State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, National Experimental Teaching Center for Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Shanghai Center for Bioinformation Technology, Shanghai, China.
FASEB J. 2020 Feb;34(2):1983-1995. doi: 10.1096/fj.201900948RR. Epub 2019 Dec 23.
Covariant residues identified by computational algorithms have provided new insights into enzyme evolutionary routes. However, the reliability and accuracy of routine statistical coupling analysis (SCA) are unable to satisfy the needs of protein engineering because SCA depends only on sequence information. Here, we set up a new SCA algorithm, SCA.SIM, by integrating structure information and MD simulation data. The more reliable covariant residues with high-quality scores are obtained from sequence alignment weighted by residual movement for eight related subfamilies, belonging to α/β hydrolase family, with Candida antarctica lipase B (CALB). The 38 predicted covariant residues are tested for function by high-throughput quantitative evaluation in combination with activity and thermostability assays of a mutant library and deep sequencing. Based on the landscapes of both activity and thermostability, most mutants play key roles in catalysis, and some mutants gain 2.4- to 6-fold increase in half-life at 50°C and 9- to 12-fold improvement in catalytic efficiency. The activity of double mutants for A225F/T103A is higher than those of A225F and T103A which means that SCA.SIM method might be useful for identifying the allosteric coupling. The SCA.SIM algorithm can be used for protein coevolution and enzyme engineering research.
计算算法鉴定的协变残基为酶的进化途径提供了新的见解。然而，常规统计耦联分析（SCA）的可靠性和准确性无法满足蛋白质工程的需求，因为 SCA 仅依赖于序列信息。在这里，我们通过整合结构信息和 MD 模拟数据，建立了一种新的 SCA 算法 SCA.SIM。我们从序列比对中获得了更可靠的协变残基，这些残基的权重是根据残基运动来确定的，涉及到属于 α/β 水解酶家族的八个相关亚家族，以南极假丝酵母脂肪酶 B（CALB）为例。通过高通量定量评估、突变文库的活性和热稳定性测定以及深度测序，对 38 个预测的协变残基进行了功能测试。基于活性和热稳定性的图谱，大多数突变体在催化中发挥关键作用，一些突变体在 50°C 时半衰期提高了 2.4 到 6 倍，催化效率提高了 9 到 12 倍。A225F/T103A 的双突变体的活性高于 A225F 和 T103A 的活性，这意味着 SCA.SIM 方法可能有助于识别变构偶联。SCA.SIM 算法可用于蛋白质共进化和酶工程研究。
