PDBsum entry 2abw

Transferase

PDB id

2abw

Contents

			Protein chains

					216 a.a.

			Ligands

					PG4

			Waters ×469

References listed in PDB file

Key reference

Title

Vitamin b6 biosynthesis by the malaria parasite plasmodium falciparum: biochemical and structural insights.

Authors

M.Gengenbacher, T.B.Fitzpatrick, T.Raschle, K.Flicker, I.Sinning, S.Müller, P.Macheroux, I.Tews, B.Kappes.

Ref.

J Biol Chem, 2006, 281, 3633-3641. [DOI no: 10.1074/jbc.M508696200]

PubMed id

16339145

Abstract

Vitamin B6 is one of nature's most versatile cofactors. Most organisms synthesize vitamin B6 via a recently discovered pathway employing the proteins Pdx1 and Pdx2. Here we present an in-depth characterization of the respective orthologs from the malaria parasite, Plasmodium falciparum. Expression profiling of Pdx1 and -2 shows that blood-stage parasites indeed possess a functional vitamin B6 de novo biosynthesis. Recombinant Pdx1 and Pdx2 form a complex that functions as a glutamine amidotransferase with Pdx2 as the glutaminase and Pdx1 as pyridoxal-5 '-phosphate synthase domain. Complex formation is required for catalytic activity of either domain. Pdx1 forms a chimeric bi-enzyme with the bacterial YaaE, a Pdx2 ortholog, both in vivo and in vitro, although this chimera does not attain full catalytic activity, emphasizing that species-specific structural features govern the interaction between the protein partners of the PLP synthase complexes in different organisms. To gain insight into the activation mechanism of the parasite bi-enzyme complex, the three-dimensional structure of Pdx2 was determined at 1.62 A. The obstruction of the oxyanion hole indicates that Pdx2 is in a resting state and that activation occurs upon Pdx1-Pdx2 complex formation.



	Figure 4. Structural analysis of Pdx2. A, ribbon representation of the x-ray structure of Pdx2. Amino acids Cys^87, His^196, and Glu^198 make up the catalytic triad. The respective residues are shown in yellow in all panels. Residues involved in the putative interface with the synthase subunit are labeled in green. Differences to the Yaa E ortholog are shown in red. B, stick representation of the active site of Pdx2. The loop carrying the nucleophilic cysteine comprises residues Gly^85, Thr^86, Cys^87, Ala^88, and Gly^89. This loop is shown together with the 2F[o] -F[c] electron density at a level of 1.2σ. The double conformation of Cys^87 is visible. The proposed binding site of the synthase subunit is indicated. C, the proposed oxyanion hole, which forms during catalysis, is obstructed in Pdx2 by the carbonyl of Gly^51. D, the oxyanion hole is formed in the apo-form of CPS (1JDB) by the peptide nitrogens of Gly^241 and Leu^270. E, in the glutamine-bound state of CPS (1A9X), this conformation is maintained. The figure was prepared with PyMOL (53).		Figure 5. Cross-species interaction between Pdx1 and YaaE. A, growth of the B. subtilis 168 (trpC2) YaaD disruptant complemented with control construct lacking the ribosomal binding site (Pdx1) (1) or the complementation construct (RBS-Pdx1) (2) on minimal plates (TMM) without and with additives (0.05 mm pyridoxal (TMM+pyridoxal) or 2% xylose (TMM+xylose)) in the presence or absence of IPTG. IPTG is required to induce the expression of the endogenous YaaE. B, growth curves of the B. subtilis 168 (trpC2) YaaD disruptant complemented with RBS-Pdx1 (filled symbols: •, ▾, and ▪) or the RBS-lacking Pdx1 control construct (open symbols: ○,▾, and □) in TMM plus IPTG without and with additives (0.05 mm pyridoxal or 2% xylose). •, RBS-Pdx1 in TMM; ○, Pdx1 in TMM; ○, RBS-Pdx1 in TMM plus 0.05 mm pyridoxal;▵, Pdx1 in TMM plus 0.05 mm mm pyridoxal; •, RBS-Pdx1 in TMM -Pdx1 expression was induced by the addition of 2% xylose; □, Pdx1 in TMM plus 2% xylose. C, PLP formation by the Pdx1-YaaE complex in the presence of ribulose 5-phosphate, G3P, and 10 mm Gln: ▴, YaaD-YaaE complex (1:1); •, Pdx1-Pdx2 complex (1:1); •, Pdx1-YaaE (1:5); ○, Pdx1-YaaE (1:1);▵, YaaD; □, Pdx1; ♦, no enzyme.

PROCHECK

	Headers