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首页> 外文期刊>Chemico-biological interactions >Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8: a hyperthermostable aldose-1-dehydrogenase with broad substrate specificity.
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Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8: a hyperthermostable aldose-1-dehydrogenase with broad substrate specificity.

机译：嗜热栖热菌HB8的短链脱氢酶/还原酶的生化和结构表征：具有广泛底物特异性的超热醛糖-1-脱氢酶。

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摘要

Thermus thermophilus HB8 is a hyperthermophilic bacterium, thriving at environmental temperature near 80 degrees C. The genomic analysis of this bacterium predicted 18 genes for proteins belonging to the short-chain dehydrogenase/reductases (SDR) superfamily, but their functions remain unknown. A SDR encoded in a gene (TTHA0369) was chosen for functional and structural characterization. Enzymatic assays revealed that the recombinant tetrameric protein has a catalytic activity as NAD(+)-dependent aldose 1-dehydroganse, which accepts various aldoses such as d-fucose, d-galactose, d-glucose, l-arabinose, cellobiose and lactose. The enzyme also oxidized non-sugar alicyclic alcohols, and was competitively inhibited by hexestrol, 1,10-phenanthroline, 2,3-benzofuran and indole. The enzyme was stable at pH 2-13 and up to 85 degrees C. We have determined the crystal structure of the enzyme-NAD(+) binary complex at 1.65A resolution. The structure provided evidence for the strict coenzyme specificity and broad substrate specificity of the enzyme. Additionally, it has unusual features, aromatic-aromatic interactions among Phe141 and Phe249 in the subunit interface and hydrogen networks around the C-terminal Asp-Gly-Gly sequence at positions 242-244. Stability analysis of the mutant D242N, F141A and F249A enzymes indicated that the two unique structural features contribute to the hyperthermostability of the enzyme. This study demonstrates that aldose 1-dehydrogenase is a member of the SDR superfamily, and provides a novel structural basis of thermostability.
机译：嗜热栖热菌HB8是一种超嗜热细菌，在80°C的环境温度下兴旺。对该细菌的基因组分析预测了18个基因属于短链脱氢酶/还原酶（SDR）超家族，但其功能尚不清楚。选择在基因（TTHA0369）中编码的SDR进行功能和结构表征。酶法测定显示重组四聚体蛋白具有作为NAD（+）依赖的醛糖1-脱氢聚糖的催化活性，该酶接受各种醛糖，例如d-岩藻糖，d-半乳糖，d-葡萄糖，1-阿拉伯糖，纤维二糖和乳糖。该酶还氧化非糖脂环族醇，并被己烯雌酚，1,10-菲咯啉，2,3-苯并呋喃和吲哚竞争性抑制。该酶在pH 2-13和高达85摄氏度的温度下均稳定。我们已在1.65A的分辨率下确定了酶-NAD（+）二元复合物的晶体结构。该结构为酶的严格辅酶特异性和广泛的底物特异性提供了证据。此外，它还具有非同寻常的功能，亚基界面中的Phe141和Phe249之间的芳香-芳族相互作用以及位置242-244处C末端Asp-Gly-Gly序列周围的氢网络。突变体D242N，F141A和F249A酶的稳定性分析表明，这两个独特的结构特征有助于该酶的超热稳定性。这项研究表明醛糖1-脱氢酶是SDR超家族的成员，并提供了新的热稳定性结构基础。

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