Functional Characterization of Cj1427, a Unique Ping-Pong Dehydrogenase Responsible for the Oxidation of GDP-d-glycero--d-manno-heptose in Campylobacter jejuni.
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abstract
The capsular polysaccharides (CPS) of Campylobacter jejuni 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-glycero--d-manno-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-glycero--d-manno-heptose has remained elusive. The enzyme Cj1427 from C. jejuni NCTC 11168 was shown to catalyze the oxidation of GDP-d-glycero--d-manno-heptose to GDP-d-glycero-4-keto--d-lyxo-heptose in the presence of -ketoglutarate using mass spectrometry and nuclear magnetic resonance spectroscopy. At pH 7.4, the apparent kcat is 0.6 s-1, with a value of kcat/Km of 1.0 104 M-1 s-1 for GDP-d-glycero--d-manno-heptose. -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.
