The assembly of the plasmodial PLP synthase complex follows a defined course.
BACKGROUND
Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers.


RESULTS
Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis.


CONCLUSIONS
Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.
Müller
Ingrid B
IB
Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Knöckel
Julia
J
Groves
Matthew R
MR
Jordanova
Rositsa
R
Ealick
Steven E
SE
Walter
Rolf D
RD
Wrenger
Carsten
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D005972:Glutaminase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D005972:Glutaminase