PHYTOBILIN BIOSYNTHESIS - CLONING AND EXPRESSION OF A GENE ENCODING SOLUBLE FERREDOXIN-DEPENDENT HEME OXYGENASE FROM SYNECHOCYSTIS SP. PCC-6803

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IX alpha, in a reaction catalyzed by heme oxygenase. A gene containing an open r eading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp. PCC 6803. This gene, named ho1, was cloned and expressed in Escherichia c oli under the control of the lacZ promoter. Cells expressing the gene became green colored due to the accumulation of biliverdin IX alpha. T he size of the expressed protein was equal to the predicted size of th e Synechocystis gene product, named HO1. Heme oxygenase activity was a ssayed in incubations containing extract of transformed E. coil cells. Incubations containing extract of induced cells, but not those contai ning extract of uninduced cells, had ferredoxin-dependent heme oxygena se activity. With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IX alpha by spectrophotometry and reverse -phase HPLC. Heme oxygenase activity was not sedimented by centrifugat ion at 100 000 g. Expression of HO1 increased several-fold during incu bation of the cells for 72 h in iron-deficient medium.