There is some debate within the literature with regards to the mechanism of cleavage by carotenoid cleavage dioxygenases. Primarily debate surrounds whether CCDs perform a monooxygenase or dioxygenase cleavage of the carotenoid. However, mechanistic studies are limited to a small number of examples performed on Z. mays NCED1, mammalian BCO and Figure 1.20 – Biosynthetic route from all-trans-β-carotene to abscisic acid. The rate limiting step, the cleavage of 9’-cis-neoxanthin, is catalysed by NCED.
Page | 23
A. thaliana CCD1. Early 18O
2 labelling experiments with Z. mays NCED1 suggested a
dioxygen mechanism, with almost 100% labelling of abscisic acid in the carboxylic acid position (Zeevaart et al. 1989). Subsequent 17O
2 labelling studies by Leuenberger et al. with
mammalian BCO gave only 50% incorporation of 17O into the retinal products (Leuenberger
et al. 2001). Consequentially this was interpreted as a monooxygenase mechanism, contradicting the findings of work on Z. mays. Labelling studies using A. thaliana CCD1, however, again showed 100% incorporation of 18O into the β-ionone and β-apo-10’-carotenal
products (Schmidt et al. 2006). Re-interpretation of the work by Leuenberger suggested that the apparent 50% incorporation on 17O could be due to exchange of isotopically labelled
oxygen with unlabelled oxygen in bulk water, as samples were left for 24 hours before analysis. Aldehydes are reactive species and can exchange rapidly with water. Beyond labelling experiments, there is no other experimental data in the literature pertaining to the CCD cleavage mechanism.
It is likely that activation of molecular oxygen in the CCD enzyme reaction occurs via single electron transfer from Fe (II) to oxygen to generate superoxide, as can be deduced by analogy to other non-heme iron dependent enzymes such as extradiol catechol dioxygenases or 2-oxo-glutarate dependent dioxygenases (Bugg 2003, Bugg & Ramaswamy 2008). Attack of superoxide on the carotenoid backbone would create a radical intermediate (Figure 1.21) which would be stabilised via conjugation along the polyene backbone. Subsequent one electron transfer back to the Fe (III) would restore the resting state Fe (II) and create a similarly highly stabilised secondary or tertiary cation, depending upon the position of cleavage. Although no evidence exists for the formation of a cation, it is consistent with inhibition of
Vigna unguiculata (cowpea) and A. thaliana NCED3 by abamine (Figure 1.21 and Section 1.8). At physiological pH, the tertiary amine of abamine is likely to be protonated, resulting in a tertiary amine cation. This cation would mimic the substrate cation and bind to the active site, inhibiting the cleavage reaction.
Page | 24 Following formation of the secondary cation, one of three reactions is possible (Figures 1.22 and 1.23). Firstly, the oxygen anion could ring close onto the cation to form a highly strained four membered dioxetane ring (path one). Breakdown of the dioxetane would result in the two aldehyde products (Schmidt et al. 2006, Harrison & Bugg 2013). The second possible mechanism involves nucleophilic attack of water on the carbocation (path two) followed by a Criegee rearrangement. This would result in a secondary carbocation which would be quenched by iron (II) hydroxide. A similar mechanism is proposed to occur in the extradiol catechol dioxygenases and the resulting hemi-acetal would break down to form the two aldehyde products (Bugg 2003). A third possible mechanism, the monooxygenase mechanism (Figure 1.23), also exists, whereby an epoxide forms following formation of the carbocation (Leuenberger et al. 2001). Formation of the epoxide would result in a high energy iron (IV) intermediate and attack on the epoxide by water would result in the formation of an α,β-diol, which breaks down to form the two aldehyde products.
Without detailed mechanistic data it is very difficult to distinguish between the possible mechanisms. Labelling experiments with 18O
2 strongly suggest a dioxygen
mechanism, incorporating both atoms of oxygen from O2. However, whether that mechanism
proceeds through a dioxetane intermediate or Criegee intermediate is unknown. It was noted in the crystal structure of VP14 that there is a lack of acidic and basic residues within the active site, which would lend support for a dioxetane mechanism (Messing et al. 2010). However, in the case of 9,10 cleavage reactions, formation of the dioxetane would require attack on a tertiary cation, which could present a steric challenge for the enzyme. In addition to this, dioxetane formation in catalysis is very rare. Very few enzymes are known to proceed Figure 1.21 – Structures of the protonated abamine and the secondary substrate cation formed during carotenoid cleavage.
Page | 25 through a dioxetane mechanism as formation of a dioxetane ring is an unfavourable endothermic process. Vitamin K-dependent carboxylase and firefly luciferase are two examples of enzymes whose mechanisms are known to proceed through dioxetane intermediates (Suttie 1985, Dowd et al. 1994, McCapra 1976, Franks & Brick 1996).
The Criegee rearrangement mechanism, however, requires the addition of water, so
18O
2 experiments would result in either one or two atoms of 18O being incorporated depending
upon the direction of the final C-O cleavage step. Only the dioxetane mechanism would Figure 1.22 – Proposed dioxygen cleavage mechanisms for CCDs based upon the NCED catalysed cleavage of 9’-cis-neoxanthin. Path one involves the formation of a dioxetane intermediate which breaks down to form two aldehyde products. Path two, however, utilises a Criegee rearrangement (which can also proceed in the reverse direction). Figure adapted from Harrison & Bugg 2013.
Page | 26 guarantee incorporation of both atoms from molecular oxygen. Another issue is the source of water to attack the substrate cation, which could be provided via the semi conserved second shell glutamic acid residues participating in catalysis, possibly by deprotonating water. This could arise through the iron cofactor acting as a Lewis acid. The sixth ligand on the iron (II), most likely water, would have its pKa lowered to between 7-10. A proton this acidic could
then be abstracted by one of the second shell glutamate residues, allowing participation in catalysis. A metal cofactor acting as a Lewis acid is not unprecedented. In the carboxypeptidase active site the zinc cofactor decreases the pKa of bound water allowing
deprotonation by a glutamate ligand (Christianson & Lipscomb 1989). If this were the case it would lend support for the Criegee rearrangement.
As discussed, there is a high degree of sequence similarity between the carotenoid cleavage dioxygenases and the lignostilbene dioxygenases. Labelling studies using 18O
2 on
two lignostilbene dioxygenases identified from Novosphingomonas aromaticivorans have shown only 50% incorporation of the 18O label into the products, suggesting a monooxygenase
mechanism for these lignostilbene dioxygenases (Marasco & Schmidt-Dannert 2008). In addition, the LSDs assayed showed no activity against carotenoids or apocarotenoids. The inability of LSD to cleave carotenoids is unsurprising, given how CCDs tend to cleave substrates of similar structures (Wirtz et al. 2001). However, the observation of a monooxygenase mechanism is interesting. It is entirely possible that the reaction is proceeding via a dioxygenase mechanism, but with a Criegee rearrangement, which eliminates the other
18O label. Such a mechanism would explain why only 50% labelling is observed.
Figure 1.23 – Possible monooxygenase cleavage mechanism of carotenoids by CCDs, proposed by Leuenberger et al. (Leuenberger et al. 2001). Figure from Harrison & Bugg 2013.
Page | 27 Computational studies have been performed examining the thermodynamics of possible CCD cleavages (Borowski et al. 2008). Such studies, which examine the lowest energy routes through the reaction, suggest that energetically the dioxetane intermediate is more likely than the epoxide intermediate. However, the study did not examine the possible Criegee rearrangement, so such a mechanism cannot be discounted.
Little insight into carotenoid cleavage is provided by model chemistry. To date, only one model complex, involving a ruthenium porphyrin, has been demonstrated (French et al. 2000). The ruthenium complex has been shown to cleave β-carotene to two equivalents of retinal in the presence of t-butyl hydroperoxide.