Structural study of a novel vitamin B6 biosynthetic pathway

Abstract

Pyridoxal 5\u27-phosphate (PLP, vitamin B6), a cofactor in many enzymatic reactions, has two distinct biosynthetic routes, which do not coexist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea, and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT produces ammonia from glutamine and PdxS combines ammonia with five- and three-carbon phosphosugars to form PLP. A 2.2-Å crystal structure of PdxS is reported in this thesis. PdxS monomer has a classic (β/α)8 barrel fold. However, PdxS monomers form a cylindrical dodecamer in the crystal. Two hexamer rings of PdxS are linked by sulfate ions between the rings with the active sites positioned on the inside of the cylinder. A novel phosphate-binding site is suggested by another bound sulfate. This sulfate, bound at the top of the barrel, and another bound molecule, methyl pentanedial (MPD), were used to model the substrate ribulose 5-phosphate, and to propose catalytic roles for residues in the active site. The aggregation states of PdxS, PdxT and PLP synthase were investigated by analytical ultracentrifugation (AUC). The hexamer and dodecamer forms of PdxS coexist in solution. PdxT is a monomer in solution. PLP synthase is composed of a PdxS dodecamer core plus 12 PdxT subunits. The structures of PdxS and PLP synthase in solution were studied by small angle X-ray scattering (SAXS). The PdxS structure in solution is consistent with the crystal structure. The structure determination of PLP synthase in solution turned to be a much more difficult case than expected and more investigations are needed. A model of PLP synthase was proposed based on the distribution of conserved surfaces in the PdxS dodecdamer, the results from AUC experiments, and the information from SAXS experiments