Characterization of cyanobacterial biliverdin reductase. Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis.
The Synechocystis sp. PCC 6803 gene (bvdR) encoding biliverdin reductase was amplified by the polymerase chain reaction, cloned, and overexpressed in Escherichia coli as the native form and as a 6-histidine-tagged amino-terminal fusion. The latter form of the enzyme was purified by affinity chromatography and shown to have the appropriate molecular weight by electrospray mass spectrometry. Both forms of the enzyme reduced biliverdin IXalpha using NADPH or NADH, with NADPH as the preferred reductant. The His-tagged enzyme has a Km for biliverdin of 1.3 microM. The pH optimum for the NADPH-dependent activity is 5.8, whereas that for rat biliverdin reductase is at pH 8.7. Absorbance spectra and high performance liquid chromatography retention times of the reaction product reaction match those of authentic bilirubin, the product of the reduction of biliverdin by the mammalian enzymes. These results provide the first evidence for the formation of bilirubin in bacteria. Fully segregated Synechocystis sp. PCC 6803 bvdR interposon mutants produce approximately 85% of the normal amount of phycobilisome cores containing allophycocyanin and other phycocyanobilin-bearing core polypeptides, but no detectable phycocyanin. Thus, surprisingly, the blockage of the conversion of biliverdin to bilirubin interferes with normal phycobiliprotein biosynthesis in cyanobacteria. Possible interpretations of this finding are presented.
Schluchter
W M
WM
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3206, USA.
Glazer
A N
AN
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis*; D001663:Bilirubin / Q000378:metabolism*; D001664:Biliverdine / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D045524:Phycobilisomes; D010860:Pigments, Biological / Q000096:biosynthesis*; D010940:Plant Proteins / Q000096:biosynthesis*; D051381:Rats; D016415:Sequence Alignment
D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D045524:Phycobilisomes; D010860:Pigments, Biological; D010940:Plant Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D001664:Biliverdine; D001663:Bilirubin