Functional Characterization of YdjH, a Sugar Kinase of Unknown Specificity in Escherichia coli K12
Functional Characterization of YdjH, a Sugar Kinase of Unknown Specificity in Escherichia coli K12
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DOI：
10.1021/acs.biochem.9b00327
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发表时间：
2019-08-06
期刊：
影响因子：
2.9
通讯作者：
Raushel, Frank M.
中科院分区：
文献类型：
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作者：
Huddleston, Jamison P.;Raushel, Frank M.
ydj 基因簇被注释为催化未知碳水化合物的分解代谢。此前，YdjI（一种 II 类醛缩酶）被证明可以催化 L-g/ycero-L-半乳糖辛醛糖醛酸-1-磷酸逆羟醛裂解为 DHAP 和 L-阿拉伯糖醛酸。在本报告中，介绍了 YdjH 的功能表征。 YdjH 催化 2-酮单糖在 C1 羟基处的磷酸化，其底物谱明显比 YdjI 更严格。与 YdjI 类似，YdjH 显示出对具有羧酸根末端的高级单糖（七至九个碳）的强烈偏好。确定最佳底物为 L-甘油-L-半乳辛醛糖醛酸，产生 L-甘油-L-半乳糖辛醛醛酸-1-磷酸，k(cat) 为 16 s(-1)，k(cat)/K-m 为 2.1 x 10(4) M-1 s(-1)。这显然是第一个报道的八碳单糖激酶活性的例子。 YdjH 的两种晶体结构先前已确定为 2.15 埃和 1.8 埃分辨率（蛋白质数据库条目 3H49 和 3IN1）。我们对活性位点布局进行了分析，并使用计算对接来识别 L-甘油-L-半乳辛醛酸结合中潜在的关键残基。
The ydj gene cluster is annotated to catalyze the catabolism of an unknown carbohydrate. Previously, YdjI, a class II aldolase, was shown to catalyze the retro-aldol cleavage of L-g/ycero-L-galacto-octuluronate-1-phosphate into DHAP and L-arabinuronate. In this report, the functional characterization of YdjH is presented. YdjH catalyzes the phosphorylation of 2-keto-monosaccharides at the C1 hydroxyl group with a substrate profile significantly more stringent than that of YdjI. Similar to YdjI, YdjH shows a strong preference for higher-order monosaccharides (seven to nine carbons) with a carboxylate terminus. The best substrate was determined to be L-glycero-L-galacto-octuluronate, yielding L-glycero-L-galacto-octuluronate-1-phosphate with a k(cat) of 16 s(-1) and a k(cat)/K-m of 2.1 x 10(4) M-1 s(-1). This is apparently the first reported example of kinase activity with eight-carbon monosaccharides. Two crystal structures of YdjH were previously determined to 2.15 and 1.8 angstrom resolution (Protein Data Bank entries 3H49 and 3IN1). We present an analysis of the active site layout and use computational docking to identify potential key residues in the binding of L-glycero-L-galacto-octuluronate.