Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation.
N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We have determined the crystal structure of the NAD(+) complex of a truncated form of the enzyme, GFORDelta, in which the first 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, refined at 2.7 A 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 the N-terminal arm also greatly increases the solvent exposure of the cofactor; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORDelta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity.
Lott
J S
JS
School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Halbig
D
D
Baker
H M
HM
Hardman
M J
MJ
Sprenger
G A
GA
Baker
E N
EN
D000595:Amino Acid Sequence; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017384:Sequence Deletion / Q000235:genetics; D012997:Solvents; D013379:Substrate Specificity; D017355:Zymomonas / Q000201:enzymology*
D012997:Solvents; D009243:NAD; D009249:NADP; D010088:Oxidoreductases; C074291:glucose-fructose oxidoreductase