Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis

Abstract

Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I₃₂₀ intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand β6 of the Pdx1 (βα)₈-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

Figure 1|. The reaction catalyzed by the Pdxl and Pdx2 subunits of the PLP synthase complex and the structure of Pdxl.

(a) Pyridoxal 5-phosphate synthase catalyzes the complex condensation reaction between ribose 5-phosphate, glyceraldehyde 3-phosphate and ammonia. (b) The overall structure of the Pdxl core. Pdxl forms a dodecamer from two interlocked hexameric rings. This core complex is shown in two orientations, with subunit boundaries and the two phosphate-binding sites for each protein monomer indicated. The phosphate-binding sites P1 and P2 are required for substrate and product binding, respectively, and are separated by 21 Å (phosphorus to phosphorus). A cartoon representation for one monomer illustrates the (βα)₈-barrel fold of Pdxl.

Figure 2|. Crystallographic structures of five covalent intermediates in PLP biosynthesis.

Pdxl is shown in cartoon representation, and catalytic lysine side chains and intermediate atoms are shown in stick representation with carbon atoms in green (for Lys98 and Lys166) and orange (for intermediates), nitrogen atoms in blue, oxygen atoms in red, and phosphorus atoms in purple. 2F_o-F_c electron density maps are shown at 1 σ for the complexes. (a) Binding of R5P (first substrate) uses P1 and occurs by covalent attachment through Schiff base formation with Lys98. (b,c) Addition of ammonia (second substrate) leads to the formation of an intermediate in P1 in the K166R mutant (b), which converts to the I₃₂₀ species in wild-type enzyme through formation of a second Schiff base with Lys166 (c). (d) Incorporation of G3P (third substrate) leads to a covalent complex with I₃₂₀, with the G3P phosphate bound in the P1 site. (e) The PLP complex shows PLP covalently bound to the enzyme through Schiff base formation with Lys166, with its phosphate bound in the P2 site.

Figure 3|. The dual-specificity binding site P1 and the product-binding site P2.

(a) The UV-Vis spectrum of a Pdx1-I₃₂₀ crystal shows an absorption maximum at 280 nm, corresponding to protein, and near 320 nm for the I₃₂₀ intermediate. (b) The intermediate I₃₂₀ is covalently bound to both Lys98 and Lys166, shown in two views rotated by 90°. The overlay with the R5P complex (faded) shows the different positioning of Lys166 and adjacent residues Thr165 and Glyl67. Selected amino acid atoms and the I₃₂₀ atoms are shown in stick representation, with carbon atoms in green (for amino acids) and orange (for intermediates), nitrogen atoms in blue, oxygen atoms in red, and phosphorus atoms in purple. Arrows indicate the different conformations of the Lys166 side chains and the Lys166-Gly167 peptide between Pdxl-I₃₂₀₎ and Pdx1-R5P complexes. Pep-flip, peptide flip. (c) The I₃₂₀-G3P complex horseshoe-like intermediate. The intermediate is covalently attached to both Lys98 and Lys166. Formation of the G3P binding site requires prior formation of the I₃₂₀ adduct to dissociate the C3-C5 atoms from the binding site, which allows the G3P phosphate to bind in the P1 site. The overlay with the R5P complex (faded) shows where the ribose and triose portions match. Atoms colored as described for Figure 3b. (d) The PLP adduct is covalently bound by a Schiff base to Lys166. 2F_o - F_c electron density map of the refined complex shown at 1 σ. Atoms colored as described for Figure 3b.

Figure 4|. The central role of I₃₂₀ intermediate transfer in vitamin B6 biosynthesis.

The proposed Pdx1 reaction mechanism is shown as a schematic. Lys98 points toward P1, and Lys166 points toward P2 in the free enzyme. Structural transitions support reorientation of the side chain of Lys166 toward the P1 site at the time of ammonia incorporation, leading to formation of I₃₂₀, creation of the G3P binding site, and the subsequent formation of the covalent I₃₂₀-G3P complex. The reversion of the structural transitions around Lys166 lead to release of the I₃₂₀-G3P intermediate from covalent attachment to Lys98 and observation of covalently bound PLP in the P2 site.