Zhang Shiqing, Liu Yongjun
Key Lab of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan, Shandong 250100, China.
Inorg Chem. 2020 Jul 20;59(14):9627-9637. doi: 10.1021/acs.inorgchem.0c00726. Epub 2020 Jul 9.
The b-type cytochrome Lcp is a latex clearing protein (Lcp), which acts as an endotype dioxygenase to catalyze the extracellular cleavage of the chemically inert aliphatic polymer poly(-1,4-isoprene), producing oligo-isoprenoids with different terminal carbonyl groups (aldehyde and ketone, -CH-CHO and -CH-COCH). On the basis of the fact that the muteins of E148A, E148Q, and E148H have substantially reduced reactivity, and the E148-initiated reaction mechanism has been previously proposed, in which a cyclic dioxetane intermediate or an epoxide intermediate may be involved, however, open questions still remain. In this paper, on the basis of the crystal structure of Lcp the enzyme-substrate reactant model was constructed, and the cleavage mechanism of the central double bond of poly(-1,4-isoprene) was elucidated by performing quantum mechanics/molecular mechanics calculations. Our calculation results revealed that the oxidative cleavage reaction is triggered by the addition of the heme-bound dioxygen to the double bond of the polymer, and E148 does not act as the catalytic base to extract the allylic proton to assist the reaction as previously suggested. Of the two considered pathways, the pathway that involves the dioxetane intermediate was calculated to be more favorable. During the catalysis, the distal oxygen first adds to the double bond of the substrate to form a radical intermediate, and then the Fe-O1 (proximal oxygen) bond cleaves to generate the dioxetane intermediate, which can easily collapse affording the final ketone and aldehyde products. In general, the cleavage mechanism of double C-C bond catalyzed by Lcp is similar to those of indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, and the nonheme stilbene cleavage oxygenase NOV1 that all depend on the iron-bound dioxygen to initiate the cleavage reaction.
b型细胞色素Lcp是一种乳胶清除蛋白（Lcp），它作为一种内型双加氧酶，催化化学惰性的脂肪族聚合物聚（-1,4-异戊二烯）的细胞外裂解，产生具有不同末端羰基（醛和酮，-CH-CHO和-CH-COCH）的低聚异戊二烯。基于E148A、E148Q和E148H突变体的反应活性大幅降低这一事实，以及之前提出的E148引发的反应机制，其中可能涉及环状二氧杂环丁烷中间体或环氧化物中间体，然而，仍存在一些未解决的问题。在本文中，基于Lcp的晶体结构构建了酶-底物反应物模型，并通过进行量子力学/分子力学计算阐明了聚（-1,4-异戊二烯）中心双键的裂解机制。我们的计算结果表明，氧化裂解反应是由与血红素结合的双加氧加到聚合物的双键上引发的，并且E148并不像之前所认为的那样作为催化碱提取烯丙基质子来辅助反应。在所考虑的两条途径中，涉及二氧杂环丁烷中间体的途径被计算为更有利。在催化过程中，远端氧首先加到底物的双键上形成自由基中间体，然后Fe-O1（近端氧）键断裂生成二氧杂环丁烷中间体，该中间体可容易地分解得到最终的酮和醛产物。一般来说，Lcp催化的C-C双键裂解机制与吲哚胺2,3-双加氧酶、色氨酸2,3-双加氧酶以及非血红素芪裂解双加氧酶NOV1的机制相似，它们都依赖于与铁结合的双加氧来引发裂解反应。
