A 'Split-Gene' Transketolase From the Hyper-Thermophilic Bacterium Carboxydothermus hydrogenoformans: Structure and Biochemical Characterization.
A 'Split-Gene' Transketolase From the Hyper-Thermophilic Bacterium Carboxydothermus hydrogenoformans: Structure and Biochemical Characterization.
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DOI：
10.3389/fmicb.2020.592353
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发表时间：
2020
影响因子：
5.2
通讯作者：
Littlechild JA
中科院分区：
文献类型：
--
作者：
James P;Isupov MN;De Rose SA;Sayer C;Cole IS;Littlechild JA
一种新的转酮醇酶是由两个独立的多肽链重组而成的，这些多肽链是由一个在超嗜热细菌（羧热菌）的基因组中发现的“分裂基因”编码的。重组的活性α2β2四聚体酶进行了生物化学表征，并以乙醇醛、苯乙醛和环己烷醛为酮受体，羟丙酮酸为供体测定了其活性。由于产物二氧化碳的释放，该反应进行到接近100%完成，并且可以用于合成制药工业感兴趣的一系列糖。这种新型重组转酮醇酶在70℃下孵育1小时后无活性损失，在50%的有机溶剂甲醇、乙醇、异丙醇、DMSO、乙腈和丙酮中孵育1小时后也保持稳定。全息重构α2β2四聚体转酮醇酶的x射线结构测定为1.4 Å分辨率。此外，失活的四聚体β4蛋白的结构已被确定为1.9 Å分辨率。对重组活性α2β2酶的结构与相关酶的结构进行了比较；丙酮酸脱氢酶复合物和d -木醛糖-5-磷酸合成酶的E1组分，试图使这些酶之间结构和底物特异性的差异合理化。这是首次对重组的“分裂基因”转酮醇酶进行生物化学和结构表征，从而评估其工业生物催化潜力。
A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a ‘split-gene’ identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α2β2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α2β2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α2β2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted ‘split-gene’ transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.
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影响因子：
3.9
作者：
Littlechild, J. A.;Guy, J.;Isupov, M.
通讯作者：
Isupov, M.
DOI：
10.1016/j.str.2008.10.010
发表时间：
2008-12-10
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Kato M;Wynn RM;Chuang JL;Tso SC;Machius M;Li J;Chuang DT
通讯作者：
Chuang DT
影响因子：
2.9
作者：
Arjunan, P;Nemeria, N;Furey, W
通讯作者：
Furey, W
影响因子：
21.8
作者：
Luedtke, Stefan;Neumann, Piotr;Tittmann, Kai
通讯作者：
Tittmann, Kai
影响因子：
5.8
作者：
Fullam E;Pojer F;Bergfors T;Jones TA;Cole ST
通讯作者：
Cole ST