Abstract:
Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30 to 42 °C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO.
摘要:
醛-去酰化加氧酶(ADO)是生产长链烷烃作为滴入生物燃料的必需酶,与现有的燃油系统兼容。最活跃的ADO存在于嗜温蓝细菌中,特别是Nostocpunctiforme鉴于热稳定酶在生物精炼厂中的潜在应用,在这里,我们基于来自几种嗜热蓝细菌菌株的ADO序列的共识产生了热稳定(Cts)-ADO。使用计算机设计管道和包含41个温泉微生物群落的宏基因组文库,我们创造了Cts-ADO.当温度从30°C升高到42°C时,Cts-ADO的十五烷产量增加了3.8倍,而来自点状N.的ADO(Np-ADO)表现出1.7倍的下降。Cts-和Np-ADO在不同温度下的3D结构建模和分子动力学模拟揭示了两种酶在这些变体的暴露环上聚集的残基中的差异。这影响了形成ADO催化核心所涉及的螺旋的构象。在Cts-ADO中,这种构象变化促进了配体与其优选的铁结合,Fe2,在较高温度下的双铁团簇中,但在Np-ADO中观察到相反的情况。赋予Cts-ADO热稳定性的残基的详细作图确定了四个氨基酸,我们在Np-ADO中单独和一起替换。在这些替代变体中,A161E在活性最佳方面与Cts-ADO非常相似,动力学参数,和结构在较高的温度。A161E位于L6环中,它连接螺旋线H5和H6,并支持在较高温度下与Fe2结合的配体,从而促进在这些温度下的最佳活性并解释Cts-ADO的热稳定性增加。