Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria.
Chembiochem. 2021 May 14;22(10):1833-1840. doi: 10.1002/cbic.202000851. Epub 2021 Mar 31.
Light-dependent enzymes are a rare type of biocatalyst with high potential for research and biotechnology. A recently discovered fatty acid photo-decarboxylase from Chlorella variabilis NC64A (CvFAP) converts fatty acids to the corresponding hydrocarbons only when irradiated with blue light (400 to 520 nm). To expand the available catalytic diversity for fatty acid decarboxylation, we reconstructed possible ancestral decarboxylases from a set of 12 extant sequences that were classified under the fatty acid decarboxylases clade within the glucose-methanol choline (GMC) oxidoreductase family. One of the resurrected enzymes (ANC1) showed activity in the decarboxylation of fatty acids, showing that the clade indeed contains several photo-decarboxylases. ANC1 has a 15 °C higher melting temperature (T ) than the extant CvFAP. Its production yielded 12-fold more protein than this wild type decarboxylase, which offers practical advantages for the biochemical investigation of this photoenzyme. Homology modelling revealed amino acid substitutions to more hydrophilic residues at the surface and shorter flexible loops compared to the wild type. Using ancestral sequence reconstruction, we have expanded the existing pool of confirmed fatty acid photo-decarboxylases, providing access to a more robust catalyst for further development via directed evolution.
依赖于光的酶是一种具有高研究和生物技术潜力的罕见生物催化剂。最近从变绿球藻 NC64A（CvFAP）中发现的一种脂肪酸光脱羧酶，只有在蓝光（400 到 520nm）照射下才会将脂肪酸转化为相应的烃类。为了扩大脂肪酸脱羧反应的可用催化多样性，我们从一组 12 种现存的序列中重建了可能的祖先脱羧酶，这些序列被归类为葡萄糖-甲醇-胆碱（GMC）氧化还原酶家族中的脂肪酸脱羧酶分支。其中一种复活的酶（ANC1）在脂肪酸脱羧反应中表现出活性，这表明该分支确实包含几种光脱羧酶。ANC1 的熔点（T ）比现存的 CvFAP 高 15°C。其产量比野生型脱羧酶高出 12 倍，这为该光酶的生化研究提供了实际优势。同源建模显示，与野生型相比，表面的氨基酸取代为更亲水的残基，并且柔性环更短。通过祖先序列重建，我们扩展了已确认的脂肪酸光脱羧酶的现有库，为通过定向进化进一步开发提供了更稳健的催化剂。
