Deciphering The Chemoselectivity Of Nickel-dependent Quercetin 2,4-Dioxygenase

Metalloenzymes play very important roles in many biological processes,and they are capable of catalyzing many different types of biological reactions with high efficiency and remarkable selectivity.Understanding the catalytic mechanism of metalloenzymes can provide theoretical guidance for the modification and application of enzymes.In recent years,with the development of quantum chemistry methods and the advancement of computer power,the combined quantum mechanics/molecular mechanics?QM/MM?method has been proved to be very successful in elucidating the reaction mechanisms and rationalizing the various selectivities in metalloenzymes.In this thesis,we used the combined QM/MM method to study the catalytic mechanism and the chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase?2,4-QueD?in bacteria.The main results of this thesis are as follows:A model of the enzyme substrate complex in aqueous solution was built from the X-ray structure of the enzyme,a proper QM region was selected,in which the first-shell residue Glu74 has been considered to be either neutral or deprotonated.From calculating the potential energy profiles for the whole reaction,a mechanism was suggested to explain the chemoselectivity.QM/MM calculations demonstrated that Glu74 must be deprotonated to reproduce chemoselectivity and to steer the 2,4-dioxygenolytic cleavage of quercetin,which harvests the experimentally-observed product 2-protocatechuoylphloroglucinol carboxylic acid coupled with the release of a carbon monoxide.If the enzyme has a neutral Glu74 residue,the undesired 2,3-dioxygenolytic cleavage of quercetin becomes the dominant pathway,leading to the formation of the side product?-keto acid.The reaction starts from the binding of the dioxygen molecule to the Ni^II ion,coupled with an electron transfer from the anionic quercetin substrate to the dioxygen moiety,which results in the formation of a Ni^II-superoxide?O₂·^-?quercetin radical complex.The following reaction takes place via three major steps:?1?attack of the superoxide on the C2 of the substrate pyrone ring to form the first C-O bond and to generate a Ni^II-peroxide intermediate.?2?formation of the second C-O bond between C4 and the peroxide to produce a peroxide bridge.Two different conformational changes take place during the reaction,leading to two different intermediates,which are the corresponding precursors for the subsequent 2,4-dioxygenolytic cleavage and 2,3-dioxygenolytic cleavage.?3?simultaneous cleavages of the C2-C3,C3-C4,and O1-O2 bonds with the formation of 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide.The third step was calculated to be rate-limiting,with a barrier of 17.4 kcal/mol,which is in very good agreement with the experimental kinetic data.For the second C-O bond formation,an alternative pathway is that the peroxide attacks the C3 of the substrate pyrone ring,leading to the formation of a four-membered ring intermediate,which then undergoes concerted C2-C3 and O1-O2 bond cleavages to produce an?-keto acid.This pathway is however associated with a barrier of 30.6 kcal/mol,which is much higher than that for the 2,4-dioxygenolytic pathway.When Glu74 is protonated,the barrier of the 2,3-dioxygenolytic pathway is 21.8 kcal/mol,which is unexpectedly lower than the 2,4-dioxygenase pathway?24.8 kcal/mol?.Therefore,the model with a neutral Glu74residue can be ruled out.