| 论文题目: | Facilitating the evolution of Esterase Activity from a Promiscuous Enzyme Mhg with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel |
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| 作者: | Yan, Xiaodan Wang, Jianjun Sun, Yu Zhu, Junge Wu, Sheng |
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| 刊物名称: | Appl Environ Microbiol |
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| 年份: | 2016 |
| 影响因子: | 4.303 |
| 论文下载: | 下载地址 |
| 摘要: | Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical alpha/beta fold hydrolase, was previously reported to have both gamma-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively-studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, not only hydrolyzing the classical p-nitrophenol esters, but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, L233G was a very specific esterase without any gamma-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities. IMPORTANCE: Promiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited a universal significance in this subset of alpha/beta fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies. |
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