Open AccessFunctional Characterization of Cj1427, a Unique Ping-Pong Dehydrogenase Responsible for the Oxidation of GDP-d -glycero-α-d -manno-heptose in Campylobacter jejuni
Author(s) -
Jamison P. Huddleston,
Frank M. Raushel
Publication year - 2020
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.0c00097
Subject(s) - heptose , campylobacter jejuni , enzyme kinetics , chemistry , epimer , stereochemistry , enzyme , dehydrogenase , alcohol oxidoreductase , biochemistry , nad+ kinase , bacteria , active site , biology , mutant , genetics , gene
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 × 10 4 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.