Structural basis for the function of pyridoxine 5 '-phosphate synthase

Background: Pyridoxal 5 ' -phosphate is the active form of vitamin B, that acts as an essential, ubiquitous coenzyme in amino acid metabolism. In Esch erichia coli, the pathway of the de novo biosynthesis of vitamin B-6 result s in the formation of pyridoxine 5 ' -phosphate (PNP), which can be regarde d as the first synthesized B-6 vitamer. PNP synthase (commonly referred to as PdxJ) is a homooctameric enzyme that catalyzes the final step in this pa thway, a complex intramolecular condensation reaction between 1-deoxy-D-xyl ulose-5 ' -phosphate and 1-amino-acetone-3-phosphate. Results: The crystal structure of E. coli PNP synthase was solved by single isomorphous replacement with anomalous scattering and refined at a resolut ion of 2.0 Angstrom. The monomer of PNP synthase consists of one compact do main that adopts the abundant TIM barrel fold. Intersubunit contacts are me diated by three additional helices, respective to the classical TIM barrel helices, generating a tetramer of symmetric dimers with 422 sym metry. in t he shared active sites of the active dimers, Arg20 is directly involved in substrate binding of the partner monomer. Furthermore, the structure of PNP synthase with its physiological products, PNP and P-i, was determined at 2 .3 Angstrom resolution, which provides insight into the dynamic action of t he enzyme and allows us to identify amino acids critical for enzymatic func tion. Conclusion: The high-resolution structures of the free enzyme and the enzym e-product complex of E. coli PNP synthase suggest essentials of the enzymat ic mechanism. The main catalytic features are active site closure upon subs trate binding by rearrangement of one C-terminal loop of the TIM barrel, ch arge-charge stabilization of the protonated Schiff-base intermediate, the p resence of two phosphate binding sites, and a water channel that penetrates the beta barrel and allows the release of water molecules in the closed st ate. All related PNP synthases are predicted to fold into a similar TIM bar rel pattern and have comparable active site architecture. Thus, a common me chanism can be anticipated.