Intersubunit Cross-talk in Pyridoxal 5′-Phosphate Synthase, Coordinated by the C Terminus of the Synthase Subunit

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.

Defining the structural requirements for ribose 5‐phosphate‐binding and intersubunit cross‐talk of the malarial pyridoxal 5‐phosphate synthase

“…The C-terminal region of Pdx1 is responsible for intersubunit cross-talk and confers cooperativity to the enzyme [18]. The full C-terminus of Pdx1 is not resolved in prokaryotic 3D structures [9,10,23] and in these structures helix a8 00 is the most C-terminal secondary structure element in the core of Pdx1 (Fig.…”

“…I 320 -specific activity increased above that of wild-type in the Pdx2-dependent assay (167% versus 100%; Table 2), while PLP-specific activity was further reduced (32%; Table 2). Thus, Pdx2 in its glutamine bound form has an influence on chromophore formation, exerting an allosteric effect [18], even when only the truncated form is considered.…”

“…The C-terminus of Pdx1, which has evaded structural determination, is essential for catalytic activity of the protein [18]. In the current study, we performed a deletion analysis and assigned specific functions to defined regions of the C-terminus.…”

“…Cooperativity between Pdx1 subunits is induced upon binding of the pentose substrate [18]. Moreover, Pdx2 acts as an allosteric effector of Pdx1 when its substrate glutamine is bound and then further enhances the affinity of Pdx1 for R5P [18].…”

“…The activity of the PLP synthase system is highly regulated [7,[14][15][16][17][18][19]. Cooperativity between Pdx1 subunits is induced upon binding of the pentose substrate [18].…”