Unraveling the mechanism of latex clearing protein LcpK30 catalysed oxidative cleavage of poly(cis-1,4-isoprene)

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)-02 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 Lcp(K30), 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 Fe-IV=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.