The University of Southampton
University of Southampton Institutional Repository

Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase

Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase
Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase
Two biosynthetic routes for the synthesis of pyridoxal 5'-phosphate (PLP), the biologically active compound of vitamin B6, have been characterized. The major pathway leads to direct formation of PLP from a pentasaccharide and a trisaccharide and is operative in plants, fungi, protozoa, and bacteria. This reaction is catalyzed by a single glutamine amidotransferase enzyme complex consisting of a pyridoxal synthase, termed Pdx1, and a glutaminase, termed Pdx2. In this complex, Pdx2 generates ammonia from L-glutamine and supplies it to Pdx1 for incorporation into PLP. The glutaminase activity of Pdx2 requires the presence of Pdx1 in a heteromeric complex, previously characterized by a crystallographic three-dimensional (3D) structure determination. Here, we give a thermodynamic description of complex formation of Bacillus subtilis PLP synthase in the absence or presence of L-glutamine. We show that L-glutamine directly affects the tightness of the protein complex, which exhibits dissociation constants of 6.9 and 0.3 microM in its absence and presence, respectively (at 25 degrees C). This result relates to the positioning of L-glutamine on the heterodimer interface as seen in the 3D structure. In an analysis of the temperature dependence of the enthalpy, negative heat capacity changes (deltaCp) agree with a protein interface governed by hydrophobic interactions. The measured heat capacity change is also a function of L-glutamine, with a negative deltaCp in the presence of L-glutamine and a more negative one in its absence. These findings suggest that L-glutamine not only affects the strength of complex formation but also determines the forces involved in complex formation, with regard to different relative contributions of hydrophobic and hydrophilic interactions.
0006-2960
5131-5139
Neuwirth, Martina
6ea2236b-a98c-48b3-a908-52fb9b318ff8
Flicker, Karlheinz
2d14a029-e083-4424-80c1-c6b83b5bd345
Strohmeier, Marco
945de88c-2eb8-4708-b91b-8f599b4b8c24
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Macheroux, Peter
e1c49266-c971-42f7-86ae-394bd128d040
Neuwirth, Martina
6ea2236b-a98c-48b3-a908-52fb9b318ff8
Flicker, Karlheinz
2d14a029-e083-4424-80c1-c6b83b5bd345
Strohmeier, Marco
945de88c-2eb8-4708-b91b-8f599b4b8c24
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Macheroux, Peter
e1c49266-c971-42f7-86ae-394bd128d040

Neuwirth, Martina, Flicker, Karlheinz, Strohmeier, Marco, Tews, Ivo and Macheroux, Peter (2007) Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxalphosphate synthase. Biochemistry, 46 (17), 5131-5139. (doi:10.1021/issn.0006-2960). (PMID:17408246)

Record type: Article

Abstract

Two biosynthetic routes for the synthesis of pyridoxal 5'-phosphate (PLP), the biologically active compound of vitamin B6, have been characterized. The major pathway leads to direct formation of PLP from a pentasaccharide and a trisaccharide and is operative in plants, fungi, protozoa, and bacteria. This reaction is catalyzed by a single glutamine amidotransferase enzyme complex consisting of a pyridoxal synthase, termed Pdx1, and a glutaminase, termed Pdx2. In this complex, Pdx2 generates ammonia from L-glutamine and supplies it to Pdx1 for incorporation into PLP. The glutaminase activity of Pdx2 requires the presence of Pdx1 in a heteromeric complex, previously characterized by a crystallographic three-dimensional (3D) structure determination. Here, we give a thermodynamic description of complex formation of Bacillus subtilis PLP synthase in the absence or presence of L-glutamine. We show that L-glutamine directly affects the tightness of the protein complex, which exhibits dissociation constants of 6.9 and 0.3 microM in its absence and presence, respectively (at 25 degrees C). This result relates to the positioning of L-glutamine on the heterodimer interface as seen in the 3D structure. In an analysis of the temperature dependence of the enthalpy, negative heat capacity changes (deltaCp) agree with a protein interface governed by hydrophobic interactions. The measured heat capacity change is also a function of L-glutamine, with a negative deltaCp in the presence of L-glutamine and a more negative one in its absence. These findings suggest that L-glutamine not only affects the strength of complex formation but also determines the forces involved in complex formation, with regard to different relative contributions of hydrophobic and hydrophilic interactions.

Text
Biochem_46-5131.pdf - Version of Record
Restricted to Repository staff only
Request a copy

More information

Published date: 1 May 2007
Additional Information: Funded by European Commission - FP6: Vitamin biosynthesis as a target for antimalarial therapy (VITBIOMAL) (12158)
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 200615
URI: http://eprints.soton.ac.uk/id/eprint/200615
ISSN: 0006-2960
PURE UUID: a7eba63d-5478-4cfb-b0a9-5974fec6f8b5
ORCID for Ivo Tews: ORCID iD orcid.org/0000-0002-4704-1139

Catalogue record

Date deposited: 01 Nov 2011 14:19
Last modified: 12 Nov 2019 01:41

Export record

Altmetrics

Contributors

Author: Martina Neuwirth
Author: Karlheinz Flicker
Author: Marco Strohmeier
Author: Ivo Tews ORCID iD
Author: Peter Macheroux

University divisions

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×