Characterization of Cyanobacterial Biliverdin Reductase: CONVERSION OF BILIVERDIN TO BILIRUBIN IS IMPORTANT FOR NORMAL PHYCOBILIPROTEIN BIOSYNTHESIS

Wendy M Schluchter,Alexander N Glazer

doi:10.1074/jbc.272.21.13562

Abstract

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.

Highlights

PCC 6803 gene 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
Heme is the biosynthetic precursor of the phycobilins [33,34,35,36], with biliverdin IX␣ as the initial product of heme cleavage in the pathway [6, 7, 36]
The cyanobacterial genes encoding the enzymes of the phycobilin biosynthetic pathway are unknown

Summary

Introduction

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. This presented the opportunity to ask if there were open reading frames (ORFs) homologous to the mammalian biliverdin reductases from rat and human whose sequences are known Such open reading frames might encode enzymes that catalyze reactions in the phycobilin biosynthetic pathway. A fully segregated bvdR interposon mutant contains approximately 6-fold less protein-bound PCB than wild-type cells This mutant produces smaller than normal amounts of phycobilisomes consisting only of core subassemblies that contain PCB-bearing polypeptides such as allophycocyanin and the core-membrane linker (LCM) [16, 17]. These results suggest that in cyanobacteria the conversion of biliverdin to bilirubin may have a regulatory as well as a degradative function

Methods

Results

Conclusion