Babkova Petra, Sebestova Eva, Brezovsky Jan, Chaloupkova Radka, Damborsky Jiri
Loschmidt Laboratories, Department of Experimental Biology and, Research Centre for Toxic Compounds in the Environment RECETOX, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00, Brno, Czech Republic.
International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91, Brno, Czech Republic.
Chembiochem. 2017 Jul 18;18(14):1448-1456. doi: 10.1002/cbic.201700197. Epub 2017 Jun 12.
Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1-5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (ΔT up to 24 °C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.
祖先序列重建（ASR）是一种用于实证检验多种蛋白质结构-功能关系的强大方法。我们采用ASR来预测来自HLD-II亚家族的五种祖先卤代烷脱卤酶（HLD）的序列。编码推断祖先序列的基因被合成并在大肠杆菌中表达，复活的祖先酶（AncHLD1-5）经过实验表征。令人惊讶的是，与现存酶相比，祖先HLD表现出显著增强的热稳定性（ΔT高达24°C），以及对短多取代卤代底物具有更高的比活性。此外，多变量统计分析揭示了AncHLD1和AncHLD2底物特异性谱的转变。这通过合理的蛋白质工程极难实现。该研究强调，ASR是开发新型生物催化剂和用于定向进化的强大模板的有效方法。
