Romsuk Jutapat, Yasumoto Shuhei, Seki Hikaru, Fukushima Ery Odette, Muranaka Toshiya
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.
Industrial Biotechnology Initiative Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka, Japan.
Front Bioeng Biotechnol. 2022 Aug 19;10:955650. doi: 10.3389/fbioe.2022.955650. eCollection 2022.
Triterpenoids constitute a group of specialized plant metabolites with wide structural diversity and high therapeutic value for human health. Cytochrome P450 monooxygenases (CYP) are a family of enzymes important for generating the structural diversity of triterpenoids by catalyzing the site-specific oxidization of the triterpene backbone. The CYP716 enzyme family has been isolated from various plant families as triterpenoid oxidases; however, their experimental crystal structures are not yet available and the detailed catalytic mechanism remains elusive. Here, we address this challenge by integrating bioinformatics approaches with data from other CYP families. CYP716A12, the first functionally characterized CYP716A subfamily enzyme, was chosen as the model for this study. We performed homology modeling, structural alignment, site-directed mutagenesis, and molecular docking analysis to search and screen key amino acid residues relevant to the catalytic activity and substrate specificity of the CYP716A subfamily enzyme in triterpenoid biosynthesis. An functional analysis using engineered yeast that endogenously produced plant-derived triterpenes was performed to elucidate the results. When the amino acids in the signature region and substrate recognition sites (SRSs) were substituted, the product profile of CYP716A12 was modified. We identified amino acid residues that control the substrate contraction of the enzyme (D292) and engineered the enzyme to improve its catalytic activity and substrate specificity (D122, I212, and Q358) for triterpenoid biosynthesis. In addition, we demonstrated the versatility of this strategy by changing the properties of key residues in SRSs to improve the catalytic activity of CYP716A1 (S356) and CYP716A2 (M206, F210) at C-28 on the triterpene backbone. This research has the potential to help in the production of desired triterpenoids in engineered yeast by increasing the catalytic activity and substrate specificity of plant CYP716A subfamily enzymes.
三萜类化合物是一类结构多样且对人类健康具有高治疗价值的特殊植物代谢产物。细胞色素P450单加氧酶（CYP）是一类通过催化三萜骨架的位点特异性氧化来产生三萜类化合物结构多样性的重要酶家族。CYP716酶家族已从多个植物科中分离出来作为三萜氧化酶；然而，它们的实验晶体结构尚未可得，详细的催化机制仍不清楚。在这里，我们通过将生物信息学方法与来自其他CYP家族的数据相结合来应对这一挑战。CYP716A12是第一个功能特征明确的CYP716A亚家族酶，被选为本研究的模型。我们进行了同源建模、结构比对、定点诱变和分子对接分析，以搜索和筛选与三萜类生物合成中CYP716A亚家族酶的催化活性和底物特异性相关的关键氨基酸残基。使用内源性产生植物来源三萜的工程酵母进行功能分析以阐明结果。当特征区域和底物识别位点（SRSs）中的氨基酸被取代时，CYP716A12的产物谱发生了改变。我们鉴定了控制酶底物收缩的氨基酸残基（D292），并对该酶进行工程改造以提高其在三萜类生物合成中的催化活性和底物特异性（D122、I212和Q358）。此外，我们通过改变SRSs中关键残基的性质来提高CYP716A1（S356）和CYP716A2（M206、F210）在三萜骨架C-28位的催化活性，证明了该策略的通用性。这项研究有可能通过提高植物CYP716A亚家族酶的催化活性和底物特异性来帮助在工程酵母中生产所需的三萜类化合物。
