Catalytic properties and reaction mechanism of the CrtO carotenoid ketolase from the cyanobacterium Synechocystis sp. PCC 6803
2013
Breitenbach, J. (Jürgen) | Gerjets, Tanja | Sandmann, Gerhard
CrtW and CrtO are two distinct non-homologous β-carotene ketolases catalyzing the formation of echinenone and canthaxanthin. CrtO belongs to the CrtI family which comprises carotene desaturases and carotenoid oxidases. The CrtO protein from Synechocystis sp. PCC 6803 has been heterologously expressed, extracted and purified. Substrate specificity has been determined in vitro. The enzyme from Synechocystis is basically a mono ketolase. Nevertheless, small amounts of diketo canthaxanthin can be formed. The poor diketolation reaction could be explained by the low relative turnover numbers for the mono keto echinenone. Also other carotenoids with an unsubstituted β-ionone ring were utilized with low conversion rates by CrtO regardless of the substitutions at the other end of the molecule. The CrtO ketolase was independent of oxygen and utilized an oxidized quinone as co-factor. In common to CrtI-type desaturases, the first catalytic step involved hydride transfer to the quinone. The stabilization reaction of the resulting carbo cation was a reaction with OH⁻ forming a hydroxy group. Finally, the keto group resulted from two subsequent hydroxylations at the same C-atom and water elimination. This reaction mechanism was confirmed by in vitro conversion of the postulated hydroxy intermediates and by their enrichment and identification as trace intermediates during ketolation.
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