Article abstract
Nature Chemical Biology 4, 425 - 430 (2008)
Published online: 30 May 2008 | doi:10.1038/nchembio.93
13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase
Jeremiah W Hanes1, Ivan Keresztes1 & Tadhg P Begley1
Abstract
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 13C- and 15N-labeled substrates as well as 15N-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.
Correspondence to: Tadhg P Begley1 e-mail: tpb2@cornell.edu
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