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Functional analysis of PDX2 from arabidopsis, a glutaminase involved in vitamin B6 biosynthesis

Functional analysis of PDX2 from arabidopsis, a glutaminase involved in vitamin B6 biosynthesis
Functional analysis of PDX2 from arabidopsis, a glutaminase involved in vitamin B6 biosynthesis
Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5'-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1.
0032-0889
915-925
Tambasco-Studart, Marina
3d16c214-ca18-4914-963e-274ee0243cfe
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Amrhein, Nikolaus
2669a3df-0561-47ae-b5ae-db36911eb618
Fitzpatrick, Teresa B
ba3eee8b-d3e5-4a18-9845-64f5e56962f5
Tambasco-Studart, Marina
3d16c214-ca18-4914-963e-274ee0243cfe
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
Amrhein, Nikolaus
2669a3df-0561-47ae-b5ae-db36911eb618
Fitzpatrick, Teresa B
ba3eee8b-d3e5-4a18-9845-64f5e56962f5

Tambasco-Studart, Marina, Tews, Ivo, Amrhein, Nikolaus and Fitzpatrick, Teresa B (2007) Functional analysis of PDX2 from arabidopsis, a glutaminase involved in vitamin B6 biosynthesis. Plant Physiology, 144 (2), 915-925. (doi:10.1104/pp.107.096784). (PMID:17144654)

Record type: Article

Abstract

Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5'-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1.

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More information

Published date: June 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: 200597
URI: http://eprints.soton.ac.uk/id/eprint/200597
ISSN: 0032-0889
PURE UUID: 20f882d8-521a-4e1b-806b-bae2da4a6a64
ORCID for Ivo Tews: ORCID iD orcid.org/0000-0002-4704-1139

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Date deposited: 01 Nov 2011 14:11
Last modified: 15 Mar 2024 03:36

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Contributors

Author: Marina Tambasco-Studart
Author: Ivo Tews ORCID iD
Author: Nikolaus Amrhein
Author: Teresa B Fitzpatrick

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