Crystal structures of the precursor form of glucose-fructose oxidoreductase from Zymomonas mobilis and its complexes with bound ligands

The NADP(H)-dependent enzyme glucose-fructose oxidoreductase (GFOR) is a cl assic example of a redox protein that is translocated across a membrane in fully folded form. GFOR is synthesized in the cytoplasm with a 52-residue s ignal peptide, giving a precursor form, preGFOR, that is fully active and h as its cofactor tightly bound. A twin-arginine motif in the signal peptide directs it to a See-independent pathway by which it is translocated, in ful ly folded form, into the periplasm where it functions to produce sorbitol f or osmoprotection. We have determined the crystal structures of four differ ent forms of preGFOR, (i) oxidized preGFOR, with succinate bound in the act ive site, (ii) oxidized preGFOR with glycerol bound, (iii) reduced preGFOR in 0.3 M glucose, and (iv) reduced preGFOR in 1.5 M sorbitol, at resolution s of 2.2, 2.05, 2.5, and 2.6 Angstrom, respectively. In all four crystal st ructures, the signal peptide is disordered, implying a flexibility that may be important for its interaction with the translocation apparatus; a facto r contributing to this disorder may be the high positive charge of the prot ein surface in the region where the signal peptide emerges. This may disfav or a stable association between the signal peptide and the rest of the prot ein. The crystal structures show that the mature enzyme portion of preGFOR is identical to native GFOR, in structure and cofactor binding, explaining the enzymatic activity of the precursor form. In the glycerol complex, preG FOR(gll), a bound glycerol molecule models the binding of the glucose subst rate, with its O1 atom hydrogen bonded to the essential acid/base catalyst, Tyr269, and Cl only 3 Angstrom from C4 of the nicotinamide. In the glucose -soaked structure, preGFOR(glu), we identify a conformational change of the nearby Lys181 that probably results from the oxidation of glucose to gluco nolactone, and functions to prevent rebinding, of glucose prior to the bind ing of fructose. In this conformational change, the Lys181 side chain moves closer to the nicotinamide ring, stabilized by its increased negative char ge.