Biliverdin Binds Covalently to Agrobacterium Phytochrome Agp1 via Its Ring A Vinyl Side Chain*

The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction. In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein. Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20). By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment. Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner. Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249). It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket. In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein. However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin. This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain.

[1]  R. Vierstra,et al.  Bacteriophytochromes: phytochrome-like photoreceptors from nonphotosynthetic eubacteria. , 1999, Science.

[2]  B. Montgomery,et al.  Phytochrome ancestry: sensors of bilins and light. , 2002, Trends in plant science.

[3]  T. Shinomura,et al.  Structural requirement of bilin chromophore for the photosensory specificity of phytochromes A and B , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[4]  T. Hirano,et al.  PHYTOCHROME PHOTOCHROMISM PROBED BY SITE-DIRECTED MUTATIONS AND CHROMOPHORE ESTERIFICATION , 1997 .

[5]  M. Rosbash,et al.  PAS is a dimerization domain common to Drosophila Period and several transcription factors , 1993, Nature.

[6]  K. Norris,et al.  DETECTION, ASSAY, AND PRELIMINARY PURIFICATION OF THE PIGMENT CONTROLLING PHOTORESPONSIVE DEVELOPMENT OF PLANTS. , 1959, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. McDowell,et al.  Phycocyanobilin Is the Natural Precursor of the Phytochrome Chromophore in the Green Alga Mesotaenium caldariorum * , 1997, The Journal of Biological Chemistry.

[8]  T. Kohchi,et al.  The Arabidopsis HY2 Gene Encodes Phytochromobilin Synthase, a Ferredoxin-Dependent Biliverdin Reductase , 2001, Plant Cell.

[9]  Eric Giraud,et al.  Bacteriophytochrome controls photosystem synthesis in anoxygenic bacteria , 2002, Nature.

[10]  R. Vierstra,et al.  The pair of bacteriophytochromes from Agrobacterium tumefaciens are histidine kinases with opposing photobiological properties , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  A. Mcdonagh.  6 – Bile Pigments: Bilatrienes and 5,15-Biladienes , 1979 .

[12]  K. Uchida,et al.  In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Hughes,et al.  Characterization of recombinant phytochrome from the cyanobacterium Synechocystis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[14]  W. Marwan,et al.  A photoreceptor with characteristics of phytochrome triggers sporulation in the true slime mould Physarum polycephalum , 1995, FEBS letters.

[15]  T. Hübschmann,et al.  Characterization of the Cph1 holo-phytochrome from Synechocystis sp. PCC 6803. , 2001, European journal of biochemistry.

[16]  W. Gärtner,et al.  A Novel Chromophore Selectively Modifies the Spectral Properties of One of the Two Stable States of the Plant Photoreceptor Phytochrome , 1998 .

[17]  J. Hughes,et al.  Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803. Purification, assembly, and quaternary structure. , 2001, European journal of biochemistry.

[18]  T. Kohchi,et al.  Functional Genomic Analysis of the HY2 Family of Ferredoxin-Dependent Bilin Reductases from Oxygenic Photosynthetic Organisms , 2001, Plant Cell.

[19]  W. Gärtner,et al.  Two independent, light-sensing two-component systems in a filamentous cyanobacterium. , 2002, European journal of biochemistry.

[20]  J. Katz,et al.  Structure of phycocyanobilin , 1967 .

[21]  M. Gilles-Gonzalez,et al.  Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Tilman Lamparter,et al.  Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Christie,et al.  LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Seth J. Davis,et al.  Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore , 2001, Nature.

[25]  K. Yeh,et al.  A cyanobacterial phytochrome two-component light sensory system. , 1997, Science.

[26]  P. Quail,et al.  Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3. , 2000, Proceedings of the National Academy of Sciences of the United States of America.