Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer.
BACKGROUND
Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro.


METHODS
Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members.


RESULTS
Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity.


CONCLUSIONS
As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer.


CONCLUSIONS
The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.
Knöckel
Julia
J
Bernhard Nocht Institute for Tropical Medicine, Department of Biochemistry, Bernhard Nocht Strasse 74, D-20359 Hamburg, Germany.
Jordanova
Rositsa
R
Müller
Ingrid B
IB
Wrenger
Carsten
C
Groves
Matthew R
MR
D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D002942:Circular Dichroism; D017124:Conserved Sequence; D004789:Enzyme Activation; D005998:Glycine / Q000378:metabolism*; D008027:Light; D008970:Molecular Weight; D050505:Mutant Proteins / Q000378:metabolism; D019884:Nitrogenous Group Transferases / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D021381:Protein Transport; D012542:Scattering, Radiation
D050505:Mutant Proteins; D019884:Nitrogenous Group Transferases; D005998:Glycine