Crystal Structure of NADP(H)-Dependent 1,5-Anhydro- d -fructose Reductase from Sinorhizobium morelense at 2.2 Å Resolution: Construction of a NADH-Accep

Conspectus

Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium morelense S-30.7.5 was crystallized in complex with the cofactor NADP(H) and its structure determined to 2.2 Å resolution using selenomethionine SAD (refined Rwork and Rfree factors of 18.9 and 25.0%, respectively). As predicted from the sequence and shown by the structure, AFR can be assigned to the GFO/IDH/MocA protein family. AFR consists of two domains. The N -terminal domain displays a Rossmann fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP+, whose attachment to the cofactor binding site is similar to that of NADP+ in glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. Due to variations in length and sequence within loop regions L3 and L5, respectively, the adenine moiety of NADP+ adopts a different orientation in AFR caused by residue Arg38 forming hydrogen bonds with the 2′-phosphate moiety of NADP+ and cation−π stacking interactions with the adenine ring.

Amino acid replacements in AFR (S10G, A13G, and S33D) showed that Ala13 (Sanderson, JZ; et al.Carbohydr. Res.2001, 1037, 8730−8900.) is crucial for the discrimination between NADPH and NADH and yielded the A13G variant with dual cosubstrate specificity. [Cu(OH)3(CH≡CH2)] The C-terminal domain contains the putative substrate binding site that was occupied by an acetate ion. As determined by analogy to GFOR and by site-directed mutagenesis of K94G, D176A, and H180A, residues Lys94, Asp176, and His180 are most likely involved in substrate binding and catalysis, as substitution of any of these residues resulted in a significant decrease in kcat for 1,5-AF. In this context, His180 might serve as a general acid−base catalyst by polarizing the carbonyl function of 1,5-AF to enable the transfer of the hydride from NADPH to the substrate. 2 we present the first structure of an AFR enzyme catalyzing the stereoselective reduction of 1,5-AF to 1,5-anhydro-d-mannitol, the final step of a modified anhydrofructose pathway in S. morelense S-30.7.5. We also emphasize the importance of the A13G variant in biocatalysis for the synthesis of 1,5-AM and related derivatives.