Functional Characterization of Cj1427, a Unique Ping-Pong Dehydrogenase Responsible for the Oxidation of GDP-d-α-d--heptose in .

The capsular polysaccharides (CPS) of contain multiple heptose residues with variable stereochemical arrangements at C3-C6. The immediate precursor to all of these possible variations is currently believed to be GDP-d--α-d--heptose. Oxidation of this substrate at C4 enables subsequent epimerization reactions at C3-C5 that can be coupled to the dehydration/reduction at C5/C6. However, the enzyme responsible for the critical oxidation of C4 from GDP-d--α-d--heptose has remained elusive. The enzyme Cj1427 from NCTC 11168 was shown to catalyze the oxidation of GDP-d--α-d--heptose to GDP-d--4-keto-α-d--heptose in the presence of α-ketoglutarate using mass spectrometry and nuclear magnetic resonance spectroscopy. At pH 7.4, the apparent is 0.6 s, with a value of / of 1.0 × 10 M s for GDP-d--α-d--heptose. α-Ketoglutarate is required to recycle the tightly bound NADH nucleotide in the active site of Cj1427, which does not dissociate from the enzyme during catalysis.