The University of Southampton
University of Southampton Institutional Repository

Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis

Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis
Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis
Pyridoxal 5-phosphate (PLP), an active form of vitamin B(6), is one of the most versatile cofactors and is involved in numerous biochemical reactions. The main pathway for de novo PLP biosynthesis leads to direct formation of PLP from a pentose and triose. This reaction is catalyzed by the heteromeric PLP synthase, consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. l-Glutamine hydrolysis by Pdx2 supplies ammonia to Pdx1 for incorporation into PLP. Autonomous glutaminase Pdx2 is inactive; however, interaction with Pdx1 leads to enzymatic activity. Oxyanion hole formation in the active site of Pdx2 is required for substrate binding and was suggested as the prime event of enzyme activation. Here, we dissect interactions required for complex formation from interactions required for catalytic activation of the glutaminase. The three-dimensional structural analysis suggested a number of invariant residues that regulate complex formation and enzyme activation. We have replaced several of these invariant residues by site-directed mutagenesis in an effort to understand their function. In addition to the biochemical characterization of enzyme activity, the generated protein variants were studied by isothermal calorimetry to investigate their role in complex formation. The assembled data describe a multistep activation mechanism. Residues of helix alphaN of Pdx1 are essential for formation of the Pdx1-Pdx2 complex and also stabilize the oxyanion hole. Thus, these interactions describe the encounter complex. On the other hand, residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 contribute to interface formation and are required for the organization of the catalytic center; thus, these interactions describe the Michaelis complex. However, the main players for formation of the Michaelis complex reside on Pdx2, as replacement of residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 leads to reduction but not complete inactivation of the glutaminase.
0006-2960
1928-1935
Wallner, Silvia
147f37c7-9e66-40f5-9296-89e910237ad6
Neuwirth, Martina
6ea2236b-a98c-48b3-a908-52fb9b318ff8
Flicker, Karlheinz
2d14a029-e083-4424-80c1-c6b83b5bd345
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Macheroux, Peter
e1c49266-c971-42f7-86ae-394bd128d040
Wallner, Silvia
147f37c7-9e66-40f5-9296-89e910237ad6
Neuwirth, Martina
6ea2236b-a98c-48b3-a908-52fb9b318ff8
Flicker, Karlheinz
2d14a029-e083-4424-80c1-c6b83b5bd345
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Macheroux, Peter
e1c49266-c971-42f7-86ae-394bd128d040

Wallner, Silvia, Neuwirth, Martina, Flicker, Karlheinz, Tews, Ivo and Macheroux, Peter (2009) Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal 5-phosphate synthase complex from Bacillus subtilis. Biochemistry, 48 (9), 1928-1935. (doi:10.1021/bi801887r). (PMID:19152323)

Record type: Article

Abstract

Pyridoxal 5-phosphate (PLP), an active form of vitamin B(6), is one of the most versatile cofactors and is involved in numerous biochemical reactions. The main pathway for de novo PLP biosynthesis leads to direct formation of PLP from a pentose and triose. This reaction is catalyzed by the heteromeric PLP synthase, consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. l-Glutamine hydrolysis by Pdx2 supplies ammonia to Pdx1 for incorporation into PLP. Autonomous glutaminase Pdx2 is inactive; however, interaction with Pdx1 leads to enzymatic activity. Oxyanion hole formation in the active site of Pdx2 is required for substrate binding and was suggested as the prime event of enzyme activation. Here, we dissect interactions required for complex formation from interactions required for catalytic activation of the glutaminase. The three-dimensional structural analysis suggested a number of invariant residues that regulate complex formation and enzyme activation. We have replaced several of these invariant residues by site-directed mutagenesis in an effort to understand their function. In addition to the biochemical characterization of enzyme activity, the generated protein variants were studied by isothermal calorimetry to investigate their role in complex formation. The assembled data describe a multistep activation mechanism. Residues of helix alphaN of Pdx1 are essential for formation of the Pdx1-Pdx2 complex and also stabilize the oxyanion hole. Thus, these interactions describe the encounter complex. On the other hand, residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 contribute to interface formation and are required for the organization of the catalytic center; thus, these interactions describe the Michaelis complex. However, the main players for formation of the Michaelis complex reside on Pdx2, as replacement of residues at the N-terminal face of the (betaalpha)(8) barrel of Pdx1 leads to reduction but not complete inactivation of the glutaminase.

Text
Biochem_48-1928.pdf - Version of Record
Restricted to Repository staff only
Request a copy

More information

e-pub ahead of print date: 16 January 2009
Published date: 10 March 2009
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: 200337
URI: http://eprints.soton.ac.uk/id/eprint/200337
ISSN: 0006-2960
PURE UUID: 5716216d-9570-4240-8e2b-21d1b6fd3af1
ORCID for Ivo Tews: ORCID iD orcid.org/0000-0002-4704-1139

Catalogue record

Date deposited: 24 Oct 2011 16:16
Last modified: 15 Mar 2024 03:36

Export record

Altmetrics

Contributors

Author: Silvia Wallner
Author: Martina Neuwirth
Author: Karlheinz Flicker
Author: Ivo Tews ORCID iD
Author: Peter Macheroux

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.

×