Defining the structural requirements for ribose 5‐phosphate‐binding and intersubunit cross‐talk of the malarial pyridoxal 5‐phosphate synthase

Abstract

a b s t r a c tMost organisms synthesise the B 6 vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I 320 , and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex.

Exploring inhibition of Pdx1, a component of the PLP synthase complex of the human malaria parasite Plasmodium falciparum

“…An RHE (R85A, H88A and E91A) mutant Pf Pdx1 was shown previously to prevent dodecameric assembly, and to be incapable of activating Pf Pdx2 subunits [24]. These observations are in agreement with functions assigned to the C-terminal region and moreover emphasized the importance of the RHE residues for R5P binding and imine adduct formation [30]. Additional experimentation with RHE Pf Pdx1 mutants and C Pdx1, lacking the 30 C-terminal residues, revealed that both proteins were unable to form I 320 ( Figure 3B).…”

“…Previous studies have revealed several important residues involved in I 320 formation that include Asp 26 , Lys 83 and Lys 151 [24] as well as residues from the C-terminus [30]. We sought to verify the importance of these residues during Pf Pdx1 I 320 formation.…”

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis

“…This phosphate-binding site is located in the typical position for the (βα) 8 -fold at the C-terminal face of the β-barrel, and makes additional contacts with the β6-α6 loop that carries Lys l66 (refs. 11,12,32). The Pdxl-pre-I 320 structure highlights the role of this phosphate group as a rigid anchor that, together with the Lys98 imine, ensures correct positioning of the intermediate for ammonia incorporation and covalent attachment to Lysl66 in the wild-type protein ( Fig.…”

“…The total loss of diffraction upon prolonged G3P exposure provides strong evidence for extensive structural changes that accompany the final steps of PLP formation. The C terminus of Pdxl is disordered in crystal structures and is known to participate in the final stages of PLP biosynthesis 32–34 . The packing of Pdxl in the crystalline state may prevent the C terminus from adopting the conformation required for Pdxl to complete catalysis of PLP biosynthesis.…”

Identification of Wood Decay Related Genes fromPiptoporus Betulinus(Bull. Fr.) Karsten Using Differential Display Reverse Transcription PCR (DDRT-PCR)

“…wood. this fungus decays wood slowly under many conditions and causes brown rot on the branch and trunk wood of weakened birch trees (6). to degrade wood, this fungus first decays the inner discolored trunk zone, and then the non-discolored wood (28,36).…”