Defining the Bilin Lyase Domain:  Lessons from the Extended Phytochrome Superfamily

Abstract

Through pattern searches of genomic databases, new members of the growing family of phytochrome-related genes were identified and used to construct a 130-180 amino acid motif that delimits the bilin lyase domain, a subdomain of the extended phytochrome family that is sufficient for covalent attachment of linear tetrapyrroles (bilins). To test this hypothesis, portions of locus sll0821, a novel phytochrome-related gene from Synechocystis sp. PCC6803 that encodes a large protein with two potential bilin binding sites, were amplified, and the recombinant apoproteins were tested for bilin binding and phytochrome photoactivity. Our experiments indicated that both sites of this protein, termed Cph2 for cyanobacterial phytochrome 2, possessed bilin lyase activity, revealing two distinct classes of bilin lyase domains--those whose bilin adducts are red, far-red reversible and a second class whose bilin adducts are nonphotochromic. Spectroscopic analysis of photochromic phycocyanobilin and fluorescent phycoerythrobilin adducts of a 24-kDa fragment of Cph2 definitively established that the motif identified by pattern searches represents a bona fide bilin lyase domain. Site-directed mutagenesis of highly conserved charged residues within bilin lyase domains of nearly all members of the extended phytochrome superfamily has identified a glutamate residue critical for bilin binding.

The Structure of Phytochrome: A Picture Is Worth a Thousand Spectra

“…Since it is not possible to ascertain the protonation state of the BV prosthetic group of DrBphP from the structure of Wagner et al (2005), the hypothesis that its chromophore may not be fully protonated remains a formal, albeit less appealing, possibility. In this regard, removal of the PHY domains of plant, cyanobacterial, and bacteriophytochromes is known to strongly alter the spectroscopic and photochemical properties of the truncated holoproteins (Vierstra, 1993;Wu and Lagarias, 2000;Karniol et al, 2005). Structural information for the PHY domain, as well as additional spectroscopic measurements, will aid in interpretation of this aspect of phytochrome structure, as would determination of the structures of PAS and GAF domains for other phytochromes.…”

“…The same is true for cyanobacterial, fungal, and bacteriophytochromes (Karniol et al, 2005). While recent work implicates the importance of the P2 domain for proper holoprotein assembly of a cyanobacterial phytochrome (Zhao et al, 2004), the more distantly related phytochromes of the Cph2 subfamily lack this domain altogether but are nevertheless able to support bilin attachment (Wu and Lagarias, 2000). This suggests that the P2 domain performs an accessory role in holoprotein assembly, possibly by stabilizing CURRENT PERSPECTIVE ESSAY Figure 1.…”

“…In contrast with the P2 and P3 domains, P4 domains of plant, cyanobacterial, and bacteriophytochromes are dispensable for covalent attachment of the bilin chromophore (Vierstra, 1993;Wu and Lagarias, 2000;Oka et al, 2004;Karniol et al, 2005). The P4 domain is however necessary both for efficient photochemistry and for the normal Pr spectrum, which are measures of bilin chromophore conformation (Falk, 1989).…”

“…The P4 domain is however necessary both for efficient photochemistry and for the normal Pr spectrum, which are measures of bilin chromophore conformation (Falk, 1989). P4 therefore seems critical for optimizing the bilin chromophore environment to enable efficient photoconversion, perhaps by conferring rigidity to the bound bilin and thereby minimizing competing radiationless deexcitation pathways (Wu and Lagarias, 2000;Fischer and Lagarias, 2004). It was previously reported that P2 and P3 are related to domains known to be involved in ligand binding and signal transduction (Montgomery and Lagarias, 2002): P2 domains of some phytochrome protein sequences have been identified by domain database searches to be PAS domains (named for period clock, ARNT, and singleminded proteins; Ponting and Aravind, 1997), while P3 is identified as a GAF domain (named for vertebrate cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and the transcription activator FhlA; Aravind and Ponting, 1997).…”

Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site

“…The discovery of phytochromes in bacteria (2,3) showed that these chromoproteins are of prokaryotic origin, which gave great insight into the evolution of phytochromes. Prokaryotic phytochromes offer advantages for biochemical and biophysical studies (4)(5)(6) and help to define the role of protein domains and single amino acids (7). Phytochromes carry a bilin chromophore, either phytochromobilin (8), phycocyanobilin (PCB; ref.…”