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

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), pp. 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.

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e-pub ahead of print date: 16 January 2009
Published date: 10 March 2009
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

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Date deposited: 24 Oct 2011 16:16
Last modified: 18 Jul 2017 11:14

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Contributors

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

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