title: Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from Plasmodium falciparum.
abstract: Pyridoxal 5'-phosphate (PLP) is required as a cofactor by many enzymes. The predominant de novo biosynthetic route is catalyzed by a heteromeric glutamine amidotransferase consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. Previously, Bacillus subtilis PLP synthase was studied by X-ray crystallography and complex assembly had been characterized by isothermal titration calorimetry. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers. These studies revealed the occurrence of an encounter complex that is tightened in the Michaelis complex when the substrate l-glutamine binds. In this study, we have characterized complex formation of PLP synthase from the malaria-causing human pathogen Plasmodium falciparum using isothermal titration calorimetry. The presence of l-glutamine increases the tightness of the interaction about 30-fold and alters the thermodynamic signature of complex formation. The thermodynamic data are integrated in a 3D homology model of P. falciparum PLP synthase. The negative experimental heat capacity (C(p)) describes a protein interface that is dominated by hydrophobic interactions. In the absence of l-glutamine, the experimental C(p) is less negative than in its presence, contrasting to the previously characterised bacterial PLP synthase. Thus, while the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characteristics concerning the relative contribution of apolar/polar surface area. In addition, we have verified the role of the N-terminal region of PfPdx1 for complex formation. A "swap mutant" in which the complete alphaN-helix of plasmodial Pdx1 was exchanged with the corresponding segment from B. subtilis shows cross-binding to B. subtilis Pdx2. The swap mutant also partially elicits glutaminase activity in BsPdx2, demonstrating that formation of the protein complex interface via alphaN and catalytic activation of the glutaminase are linked processes.
authors.affiliation: Technische Universität Graz, Institut für Biochemie, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Karlheinz
authors.initials: K
authors.lastname: Flicker
authors.forename: Martina
authors.initials: M
authors.lastname: Neuwirth
authors.forename: Marco
authors.initials: M
authors.lastname: Strohmeier
authors.forename: Barbara
authors.initials: B
authors.lastname: Kappes
authors.forename: Ivo
authors.initials: I
authors.lastname: Tews
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
mesh_terms: D000818:Animals; D002151:Calorimetry; D018360:Crystallography, X-Ray; D005972:Glutaminase / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D010963:Plasmodium falciparum / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D011994:Recombinant Proteins; D005973:Glutamine; D005972:Glutaminase