It Takes Two to Tango: Defining an Essential Second Active Site in Pyridoxal 5′-Phosphate Synthase

Abstract

The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2) that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5′-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5′-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.

Structural definition of the lysine swing in Arabidopsis thaliana PDX1: Intermediate channeling facilitating vitamin B 6 biosynthesis

“…Moreover, the network of intrasubunit and intersubunit interactions zippering the C-terminal region in place would appear to be poised to coordinate cross-talk between two neighboring subunits. We have previously demonstrated that PDX1 displays high cooperativity in relation to the binding of the R5P substrate for which the C terminus is essential (12,14). Therefore, this structure of PDX1.3 serves to capture the fundamental essence of the C terminus in linking neighboring catalytically poised subunits.…”

“…Loss of phosphate and water from R5P as well as the incorporation of ammonia (from glutamine in the presence of PDX2) result in a chromophoric intermediate likely to be unique to PLP synthase, which has been observed spectroscopically (8)(9)(10) but has eluded precise structural characterization. The reaction can only proceed further in the presence of G3P, which is assumed to take place in the P2 site and is where the final product PLP has been observed in X-ray crystal structures (10,12,18). A pertinent question is how the intermediate is transferred from the P1 to the P2 site.…”

“…Two active sites, annotated P1 and P2, have been definitively located in PDX1 enzymes (16,17). These sites were originally defined by bound chloride, phosphate, or sulfate ions in X-ray crystallographic structures (16,17,29) and more recently by the bound substrate R5P (P1) and product PLP (P2) (11,18,32) and have been validated through several biochemical studies (10,12,16). In PDX1.3, the P1 site is located at the C-terminal mouth of the β-barrel and contains density for a sulfate ion surrounded by the characteristic phosphate-binding loop that includes Gly-ThrGly, as well as other established conserved residues of this active site (16,29) (Fig.…”

“…Although PDX2 is a classic glutaminase hydrolyzing glutamine to ammonia and glutamate (4)(5)(6), the remarkable reaction mechanism of PDX1 that involves pentose and triose isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization to produce the PLP molecule has received considerable attention but remains enigmatic (7)(8)(9)(10). Both biochemical and structural studies have served to capture snapshots of the PDX1 enzyme in action at certain stages along the catalytic trajectory (10)(11)(12)(13)(14)(15)(16)(17)(18). Nonetheless, the sequence of events cannot yet be stitched together and is essential to provide a complete overview of one of nature's most complicated enzymes from a fundamental perspective, as well as its potential as a drug target.…”

Molecular mechanism of PdxR – a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii

“…Although PLP is essential for all living beings, only plants and microorganisms are able to synthesize it de novo. Two mutually exclusive biosynthetic routes are known [3]: (a) the deoxyxylulose 5-phosphate (DXP)-dependent pathway, present in Escherichia coli and in a restricted number of other eubacteria [4], which is a multistep process involving seven enzymes, and (b) the so-called DXP-independent pathway, which is present in plants and most bacteria and relies on the action of the PLP synthase complex, formed by subunits encoded by pdxS and pdxT genes [5][6][7]. Many bacteria (e.g.…”