Functional Characterization of Cj1427, a Unique Ping-Pong Dehydrogenase Responsible for the Oxidation of GDP‑d-glycero-α‑d-manno-heptose in Campylobacter jejuni

• Published in: Biochemistry (Easton) 2020-04, Vol.59 (13), p.1328-1337
• Main Authors: Huddleston, Jamison P, Raushel, Frank M
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Campylobacter jejuni - chemistry; Campylobacter jejuni - enzymology; Campylobacter jejuni - genetics; Campylobacter jejuni - metabolism; Catalytic Domain; Guanosine Diphosphate - chemistry; Guanosine Diphosphate - metabolism; Heptoses - chemistry; Heptoses - metabolism; Ketoglutaric Acids - chemistry; Ketoglutaric Acids - metabolism; Kinetics; NAD - chemistry; NAD - metabolism; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.0c00097

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-α-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-α-d-manno-heptose has remained elusive. The enzyme Cj1427 from C. jejuni NCTC 11168 was shown to catalyze the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose in the presence of α-ketoglutarate using mass spectrometry and nuclear magnetic resonance spectroscopy. At pH 7.4, the apparent k cat is 0.6 s–1, with a value of k cat/K m of 1.0 × 104 M–1 s–1 for GDP-d-glycero-α-d-manno-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.