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Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
Biomolecules ( IF 4.8 ) Pub Date : 2020-10-06 , DOI: 10.3390/biom10101411
Giacomo Parisi 1, 2 , Ida Freda 1 , Cécile Exertier 1 , Cristina Cecchetti 1, 3 , Elena Gugole 1 , Gabriele Cerutti 1, 4 , Lucia D'Auria 5 , Alberto Macone 5 , Beatrice Vallone 1, 6 , Carmelinda Savino 6 , Linda Celeste Montemiglio 6
Affiliation  

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound.

中文翻译:

剖析细胞色素 P450 OleP 底物特异性:优先底物的证据

细胞色素 P450 OleP 催化 oleandomycin 生物合成的苷元 8.8a-deoxyoleandolide (DEO) 和单糖基化 L-olivosyl-8.8a-deoxyoleandolide (LO-DEO) 中间体上脂肪族碳的环氧化。我们研究了酶的底物多功能性。X 射线和平衡结合数据显示,糖苷配基 DEO 与 OleP 活性位点松散匹配,触发关闭,使其仅在少量酶上催化。打开到关闭的状态转换允许溶剂分子积聚在关闭时形成的空腔中,介导蛋白质-底物相互作用。电脑模拟单糖基化 LO-DEO 在封闭 OleP-DEO 结构中的对接表明,L-橄榄糖基部分可以位于同一空腔中,取代溶剂分子并直接接触参与转变的结构元素。在 L-鼠李糖存在下,糖苷配基结合的 OleP 的 X 射线结构证实该腔是糖结合的潜在位点。综合考虑,我们建议 LO-DEO 作为 OleP 的最佳底物,L-橄榄糖基部分可能代表分子楔,在底物结合时触发更有效的结构响应,有利于和稳定催化前的酶关闭。OleP 底物的多功能性得到了结构溶剂分子的支持,这些分子在结合苷元时补偿了糖基单元的缺失。
更新日期:2020-10-06
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