenase (GatDH; PDB entry 2wdz; Carius et al, 2010) and sorbitol dehydrogenase from R. sphaeroides (PDB entry 1k2w; Philippsen et al, 2005) shows that the conserved amino acids forming the catalytic tetrad (Asn112, Ser140, Tyr153 and Lys157 in BjSDH) superpose well (Fig. 4).…”

Characterization of Sorbitol Dehydrogenase SmoS fromSinorhizobium meliloti1021

“…Similar to [33]. However the enzyme was later predicted to function as a tetramer based on predicted surface area exposure [24], and these results were supported by size exclusion chromatography and light scattering experiments [41]. BjSDH had been…”

Coupled Enzymatic Alcohol‐to‐Amine Conversion of Isosorbide using Engineered Transaminases and Dehydrogenases

“…At this stage nine side‐chain substitutions (S30G, K31S, C51V, S52D, R53V, N54S, E55S, K56E, E57G) were introduced into TRI in an attempt to change its coenzyme specificity from NADPH to NADH, which resulted in the mutant enzyme TRm. These amino acid replacements were rationally designed based on a comparison between the 3D structures of TRI and LR (Figure 2 A) as well as those of some other NADH‐dependent ADHs, in particular, galactitol dehydrogenase from Rhodobacter sphaeroides 27 (PDB code 2WSB), acetoin reductase from Klebsiella pneumoniae 28 (PDB code 1GEG), and 3β/17β‐hydroxysteroid dehydrogenase from Comamonas testosteroni 29 (PDB code 1HXH). Both TRm and LR were produced as soluble proteins in E. coli and purified to homogeneity.…”