CrtW is responsible for the final stage of myxobacton creation, the primaryM. xanthus carotenoid product. Similarly in each of the previous organisms mentioned, with the sole exception ofS. coelicolor, which lacks both genes, the products of crtW or crtZ are believed to be responsible for the final carotenoid conversion step. In Algoriphagussp. KK10202C, the organism that displays the strongest CrtW match to theM. xanthusprotein, the gene is encoded at the end of the biosynthetic gene cluster. All of the genes in the cluster are transcribed in the same direction, though this has not proven universal in carotenoid producers. X. autotrophicus Py2 shows that if neither crtW nor crtZ are present, it is still possible to produce a functioning carotenoid. It has already been recorded that CrtZ is capable of acting on a substrate previously altered by CrtW, as is evident from the gene content of the Paracoccus species N81106. The Bradyrhizobium species ORS278, like M. xanthus, has no genetic equivalent of crtZ, so CrtW must also be able to operate exclusively and without subsequent substrate alteration by CrtZ. A number of organisms containcrtZ withoutcrtW, in particularSo. cellulosum. Therefore it must also be possible for CrtZ to function alone without CrtW present. Given the similar systems of carotenoid creation that exist in all of the organisms studied, the primary difference appears to be the presence of CrtW, CrtZ or neither, at the final biosynthetic stage. To establish reasons for this, the basic activity of both needs to be studied further.
It is possible to study the activity of both CrtZ and CrtW separately using a β- carotene substrate. When hydroxylated, β-carotene is converted into zeaxanthin, with
a hydroxyl group added to the third carbon of each of the two carbon ring structures. If the carotenoid is exposed to a ketolase, a ketone group is added to the fourth carbon of each ring creating canthaxanthin. These modifications have a direct effect on the ability of the compounds to function in cell protection. A study has been carried out looking specifically at mechanisms of carotenoid antioxidant behaviour (Di Mascioet al., 1990). One of the primary observations was that canthaxanthin was faster at quenching singlet oxygen species than zeaxanthin. The values recorded (in L mol-1s- 1
) were 2.1x1010 and 1.0x1010, respectively, so it was over twice as effective. This is a large difference when considering the actual size of the rate constants. The figures do differ when carotenoid activity in the presence of other specific radicals is studied.
Another study looked at the capability of carotenoids to react with nitrogen dioxide radicals, mercaptoethanol thiyl radicals, glutathione thiyl radicals and methanesulfonyl thiyl radicals (Mortensen et al., 1997). Zeaxanthin was far more capable of countering nitrogen-base radicals, being almost three times as effective as canthaxanthin. In contrast the opposite was true when exposed to mercaptoethanol thiyl radicals. The latter two listed radicals were controlled with similar effectiveness by each substrate. This shows that carotenoid structure has an effect on how effectively it can quench particular radical species. Highlighting this fact, a separate study (Woodallet al., 1997) looked at the effectiveness of a set of carotenoids to react to the generation of peroxyl radicals. Canthaxanthin was least effective in standard comparisons, functioning at half the level of zeaxanthin. In a reaction where each substrate was exposed to a large amount of peroxyl oxidants at once, zeaxanthin was almost seven times more efficient at quenching than canthaxanthin. The distinct differences offer an insight into why different bacterial species from different environments would need to generate various carotenoids.
In terms of carbon bonding it has been recorded that the more alternate carbon-carbon double bonds present in a carotenoid, the more effective it is at quenching singlet oxygen species (Footeet al., 1970). In the examples of zeaxanthin and canthaxanthin the latter was far more effective, despite both possessing a similar carbon double bond number. In this case the addition of the two double bonds in the ketone group (which also fall into the alternate bonding pattern when combined with the other carbon double bonds) act to increase its effectiveness. This gives it thirteen double bonds in total, compared to the eleven present in zeaxanthin. The addition of a double bonded oxygen atom may also provide the molecule with added stability compared to that of zeaxanthin. This may explain why the two react differently to different radicals – clearly the presence of an OH group enables the carotenoid to quench peroxyl radicals easier and vice versa for glutathione thiyl radicals. In conclusion, the presence of both additional groups together in a carotenoid molecule should lead to the generation of an ester that is effective at quenching all of the
reactive species from the canthaxanthin and zeaxanthin studies. When β-carotene is
both hydroxylated and undergoes ketonisation, a structure is obtained which has a C4 ketone and a C3hydroxyl group; astaxanthin.
Overall astaxanthin is a better quencher of singlet oxygen species, with a rate constant of 2.4x1010 (L mol-1 s-1), than either hydroxylated or ketonised carotenoids.
The increased molecular stability provided by the hydroxyl groups appears to increase its effectiveness. Similarly with each of the previously tested nitrogen, glutamine thiyl and methanesulfonyl radicals; astaxanthin displays overall increased or at least similar maximum levels of quenching as for zeaxanthin and canthaxanthin. The sole exception is the mercaptoethanol thiyl radicals which are quenched at a reduced rate compared to either of the previously studied carotenoids. The reduction is not considerable, with approximately a 10% drop off in activity. When studying the peroxyl radicals, astaxanthin behaved in a similar way to canthaxanthin, with zeaxanthin still far more effective at quenching. This suggests that the presence of the ketone group leads to a decrease in the ability of the carotenoid to deal with these particular radicals. By looking at the three different carotenoids that can be produced
from β-carotene using just hydroxylase and ketolase activity, it is obvious that each
function has a significant effect. The most effective overall is astaxanthin, created through the combined activity of both CrtW and CrtZ, and the least effective zeaxanthin, formed by CrtZ alone, depending on which radical is being encountered by the carotenoid.
The myxobacton of M. xanthus has twelve alternating carbon double bonds and one additional oxygen double bond, as there is one cyclic carbon ring in the molecule. Without the ketone group the molecule should be a more effective
quencher of singlet oxygen species than each of the previously mentioned β-carotene-
derived carotenoids, due to its longer central twelve carbon double bond spine. Despite an increased stability associated with further hydroxylation of such a molecule, thus making it more effective at radical quenching, a CrtZ hydroxylase is not encoded within theM. xanthusgenome. This result is also observed when looking at either the closely relatedS. aurantiacumorBradyrhizobiumsp. ORS278. Similarly when considering the increased instability and ineffectiveness of a hydroxylated species to deal with radical generation, there is no crtW gene in So. cellulosum, Streptomyces coelicolororPantoea ananatis(Erwinia uredovora20D3).