The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-D-rhamnose biosynthesis pathway.
The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP-D-rhamnose biosynthesis pathway.
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DOI：
10.1111/j.1742-4658.2009.06993.x
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发表时间：
2009-05
期刊：
影响因子：
--
通讯作者：
Lam JS
中科院分区：
文献类型：
--
作者：
King JD;Poon KKH;Webb NA;Anderson EM;McNally DJ;Brisson JR;Messner P;Garavito RM;Lam JS
稀有的6-脱氧糖d-鼠李糖是细菌细胞表面聚糖的组成部分，包括铜绿假单胞菌产生的d-鼠李糖均聚物，称为A-带O多糖。由 GDP-d-甘露糖合成 GDP-d-鼠李糖是由两种酶催化的。第一个是 GDP-d-甘露糖-4,6-脱水酶 (GMD)。第二种酶 RMD 将 GMD 产物 (GDP-6-deoxy-d-lyxo-hexos-4-ulose) 还原为 GDP-d-鼠李糖。编码 GMD 和 RMD 的基因存在于铜绿假单胞菌中，遗传证据表明它们在 A 带 O-多糖生物合成中发挥作用。然而，它们的酶功能的细节之前尚未阐明。我们的目的是对这些酶进行生化表征，并确定 RMD 的结构，以更好地了解决定这些酶的底物特异性和催化活性的因素。我们使用反应产物的毛细管电泳和核磁共振分析来精确定义铜绿假单胞菌 GMD 和 RMD 功能。铜绿假单胞菌GMD具有双功能，可以催化GDP-d-甘露糖4,6-脱水和随后的还原反应，产生GDP-d-鼠李糖。正如预测的那样，RMD 催化 GDP-6-deoxy-d-lyxo-hexos-4-ulose 的立体定向还原。 GDP-d-鼠李糖体外生物合成的重建表明，铜绿假单胞菌途径可能受到细胞内反馈抑制的调节。我们以 1.8 Å 的分辨率确定了嗜热动神经杆菌的 RMD 结构。 A.thermoaerophilus RMD 的结构与铜绿假单胞菌 GMD 非常相似，这解释了为什么铜绿假单胞菌 GMD 也能够催化 RMD 反应。活性位点和氨基酸序列的比较表明，保守的氨基酸侧链（铜绿假单胞菌 GMD 中的 Arg185）可能对于 GMD 酶中底物和辅因子的定向至关重要。
The rare 6-deoxysugar d-rhamnose is a component of bacterial cell surface glycans, including the d-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-d-rhamnose synthesis from GDP-d-mannose is catalyzed by two enzymes. The first is a GDP-d-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-d-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-d-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-d-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-d-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 Å resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.
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影响因子：
9.4
作者：
LAM, MYC;MCGROARTY, EJ;LAM, JS
通讯作者：
LAM, JS
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1073/pnas.94.5.2085
发表时间：
1997-03-04
影响因子：
11.1
作者：
Bonin, CP;Potter, I;Reiter, WD
通讯作者：
Reiter, WD
影响因子：
4.8
作者：
Kanipes, MI;Ribeiro, AA;Raetz, CRH
通讯作者：
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影响因子：
5.7
作者：
Blankenfeldt, W;Kerr, ID;Naismith, JH
通讯作者：
Naismith, JH