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Intersubunit cross-talk in pyridoxal 5'-phosphate synthase, coordinated by the C terminus of the synthase subunit


Raschle, T; Speziga, D; Kress, W; Moccand, C; Gehrig, P; Amrhein, N; Weber-Ban, E; Fitzpatrick, T B (2009). Intersubunit cross-talk in pyridoxal 5'-phosphate synthase, coordinated by the C terminus of the synthase subunit. Journal of Biological Chemistry, 284(12):7706-7718.

Abstract

Vitamin B(6) is essential in all organisms, due to its requirement as a cofactor in the form of pyridoxal 5'-phosphate (PLP) for key metabolic enzymes. It can be synthesized de novo by either of two pathways known as deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent. The DXP-independent pathway is the predominant pathway and is found in most microorganisms and plants. A glutamine amidotransferase consisting of the synthase Pdx1 and its glutaminase partner, Pdx2, form a complex that directly synthesizes PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate, and glutamine. The protein complex displays an ornate architecture consisting of 24 subunits, two hexameric rings of 12 Pdx1 subunits to which 12 Pdx2 subunits attach, with the glutaminase and synthase active sites remote from each other. The multiple catalytic ability of Pdx1, the remote glutaminase and synthase active sites, and the elaborate structure suggest regulation of activity on several levels. A missing piece in deciphering this intricate puzzle has been information on the Pdx1 C-terminal region that has thus far eluded structural characterization. Here we use fluorescence spectrophotometry and protein chemistry to demonstrate that the Pdx1 C terminus is indispensable for PLP synthase activity and mediates intersubunit cross-talk within the enzyme complex. We provide evidence that the C terminus can act as a flexible lid, bridging as well as shielding the active site of an adjacent protomer in Pdx1. We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx1, and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx2 and glutamine.

Vitamin B(6) is essential in all organisms, due to its requirement as a cofactor in the form of pyridoxal 5'-phosphate (PLP) for key metabolic enzymes. It can be synthesized de novo by either of two pathways known as deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent. The DXP-independent pathway is the predominant pathway and is found in most microorganisms and plants. A glutamine amidotransferase consisting of the synthase Pdx1 and its glutaminase partner, Pdx2, form a complex that directly synthesizes PLP from ribose 5-phosphate, glyceraldehyde 3-phosphate, and glutamine. The protein complex displays an ornate architecture consisting of 24 subunits, two hexameric rings of 12 Pdx1 subunits to which 12 Pdx2 subunits attach, with the glutaminase and synthase active sites remote from each other. The multiple catalytic ability of Pdx1, the remote glutaminase and synthase active sites, and the elaborate structure suggest regulation of activity on several levels. A missing piece in deciphering this intricate puzzle has been information on the Pdx1 C-terminal region that has thus far eluded structural characterization. Here we use fluorescence spectrophotometry and protein chemistry to demonstrate that the Pdx1 C terminus is indispensable for PLP synthase activity and mediates intersubunit cross-talk within the enzyme complex. We provide evidence that the C terminus can act as a flexible lid, bridging as well as shielding the active site of an adjacent protomer in Pdx1. We show that ribose 5-phosphate binding triggers strong cooperativity in Pdx1, and the affinity for this substrate is substantially enhanced upon interaction with the Michaelis complex of Pdx2 and glutamine.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
07 Faculty of Science > Department of Plant and Microbial Biology
08 University Research Priority Programs > Systems Biology / Functional Genomics
Dewey Decimal Classification:570 Life sciences; biology
580 Plants (Botany)
610 Medicine & health
Language:English
Date:2009
Deposited On:07 Dec 2009 09:21
Last Modified:26 Aug 2016 07:32
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Additional Information:This research was originally published in Raschle, T; Speziga, D; Kress, W; Moccand, C; Gehrig, P; Amrhein, N; Weber-Ban, E; Fitzpatrick, T B (2009). Intersubunit cross-talk in pyridoxal 5'-phosphate synthase, coordinated by the C terminus of the synthase subunit. Journal of Biological Chemistry, 284(12):7706-7718. © the American Society for Biochemistry and Molecular Biology.
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/jbc.M804728200
PubMed ID:19074821
Permanent URL: https://doi.org/10.5167/uzh-25061

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