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

Abstract

Vitamin B 6 is indispensible for all organisms, notably as the coenzyme form pyridoxal 5′-phosphate. Plants make the compound de novo using a relatively simple pathway comprising pyridoxine synthase (PDX1) and pyridoxine glutaminase (PDX2). PDX1 is remarkable given its multifaceted synthetic ability to carry out isomerization, imine formation, ammonia addition, aldol-type condensation, cyclization, and aromatization, all in the absence of coenzymes or recruitment of specialized domains. Two active sites (P1 and P2) facilitate the plethora of reactions, but it is not known how the two are coordinated and, moreover, if intermediates are tunneled between active sites. Here we present X-ray structures of PDX1.3 from Arabidopsis thaliana, the overall architecture of which is a dodecamer of (β/α) 8 barrels, similar to the majority of its homologs. An apoenzyme structure revealed that features around the P1 active site in PDX1.3 have adopted inward conformations consistent with a catalytically primed state and delineated a substrate accessible cavity above this active site, not noted in other reported structures. Comparison with the structure of PDX1.3 with an intermediate along the catalytic trajectory demonstrated that a lysine residue swings from the distinct P2 site to the P1 site at this stage of catalysis and is held in place by a molecular catch and pin, positioning it for transfer of serviced substrate back to P2. The study shows that a simple lysine swinging arm coordinates use of chemically disparate sites, dispensing with the need for additional factors, and provides an elegant example of solving complex chemistry to generate an essential metabolite.Arabidopsis thaliana | vitamin B6 biosynthesis | crystal structure | lysine swing

Vitamin B6 and Its Role in Cell Metabolism and Physiology

“…The PDX1/PDX2 protein pair, which forms a PLP synthase, is widely distributed among archaea, bacteria, fungi, protists, and plants, and the corresponding complexes have been characterized in a variety of pro- and eukaryotes, including Geobacillus stearothermophilus (PdxS/PdxT) [ 48 , 49 ], B. subtilis (Pdx1/Pdx2 or PdxS/PdxT) [ 50 , 51 ], Thermotoga maritima (YaaD/YaaE) [ 52 ], S. cerevisiae [ 52 , 53 ], A. thaliana [ 54 ], and Plasmodium falciparum [ 55 ].…”

“…In plants, two recent studies have shown that PLP synthases contain a lysine swing or lysine relay mechanism that allows intermediate channeling of the substrate in the process of vitB 6 biosynthesis [ 54 , 56 ]. Essentially, the lysine residues anchor the substrate to the PLP synthase and facilitate transfer of the substrate between the two active sites efficiently without extra domains or coenzymes [ 54 ]. These two sites are present in PDX1 proteins and are designated as P1 and P2 [ 56 ].…”

“…The pentose phosphate is dephosphorylated, and together with water and ammonia from glutamine, form a chromophoric I 320 intermediate with an absorbance maximum at 320 nm. This is shuttled to the P2 site, where it is condensed with a 3-carbon sugar to yield PLP [ 54 , 56 ].…”

SNZ3 Encodes a PLP Synthase Involved in Thiamine Synthesis in Saccharomyces cerevisiae

“…Several PLP synthase enzymes from bacteria, archaea, yeast and plants, have been characterized biochemically with and without the associated glutaminase (Dong et al 2004; Burns et al 2005; Raschle et al 2005; Gengenbacher et al 2006; Strohmeier et al 2006; Zein et al 2006; Raschle et al 2007; Hanes et al 2008; Neuwirth et al 2009; Raschle et al 2009; Moccand et al 2011; Robinson et al 2016; Rodrigues et al 2017). A variety of names have been used for the genes encoding PLP synthase and glutaminase enzymes, e.g.…”

Tunable hetero-assembly of a plant pseudoenzyme-enzyme complex

“…183 PDX1.3 implements an extremely efficient lysine relay mechanism where all catalytic action 184 happens within a single domain (catalytic sites P1 and P2). Two key residues K98 and K166 trap the 185 substrates, covalently tether the intermediates and then shuttle the product out5,20 . We compared the 186 obtained Cryo-EM structure of inactive PDX1.2 with several crystallographic structures of PDX1.3 187 available at different stages of such action: precursor binding stage PDX1.3-R5P (PDB:5lns), 188 intermediate stage PDX1.3-I 320 (PDB:5lnu), post-intermediate PDX1.3-I 320 -G3P (PDB:5lnw) and final 189 product stage where PLP is still covalently bound to K166 (PDB:5lnr) (Fig.3a).…”