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Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from plasmodium falciparum

Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from plasmodium falciparum
Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from plasmodium falciparum
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.
0022-2836
732-748
Flicker, Karlheinz
2d14a029-e083-4424-80c1-c6b83b5bd345
Neuwirth, Martina
6ea2236b-a98c-48b3-a908-52fb9b318ff8
Strohmeier, Marco
945de88c-2eb8-4708-b91b-8f599b4b8c24
Kappes, Barbara
10edf779-dc81-4973-878b-d3177273f86a
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Macheroux, Peter
e1c49266-c971-42f7-86ae-394bd128d040
Flicker, Karlheinz
2d14a029-e083-4424-80c1-c6b83b5bd345
Neuwirth, Martina
6ea2236b-a98c-48b3-a908-52fb9b318ff8
Strohmeier, Marco
945de88c-2eb8-4708-b91b-8f599b4b8c24
Kappes, Barbara
10edf779-dc81-4973-878b-d3177273f86a
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Macheroux, Peter
e1c49266-c971-42f7-86ae-394bd128d040

Flicker, Karlheinz, Neuwirth, Martina, Strohmeier, Marco, Kappes, Barbara, Tews, Ivo and Macheroux, Peter (2007) Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from plasmodium falciparum. Journal of Molecular Biology, 374 (3), 732-748. (doi:10.1016/j.jmb.2007.09.038). (PMID:17950752)

Record type: Article

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.

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Published date: 30 November 2007
Organisations: Centre for Biological Sciences

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Local EPrints ID: 200557
URI: http://eprints.soton.ac.uk/id/eprint/200557
ISSN: 0022-2836
PURE UUID: 7ff94834-9690-4e50-b65b-812bfa2dae17
ORCID for Ivo Tews: ORCID iD orcid.org/0000-0002-4704-1139

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Date deposited: 01 Nov 2011 13:55
Last modified: 17 Dec 2019 01:41

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