THE STRUCTURE OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM ZYMOMONAS-MOBILIS - AN OSMOPROTECTIVE PERIPLASMIC ENZYME-CONTAINING NON-DISSOCIABLE NADP

Background: The organism Zymomonas mobilis occurs naturally in sugar-r ich environments. To protect the bacterium against osmotic shock, the periplasmic enzyme glucose-fructose oxidoreductase (GFOR) produces the compatible, solute sorbitol by reduction of fructose, coupled with th e oxidation of glucose to gluconolactone. Hence, Z, mobilis can tolera te high concentrations of sugars and this property may be useful in th e development of an efficient microbial process for ethanol production , Each enzyme subunit contains tightly associated NADP which is not re leased during the catalytic cycle. Results: The structure of GFOR was determined by X-ray crystallography at 2.7 Angstrom resolution, Each s ubunit of the tetrameric enzyme comprises two domains, a classical din ucleotide-binding domain, and a C-terminal domain based on a predomina ntly antiparallel nine-stranded beta sheet. In the tetramer, the subun its associate to form two extended 18-stranded beta sheets, which pack against each other in a face to face fashion, creating an extensive i nterface at the core of the tetramer, An N-terminal arm from each subu nit wraps around the dinucleotide-binding domain of an adjacent subuni t, covering the adenine ring of NADP. Conclusions: In GFOR, the NADP i s found associated with a classical dinucleotide-binding domain in a c onventional fashion, The NADP is effectively buried in the protein-sub unit interior as a result of interactions with the N-terminal arm from an adjacent subunit in the tetramer, and with a short helix from the C-terminal domain of the protein, This accounts for NADP's inability t o dissociate, The N-terminal arm may also contribute to stabilization of the tetramer, The enzyme has an unexpected structural similarity wi th the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PD), We hypothesize that both enzymes have diverged from a common ancestor,Th e mechanism of catalysis is still unclear, but we have identified a co nserved structural motif (Glu-Lys-Pro) in the active site of GFOR and G6PD that may be important for catalysis.