Unique active site formation in a novel galactose 1‐phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum
Unique active site formation in a novel galactose 1‐phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum
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来自超嗜热古细菌 Pyrobaculum aerophilum 的新型半乳糖 1-磷酸尿苷酰转移酶中独特的活性位点形成
DOI：
10.1002/prot.25848
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Sakuraba Haruhiko
中科院分区：
文献类型：
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作者：
Ohshida Tatsuya;Hayashi Junji;Yoneda Kazunari;Ohshima Toshihisa;Sakuraba Haruhiko
在嗜热古细菌Pyrobaculum aerophilum 中鉴定出编码半乳糖1-磷酸尿苷酰转移酶（GalT）的基因。该基因在大肠杆菌中过表达，随后对其产物进行纯化和表征。表达的酶具有高度热稳定性，在高达 90°C 的温度下孵育 10 分钟后仍保留约 90% 的活性。 P的两种不同晶体结构。 aerophilumGalT 被测定：无底物酶在 2.33 Å 处，UDP 结合的 H140F 突变酶在 1.78 Å 处。 P的主链坐标。 aerophilumGalT单体与E的结构相似。大肠杆菌和人类 GalT，二聚体排列也是如此。然而，P之间存在显着的拓扑差异。 aerophilumGalT 和其他两种酶。在E.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袋的一部分。相比之下，P 中的 N 端链。 aerophilumGalT 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 端区域较短的表面环有助于 P 的独特拓扑结构。嗜气菌GalT。无底物酶与 UDP 结合的 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。这反过来可以为酶反应提供合适的环境。
A gene encoding galactose 1‐phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeonPyrobaculum aerophilum. The gene was overexpressed inEscherichia 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 ofP. aerophilumGalT 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 theP. aerophilumGalT monomer were similar to those in the structures of theE. coliand human GalTs, as was the dimeric arrangement. However, there was a striking topological difference betweenP. aerophilumGalT and the other two enzymes. In theE. coliand 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 inP. aerophilumGalT 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 ofP. aerophilumGalT. 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.