乳胶清除蛋白LcpK30催化氧气裂解聚顺-1,4异戊二烯机理

Unraveling the mechanism of latex clearing protein Lcp catalysed oxidative cleavage of poly(cis-1,4-isoprene)

乳胶清除蛋白LcpK30是一种内型双加氧酶,其通过活化分子氧催化裂解化学惰性的聚顺-1,4异戊二烯,进而生成醛和酮.实验发现活性区域的谷氨酸148对活性有重大影响,然而其作用机制和整个反应机理仍然存在争议.本研究使用量子力学/分子力学组合方法研究了乳胶清除蛋白催化分子氧氧化裂解天然橡胶模型化合物的反应机理.计算结果显示谷氨酸148在反应过程中为质子化态,并与铁(Ⅲ)-超氧物种形成稳定氢键,因此其作用可能与调节铁(Ⅲ)-超氧物种与底物的位置有关.可能反应机理为:(1)铁(Ⅲ)-超氧物种的远端氧进攻底物碳碳双键,形成过氧烃自由基中间体;(2)近端氧回弹到碳自由基,形成过氧化物中间体和铁(Ⅱ)中心;(3)铁(Ⅱ)调节的过氧化物中间体O–O键还原裂解,生成邻二醇自由基负离子中间体;(4)邻二醇自由基负离子C–C键断裂,生成产物醛和酮.此外,计算发现,过氧化物中间体O–O键直接断裂反应能垒超过160 kJ/mol,表明铁(Ⅱ)的催化对于过氧化物中间体的后续转化至关重要.因此,本研究结果不仅加深对于Lcp_(K30)反应机理的理解,而且为Lcp酶降解合成橡胶材料的设计和工程化提供有用信息.

Natural rubber or synthetic rubber materials have been extensively used in our daily life and industrial production for more than 100 years due to their unique physical properties.However,a amount of rubber waste has been released into the environment,posing great challenges to the disposal of these polymers.In contrast to the disposal strategies such as combustion or stockpiling,enzyme catalyzed rubber degradation constitutes an environmentally friendly strategy.Latex clearing protein(Lcp)is an iron-heme containing endotype dioxygenase that catalyses the oxidative cleavage of the chemically inert poly(cis-1,4-isoprene)which is the main component of rubber,affording the products of aldehyde and ketone.It was found that active site E148 residue is key to the reactivity of Lcp based on the mutation experiments.However,the role of E148 and the overall mechanism are still controversial.It is generally accepted that both of dioxygen atoms are incorporated into the substrate.In previous studies,two possible mechanisms of Lcp catalyzed cleavage of poly(cis-1,4-isoprene)are proposed.In the first one,the distal oxygen atom of Fe(Ⅲ)-O_(2)^(-)complex attacks onto the C=C double bond,leading to a three-membered epoxide intermediate.This is followed by the attack of Fe=O complex on the epoxide intermediate and subsequent C-C bond cleavage,affording the final ketone and aldehyde products.While in the second one,the proximal oxygen atom of Fe(Ⅲ)-O_(2)^(-)complex attacks onto the C=C double bond of the substrate,which is followed by the attack of the distal oxygen atom onto the substrate radical to generate a four-membered cyclic dioxetane intermediate.The final O-O as and C-C bond cleavage in the dioxetane intermediate would afford the final products.In this study,based on the crystal structure of LcpK30,the combined molecular dynamic(MD)simulations and quantum mechanics/molecular mechanics(QM/MM)calculations are performed to elucidate the catalytic mechanism of Lcp catalysed cleavage of central double bond of poly(cis-1,4-isoprene).Our study shows E148 is protonated during the reaction and forms stable H bond interactions with Fe(Ⅲ)-O_(2)^(-)species.Thus,the plausible role of E148 is related to the positioning of both Fe(Ⅲ)-O_(2)^(-)species and the substrate.Moreover,it can be ruled out that the E148 acts as a base to abstract the proton from the substrate.The proposed mechanism of Lcp works as follows.Firstly,the distal oxygen of Fe(Ⅲ)-O_(2)^(-)attacks onto the carbon-carbon double bond to generate the peroxide alky radical intermediate.This is followed by the rebound of the proximal oxygen to the carbon radical to generate the dioxetane intermediate,with Fe(Ⅲ)being reduced to Fe(Ⅱ).The following O-O bond cleavage occurs via the Fe(Ⅱ)-mediated reductive cleavage of O-O bond of the dioxetane intermediate,leading to the vicinal diol radical anion intermediate.Finally,a facile C-C cleavage is undergone to yield the ketone and aldehyde products.In particular,our calculations show that the direct O-O cleavage of the dioxetane intermediate in the absence of Fe(Ⅱ)catalysis requires a remarkably high barrier of 160 kJ/mol,indicating the Fe(Ⅱ)catalysis is vital for the further transformation of the dioxetane intermediate.For comparison,we also investigated the previously proposed mechanism involving the three-membered epoxide intermediate.However,the attack of the epoxide by FeⅣ=O complex is found to be highly unfavourable thermodynamically and thus can be ruled out.As such,this study may not only expand our understanding of the catalytic mechanism of Lcp_(K30),but also provide useful information for rational design or engineering of Lcp enzyme toward the degradation of synthetic rubber materials.