A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.

中文翻译：

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独特的活性位点形成在一种新型的半乳糖1-磷酸尿嘧啶转移酶中，它来自嗜热古细菌嗜热菌。

在超嗜热古细菌嗜热菌中鉴定出编码半乳糖1-磷酸尿酸基转移酶（GalT）的基因。该基因在大肠杆菌中过表达，然后对其产物进行纯化和鉴定。所表达的酶是高度热稳定的，并且在高达90°C的温度下孵育10分钟后，保留了其活性的90％左右。确定了P. aerophilum GalT的两种不同晶体结构：2.33Å处无底物的酶和1.78ÅUDP结合的H140F突变酶。嗜氧假单胞菌GalT单体的主链坐标与大肠杆菌和人GalTs的结构相似，二聚体排列也是如此。但是，嗜氧假单胞菌GalT与其他两种酶之间存在明显的拓扑差异。在大肠杆菌和人类酶中，N-末端链从一个亚基延伸到另一个亚基，并形成相邻亚基中底物结合口袋的一部分。相比之下，嗜氧假单胞菌GalT中的N端链延伸至同一亚基中的底物结合位点。氨基酸序列比对表明，N-末端区域中较短的表面环有助于嗜氧假单胞菌GalT的独特拓扑。无底物的酶与结合UDP的H140F的结构比较表明，底物的葡萄糖部分而不是UDP部分的结合会在活性位点周围引起较大的结构变化。反过来，这可以为酶反应提供合适的环境。在P. aerophilum GalT中，N端链延伸到同一亚基中的底物结合位点。氨基酸序列比对表明，N-末端区域中较短的表面环有助于嗜氧假单胞菌GalT的独特拓扑。无底物的酶与结合UDP的H140F的结构比较表明，底物的葡萄糖部分而不是UDP部分的结合会在活性位点周围引起较大的结构变化。反过来，这可以为酶反应提供合适的环境。在P. aerophilum GalT中，N端链延伸到同一亚基中的底物结合位点。氨基酸序列比对表明，N-末端区域中较短的表面环有助于嗜氧假单胞菌GalT的独特拓扑。无底物的酶与结合UDP的H140F的结构比较表明，底物的葡萄糖部分而不是UDP部分的结合会在活性位点周围引起较大的结构变化。反过来，这可以为酶反应提供合适的环境。无底物的酶与结合UDP的H140F的结构比较表明，底物的葡萄糖部分而不是UDP部分的结合会在活性位点周围引起较大的结构变化。反过来，这可以为酶反应提供合适的环境。无底物的酶与结合UDP的H140F的结构比较表明，底物的葡萄糖部分而不是UDP部分的结合会在活性位点周围引起较大的结构变化。反过来，这可以为酶反应提供合适的环境。