CpeF is the bilin lyase that ligates the doubly linked phycoerythrobilin on β-phycoerythrin in the cyanobacterium Fremyella diplosiphon

Abstract

Edited by Chris Whitfield Phycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using in silico analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, cpeF, in the cyanobacterium Fremyella diplosiphon. Analyzing the phenotype of the cpeF deletion, we found that cpeF is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE. We also show that in a heterologous host, CpeF can attach PEB to Cys-48/ Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes. This work was supported by National Science Foundation Grants MCB-1029414 and MCB-1818187 (to D. M. K.) and MCB-1244339 and MCB-0843664 (to W. M. S.) and by a University of New Orleans Graduate School performance and accountability fellowship (to C. M. K.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S3 and Tables S1-S4.

MpeV is a lyase isomerase that ligates a doubly linked phycourobilin on the β-subunit of phycoerythrin I and II in marine Synechococcus

“…S1 ; closest characterized structure PDB: bgfp-a) ( 48 ), suggesting that these enzymes may have a similar substrate specificity for β-PE subunits. Yet, RS9916 has PUB doubly linked at C50, 61 of CpeB and MpeB ( 31 ), rather than PEB at the equivalent C48, 59 positions of F. diplosiphon CpeB ( 46 ). This led us to hypothesize that MpeV could be a lyase isomerase acting at C50, 61 on CpeB and perhaps also on MpeB.…”

“…A notable exception is BIOS-E4-1, a strain that lacks the CA4 regulators FciA and FciB and is a natural CA4-incapable mutant ( 35 , 40 ). Besides the CpeF lyase found both in phycoerythrin-containing freshwater cyanobacteria, like F. diplosiphon ( 46 ), and its functional homologs in “green light specialists” (see above), the MpeV family also includes MpeU, which was recently partially characterized as a PE-II-specific lyase isomerase in RS9916 ( 43 , 45 ). Like mpeV , the mpeU gene was first reported from the PBS rod genomic region of WH8020 ( 47 ).…”

“…RS9916 genes of interest were expressed using compatible vectors as outlined in Table S1 . All coexpressions analyzing β-subunits as substrate were designed to also express α-subunits in an effort to increase solubility of β-subunits ( 46 , 51 ). Previous work with F. diplosiphon showed that CpeS and CpeZ were required to obtain enough chromophorylated, soluble CpeB substrate to allow for measurable CpeF activity ( 46 ).…”

“…In these recent studies, Kronfel et al. ( 46 ) also characterized the chromophorylation pattern for the doubly linked PEB at C50, 61 in F. diplosiphon CpeB by CpeF and demonstrated that linkage along the A ring likely occurs first and is important for subsequent attachment to the D ring. The closest homolog to the cpeF gene from F. diplosiphon in RS9916 is mpeV , first described in the genome of marine Synechococcus strain WH8020 ( 47 ), though WH8020 MpeV has not yet been characterized.…”

“…MpeU is responsible for PUB attachment in BL ( 43 , 45 ); however, more research is needed to fully understand its function and potential role in chromatic acclimation. To date, no lyases for β-PEI or β-PEII have been characterized in RS9916, but some studies on the biosynthesis of CpeB from the freshwater cyanobacterium Fremyella diplosiphon have been completed ( 46 ). In F. diplosiphon , CpeS is the PEB lyase for C82 on CpeB and CpeF is the PEB lyase for the doubly ligated PEB at C50,61, and both of these enzymes require the chaperone-like protein CpeZ to keep the CpeB substrate soluble ( 34 , 46 ).…”