Unusual ring D fixation by three crucial residues promotes phycoviolobilin formation in the DXCF-type cyanobacteriochrome without the second Cys.

Keiji Fushimi,Rei Narikawa

doi:10.1042/bcj20210013

Abstract

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. Among them, dual Cys-type cyanobacteriochrome GAF domains possessing not only a highly conserved 'first Cys' but also a 'second Cys' are found from multiple lineages. The first Cys stably attaches to C31 of the A-ring, while the second Cys mostly shows reversible ligation to the C10 of the chromophore. Notably, the position of the second Cys in the primary sequence is diversified, and the most abundant dual Cys-type GAF domains have a 'second Cys' within the DXCF motif, which are called DXCF GAF domains. It has been long known that the second Cys in the DXCF GAF domains not only shows the reversible ligation but also is involved in isomerization activity (reduction in C4=C5 double bond) from the initially incorporated phycocyanobilin to phycoviolobilin. However, comprehensive site-directed mutagenesis on the DXCF GAF domains, AM1_6305g1 and AM1_1499g1, revealed that the second Cys is dispensable for isomerization activity, in which three residues participate by fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is necessary, even though one side of the fixation site is far from this bridge, with the other side at C31 fixed by the first Cys.

Highlights

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole -binding photoreceptors that, to date, have been found only in cyanobacteria (Fushimi and Narikawa 2019)
It has been long known that the second Cys is crucial for two color-tuning events: isomerization activity from the initially incorporated phycocyanobilin to phycoviolobilin and reversible ligation activity to the C10 of the chromophore
We firstly identified a novel CBCR GAF domain, AM1_1499g1, which lacks the second Cys, the domain belongs to a specific DXCF CBCR lineage

Summary

Introduction

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole (bilin) -binding photoreceptors that, to date, have been found only in cyanobacteria (Fushimi and Narikawa 2019). The second Cys reversibly attaches to or detaches from the C10 position of PVB during the photoconversion process (i.e., reversible ligation activity)(Rockwell et al 2008; Ishizuka et al 2011; Burgie et al 2013; Cornilescu et al 2014; Narikawa et al 2013). In this context, the second Cys residue possesses dual functions involved in isomerization activity and reversible ligation activity. We have succeeded in evolution-inspired color-tuning based on the AM1_1499g1 scaffold; the introduction of the second Cys (S118C variant) results in the acquisition of isomerization activity but not reversible ligation activity, with the exhibition of a yellow/teal photocycle (Fig. S1C). These findings provide a general concept for the isomerization mechanism

Results and discussion

Materials and Methods

442 Acknowledgments