İzmir Institute of Technology, Faculty of Engineering, Department of Bioengineering, 35430, Urla, Izmir, Turkey.
İzmir Institute of Technology, Faculty of Engineering, Department of Bioengineering, 35430, Urla, Izmir, Turkey.
J Mol Graph Model. 2023 Jan;118:108323. doi: 10.1016/j.jmgm.2022.108323. Epub 2022 Sep 11.
Steroid-based chemicals can affect the metabolism, immune functions, and development of sexual characteristics. Because of these effects, steroid derivatives are widely used in the pharmaceutical industry. Progesterone is a steroid-based hormone that mainly controls the ovulation period of women but is also a precursor molecule for the synthesis of important hormones like testosterone and cortisone. Cytochrome P450 (CYP) enzymes are important for the production of hydroxyprogesterones in the industry since they can catalyze regio- and enantioselective hydroxylation reactions. Although human CYP enzymes can catalyze hydroxyprogesterone synthesis with high selectivity, these enzymes are membrane bound, which limits their application for industrial production. CYP119 is a soluble and thermophilic enzyme from the archaea Sulfolobus acidocaldarius. Even though the native substrate of the enzyme is not known, CYP119 can catalyze styrene epoxidation, lauric acid hydroxylation, and Amplex®Red peroxidation. In this work, an in silico mutagenesis approach was used to design CYP119 mutants with high progesterone affinity. Energy scores of progesterone docking simulations were used for the design and elimination of single, double, and triple mutants of CYP119. Among designed 674 mutants, five of them match the criteria for progesterone hydroxylation. The most common mutation of these five mutants, L69G mutant was analyzed using independent molecular dynamics (MD) simulations in comparison with the wild-type (WT) enzyme. L69G CYP119, was expressed and isolated from Escherichia coli; it showed 800-fold higher affinity for progesterone compared to WT CYP119. L69G CYP119 also catalyzed progesterone hydroxylation. The novel designed enzyme L69G CYP119 is a potential versatile biocatalyst for progesterone hydroxylation that is expected to be stable under industrial production conditions.
基于类固醇的化学物质会影响新陈代谢、免疫功能和性特征的发育。由于这些作用，类固醇衍生物被广泛应用于制药行业。孕酮是一种基于类固醇的激素，主要控制女性的排卵周期，但它也是合成睾酮和皮质醇等重要激素的前体分子。细胞色素 P450（CYP）酶在工业中对于生产羟孕酮非常重要，因为它们可以催化区域和对映选择性羟化反应。虽然人类 CYP 酶可以高度选择性地催化羟孕酮的合成，但这些酶是膜结合的，这限制了它们在工业生产中的应用。CYP119 是一种来自古菌 Sulfolobus acidocaldarius 的可溶性和嗜热酶。尽管该酶的天然底物未知，但 CYP119 可以催化苯乙烯环氧化、月桂酸羟化和 Amplex®Red 过氧化物酶反应。在这项工作中，使用了一种计算机模拟诱变方法来设计具有高孕酮亲和力的 CYP119 突变体。孕酮对接模拟的能量评分用于 CYP119 的单、双和三突变体的设计和消除。在所设计的 674 个突变体中，有 5 个符合孕酮羟化的标准。对这 5 个突变体中最常见的突变体 L69G 进行了分析，与野生型（WT）酶相比，使用独立的分子动力学（MD）模拟对其进行了分析。从大肠杆菌中表达和分离了 L69G CYP119，与 WT CYP119 相比，其对孕酮的亲和力提高了 800 倍。L69G CYP119 还催化了孕酮的羟化。新型设计酶 L69G CYP119 是一种潜在的多功能生物催化剂，可用于孕酮羟化，预计在工业生产条件下稳定。
