Phycobilin biosynthetic reactions in extracts of cyanobacteria

Abstract

Phycobilins are the chromophores of phycobiliproteins, the light-harvesting pigments of cyanobacteria, red algae and cryptophytes. Phycobilins are biosynthesized from heme by the action of heme oxygenase, which converts heme to biliverdin, followed by the action of other enzymes that convert biliverdin to the phycobilins. We previously reported on the enzymes and biosynthetic intermediates of phycobilin formation in extracts of the unicellular red alga Cyanidium caldarium. Heme oxygenase activity has now been obtained from extracts of the cyanobacterium Synechocystis sp. PCC 6701. The reaction requirements are similar to those for the C. caldarium enzyme: heme substrate, reduced ferredoxin, and a second reductant such as ascorbate or Trolox. The enzymatic nature of the reaction was verified by two criteria in addition to the requirement for cell extract: production of only the IXα isomer of the bilin product and inhibition by the substrate analog Sn-protoporphyrin IX. The enzyme was partially purified by high-speed centrifugation, 35–75% differential (NH4)2SO4 precipitation, and DEAE-cellulose anion exchange chromatography. In addition, extract capable of converting biliverdin IXα to phycobilins has been obtained from Synechocystis sp. PCC 6701 and another cyanobacterium, Synechocystis sp. PCC 6803. Only the (3Z) isomers of the phycobilins accumulated in the incubations containing unfractionated cell extracts, in contrast to incubations with unfractionated C. caldarium extracts which produce both the (3Z) and (3E) isomers. Phycocyanobilin and phycoerythrobilin were produced in comparable amounts by Synechocystis sp. PCC 6701 extracts, but only phycocyanobilin accumulated in Synechocystis sp. PCC 6803 extracts. This difference in in vitro product accumulation correlates with the phycobilins that are found in vivo in these two cell types.