Dorfner Maja, Klein Jan, Senkleiter Katharina, Lanig Harald, Kreis Wolfgang, Munkert Jennifer
Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Biology, Pharmaceutical Biology, Staudtstraße 5, 91058 Erlangen, Germany.
Department of Plant Physiology, Matthias Schleiden Institute for Genetics, Bioinformatics and Molecular Botany Friedrich-Schiller-Universität Jena, Dornburger Str. 159, 07743 Jena, Germany.
Molecules. 2024 Dec 7;29(23):5788. doi: 10.3390/molecules29235788.
Enzymes capable of processing a variety of compounds enable plants to adapt to diverse environmental conditions. PRISEs (progesterone-5β-reductase/iridoid synthase-like enzymes), examples of such substrate-promiscuous enzymes, are involved in iridoid and cardenolide pathways and demonstrate notable substrate promiscuity by reducing the activated C=C double bonds of plant-borne and exogenous 1,4-enones. In this study, we identified PRISE genes in () and (), and the corresponding enzymes were determined to share a sequence identity of 95%. Despite the high sequence identity, recombinant expressed P5βR1 was 70 times more efficient than P5βR1 for converting progesterone. In order to investigate the underlying reasons for this significant discrepancy, we focused on specific residues located near the substrate-binding pocket and adjacent to the conserved phenylalanine "clamp". This clamp describes two phenylalanines influencing substrate preferences by facilitating the binding of smaller substrates, such as 2-cyclohexen-1-one, while hindering larger ones, such as progesterone. Using structural analysis based on templates PDB ID: 5MLH and 6GSD from PRISE of , along with in silico docking, we identified positions 156 and 346 as hot spots. In P5βR1 amino acid residues, A156 and F346 seem to be responsible for the diminished ability to reduce progesterone. Moreover, the double mutant P5βR_F156L_A346L, which contains the corresponding amino acids from P5βR1, showed a 15-fold increase in progesterone 5β-reduction. Notably, this modification did not significantly alter the enzyme's ability to convert other substrates, such as 8-oxogeranial, 2-cyclohexen-1-one, and methyl vinyl ketone. Hence, a rational enzyme design by reducing the number of hotspots selectively, specifically improved the substrate preference of P5βR1 for progesterone.
能够处理多种化合物的酶使植物能够适应各种环境条件。PRISEs（孕酮-5β-还原酶/环烯醚萜合酶样酶）就是这类底物混杂酶的例子，它们参与环烯醚萜和强心苷途径，并通过还原植物源性和外源性1,4-烯酮的活化碳碳双键表现出显著的底物混杂性。在本研究中，我们在（）和（）中鉴定出PRISE基因，并且确定相应的酶具有95%的序列同一性。尽管序列同一性很高，但重组表达的P5βR1在转化孕酮方面的效率比P5βR1高70倍。为了研究这种显著差异的潜在原因，我们聚焦于位于底物结合口袋附近且与保守的苯丙氨酸“夹子”相邻的特定残基。这个夹子描述了两个苯丙氨酸通过促进较小底物（如2-环己烯-1-酮）的结合来影响底物偏好，同时阻碍较大底物（如孕酮）的结合。利用基于来自PRISE的模板PDB ID：5MLH和6GSD的结构分析以及计算机对接，我们确定156位和346位为热点。在P5βR1氨基酸残基中，A156和F346似乎是导致还原孕酮能力下降的原因。此外，包含来自P5βR1相应氨基酸的双突变体P5βR_F156L_A346L在孕酮5β-还原方面表现出15倍的增加。值得注意的是，这种修饰并没有显著改变该酶转化其他底物（如8-氧代香叶醛、2-环己烯-1-酮和甲基乙烯基酮）的能力。因此，通过选择性减少热点数量进行合理的酶设计，特别提高了P5βR1对孕酮的底物偏好。
