Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas 77843, United States.
Biochemistry. 2020 Apr 7;59(13):1328-1337. doi: 10.1021/acs.biochem.0c00097. Epub 2020 Mar 18.
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.
含有多种具有可变立体化学排列的庚糖残基。目前认为所有这些可能变化的直接前体都是 GDP-d--α-d--庚糖。该底物在 C4 上的氧化使 C3-C5 上的后续差向异构化反应成为可能，这些反应可以与 C5/C6 的脱水/还原偶联。然而，负责 GDP-d--α-d--庚糖 C4 关键氧化的酶仍然难以捉摸。来自 NCTC 11168 的 Cj1427 酶在α-酮戊二酸存在下使用质谱和核磁共振波谱显示出催化 GDP-d--α-d--庚糖氧化为 GDP-d--4-酮-α-d--庚糖的作用。在 pH 7.4 下，表观 Km 为 0.6 s，对于 GDP-d--α-d--庚糖， 值为 1.0 × 10 M s。α-酮戊二酸是在 Cj1427 的活性位点中循环紧密结合的 NADH 核苷酸所必需的，该核苷酸在催化过程中不会从酶中解离。
