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The assembly of the plasmodial PLP synthase complex follows a defined course.

Müller IB, Knöckel J, Groves MR, Jordanova R, Ealick SE, Walter RD, Wrenger C - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH
Reaction scheme of the Pdx1 and Pdx2 proteins.The PfPdx2 enzyme depends on its interaction with PfPdx1 for glutaminase activity. The Pdx1 enzyme is responsible for the de novo synthesis of PLP by utilizing the substrates ribose 5-phosphate, glyceraldehyde 3-phosphate and ammonia, whereas the latter is provided by deaminating of glutamine by Pdx2 via substrate channelling to the attached PfPdx1 protein. The reactions of each enzyme are indicated.
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pone-0001815-g001: Reaction scheme of the Pdx1 and Pdx2 proteins.The PfPdx2 enzyme depends on its interaction with PfPdx1 for glutaminase activity. The Pdx1 enzyme is responsible for the de novo synthesis of PLP by utilizing the substrates ribose 5-phosphate, glyceraldehyde 3-phosphate and ammonia, whereas the latter is provided by deaminating of glutamine by Pdx2 via substrate channelling to the attached PfPdx1 protein. The reactions of each enzyme are indicated.

Mentions: Currently two different pathways for the biosynthesis of PLP are known. The E. coli- (DOXP-dependent) creates from the substrates 4-phosphohydroxy-L-threonine, 1-deoxyxylulose 5-phosphate (DOXP) and glutamate pyridoxine. In contrast the fungi-like- (DOXP-independent) pathway, which has been firstly described in the fungus Cercospora nicotianae, synthesises the active cofactor PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate and glutamine by an enzyme complex consisting of two proteins - Pdx1 and Pdx2 [4]–[11]. The Pdx2 protein exhibits glutaminase activity and delivers ammonia to Pdx1 [5], [6] (Fig. 1). The crystal structures of Pdx1 (YaaD or PdxS), and Pdx2 (YaaE or PdxT) from various organisms have been analyzed [12]–[14]. Only recently the structures of the B6 biosynthesis complex (PLP synthase) from T. maritima and B. subtilis have been solved. Pdx1 assembles into a dodecamer, consisting of two hexameric crowns, each decorated by six Pdx2 molecules [15], [16].


The assembly of the plasmodial PLP synthase complex follows a defined course.

Müller IB, Knöckel J, Groves MR, Jordanova R, Ealick SE, Walter RD, Wrenger C - PLoS ONE (2008)

Reaction scheme of the Pdx1 and Pdx2 proteins.The PfPdx2 enzyme depends on its interaction with PfPdx1 for glutaminase activity. The Pdx1 enzyme is responsible for the de novo synthesis of PLP by utilizing the substrates ribose 5-phosphate, glyceraldehyde 3-phosphate and ammonia, whereas the latter is provided by deaminating of glutamine by Pdx2 via substrate channelling to the attached PfPdx1 protein. The reactions of each enzyme are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266796&req=5

pone-0001815-g001: Reaction scheme of the Pdx1 and Pdx2 proteins.The PfPdx2 enzyme depends on its interaction with PfPdx1 for glutaminase activity. The Pdx1 enzyme is responsible for the de novo synthesis of PLP by utilizing the substrates ribose 5-phosphate, glyceraldehyde 3-phosphate and ammonia, whereas the latter is provided by deaminating of glutamine by Pdx2 via substrate channelling to the attached PfPdx1 protein. The reactions of each enzyme are indicated.
Mentions: Currently two different pathways for the biosynthesis of PLP are known. The E. coli- (DOXP-dependent) creates from the substrates 4-phosphohydroxy-L-threonine, 1-deoxyxylulose 5-phosphate (DOXP) and glutamate pyridoxine. In contrast the fungi-like- (DOXP-independent) pathway, which has been firstly described in the fungus Cercospora nicotianae, synthesises the active cofactor PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate and glutamine by an enzyme complex consisting of two proteins - Pdx1 and Pdx2 [4]–[11]. The Pdx2 protein exhibits glutaminase activity and delivers ammonia to Pdx1 [5], [6] (Fig. 1). The crystal structures of Pdx1 (YaaD or PdxS), and Pdx2 (YaaE or PdxT) from various organisms have been analyzed [12]–[14]. Only recently the structures of the B6 biosynthesis complex (PLP synthase) from T. maritima and B. subtilis have been solved. Pdx1 assembles into a dodecamer, consisting of two hexameric crowns, each decorated by six Pdx2 molecules [15], [16].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.

ABSTRACT

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH