13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase.
The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5'-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using (13)C- and (15)N-labeled substrates as well as (15)N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5'-phosphate.
Department of Chemistry and Chemical Biology, Cornell University, 120 Baker Laboratory, Ithaca, New York 14853, USA.
Jeremiah W
JW
Hanes
Ivan
I
Keresztes
Tadhg P
TP
Begley
D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D002247:Carbon Isotopes; D002384:Catalysis; D005972:Glutaminase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011732:Pyridoxal Phosphate / Q000096:biosynthesis* / Q000737:chemistry; D012267:Ribosemonophosphates / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D025101:Vitamin B 6 / Q000096:biosynthesis* / Q000737:chemistry
D002247:Carbon Isotopes; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; D011732:Pyridoxal Phosphate; D025101:Vitamin B 6; D005972:Glutaminase