Alkene Cleavage Catalysed by Heme and Nonheme Enzymes: Reaction Mechanisms and Biocatalytic Applications

Abstract

<jats:p>The oxidative cleavage of alkenes is classically performed by chemical methods, although they display several drawbacks. Ozonolysis requires harsh conditions (−78°C, for a safe process) and reducing reagents in a molar amount, whereas the use of poisonous heavy metals such as Cr, Os, or Ru as catalysts is additionally plagued by low yield and selectivity. Conversely, heme and nonheme enzymes can catalyse the oxidative alkene cleavage at ambient temperature and atmospheric pressure in an aqueous buffer, showing excellent chemo- and regioselectivities in certain cases. This paper focuses on the alkene cleavage catalysed by iron cofactor-dependent enzymes encompassing the reaction mechanisms (in case where it is known) and the application of these enzymes in biocatalysis.</jats:p>

Utilization of Dioxygen by Carotenoid Cleavage Oxygenases

“…By contrast, labeling experiments performed on resveratrol and isoeugenol-cleaving CCOs, NOV2 (13) and isoeugenol monooxygenase (15), from the non-carotenogenic bacteria Novosphingobium aromaticivorans and Pseudomonas nitroreducens, respectively, suggested a monooxygenase pattern of oxygen incorporation for both enzymes. Thus, the labeling studies performed to date seem to indicate that perhaps both monooxygenase and dioxygenase catalytic mechanisms may exist within the CCO enzyme family (18), a possibility previously suggested by a theoretical study of the Synechocystis ACO catalytic mechanism (19).…”

“…To determine the background oxygen exchange for ACO-and NOV2-catalyzed reaction products, the exchange reactions were performed in H 2 18 O. For ACO reaction products, the reaction mixture consisted of 192 l of H 2 18 O (99% 18 O atom, Sigma), 4 l of 1 M HEPES-NaOH, pH 7.0, 1 l of 10% (w/v) Triton X-100, and 3 l of 1 g/l freshly purified ACO. Then either authentic alltrans-retinal (TRC, Toronto, Canada, Ͼ 95% purity) or HPLCpurified 8Ј-hydroxy-15Ј-apocarotenal generated in-house by a large scale ACO-catalyzed reaction were added to the reaction mixture.…”

“…Background oxygen exchange for the NOV2 reaction products was determined in a similar manner. Briefly, the reaction mixture contained 150 l of H 2 18 O (97% 18 O atom, Sigma), 3 l of 1 M HEPES-NaOH, pH 7.0, and 2.3 l of freshly purified NOV2 at 4.4 g/l. Authentic 4-hydroxybenzaldehyde (4-HBA) or 3,5-dihydroxybenzaldehyde (3,5-DHBA) (Sigma, Ͼ95% purity for both compounds) dissolved in DMSO was added to the reaction mixture.…”

“…Specifically, 3 l of purified ACO at 1 g/l were added to a reaction mixture containing 192 l of H 2 18 O (99% 18 O atom, Sigma), 4 l of 1 M HEPES-NaOH, pH 7.0, and 1 l of 10% (w/v) Triton X-100. The cleavage reaction was initiated by adding 5 l of 4 mM all-trans-8Ј-apocarotenol dissolved in ethanol.…”

“…Then the reaction was quenched with 200 l of methanol, and 400 l of diethyl ether/hexanes (4:1, v/v) was added to extract the cleavage products. For the NOV2-catalyzed reaction, 2 l of 25 mM resveratrol in DMSO was added to a 200-l reaction mixture consisting of 194 l of H 2 18 O (97% 18 O atom, Sigma), 4 l of 1 M HEPES-NaOH, pH 7.0, and 2.3 l of 4.4 g/l purified NOV2, and this reaction was allowed to proceed for 5 min at 28°C with 500 rpm shaking. The 4-HBA and 3,5-DHBA products of the reaction were then extracted with 500 l of ethyl acetate and immediately purified by normal phase HPLC as described below.…”