Crystal structure of a truncated mutant of glucose-fructose oxidoreductaseshows that an N-terminal arm controls tetramer formation

N-terminal or C-terminal arms that extend from folded protein domains can p lay a critical role in quaternary structure and other intermolecular associ ations and/or in controlling biological activity. We have tested the role o f an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We h ave determined the crystal structure of the NAD(+) complex of a truncated f orm of the enzyme, GFOR Delta, in which the first 22 residues of the N-term inal arm of the mature protein have been deleted. The structure, refined at 2.7 Angstrom resolution (R-cryst = 24.1%, R-free = 28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of th e N-terminal arm also greatly increases the solvent exposure of the cofacto r; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this muta nt results from dissociation of the cofactor. The N-terminal arm thus deter mines the quaternary structure and the retention of the cofactor for GFOR a ctivity and during translocation into the periplasm. The structure of GFOR Delta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity. (C) 2000 Academic Press.