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. 2019 Mar 15;294(11):3987-3999.
doi: 10.1074/jbc.RA118.007221. Epub 2019 Jan 22.

CpeF Is the Bilin Lyase That Ligates the Doubly Linked Phycoerythrobilin on β-Phycoerythrin in the Cyanobacterium Fremyella diplosiphon

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Free PMC article

CpeF Is the Bilin Lyase That Ligates the Doubly Linked Phycoerythrobilin on β-Phycoerythrin in the Cyanobacterium Fremyella diplosiphon

Christina M Kronfel et al. J Biol Chem. .
Free PMC article

Abstract

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.

Keywords: bilin lyase; cyanobacteria; fluorescence; light-harvesting complex (antenna complex); mass spectrometry (MS); photosynthesis; photosynthetic pigment; phycobiliprotein; phycobilisome; post-translational modification (PTM).

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Analyses of PBSs purified from WT and ΔcpeF. The WT PBS structure is illustrated above the sucrose gradient (30). A, sucrose gradients of WT (left) and ΔcpeF (right) cellular extracts from cells grown in green light. Fractions are labeled top, middle, and bottom. B, absorbance (solid lines) and fluorescence emission (dashed lines; excitation set at 490 nm) spectra of PBS fractions purified from WT (bottom fraction, black lines) and ΔcpeF (middle fraction, cyan lines; bottom fraction, blue lines) F. diplosiphon cells grown in green light. C, the Coomassie-stained SDS-polyacrylamide gel for PBSs purified from WT (bottom fraction) and ΔcpeF (middle and bottom fractions). The gel was loaded with 5 μg of total protein per sample. D and E, the Zn-enhanced fluorescence of the gel in C excited at 532 (D) and 635 nm (E). Lane Std indicates the molecular mass standard. F–H, Western blotting analyses of purified PBS samples from WT (bottom fraction) and ΔcpeF (bottom and middle fractions) using anti-CpeA (F), anti-CpeB (G), or anti-CpcA (H) antibodies. The gel was loaded with 5 μg of total protein per sample. These results are representative of three independent replicates.
Figure 2.
Figure 2.
Analyses of PE purified from WT and ΔcpeF. A, absorbance (solid lines) and fluorescence emission (dashed lines; excitation set at 490 nm) spectra of PE purified from WT (black lines) and ΔcpeF (cyan lines) F. diplosiphon cells grown in green light. B, the Coomassie-stained SDS-polyacrylamide gel for PE purified from WT and ΔcpeF. Lane Std indicates the molecular mass standard. The gel was loaded with 10 μl of each sample. C, the Zn-enhanced fluorescence of the gel in B excited at 532 nm. D and E, Western blotting analyses of PE purified from WT and ΔcpeF using anti-CpeA (D) and anti-CpeB (E) antibodies. These results are representative of three independent replicates.
Figure 3.
Figure 3.
Extracted ion chromatograms and LC-MS spectra for trypsin-digested peptides from ΔcpeF PE. A, combined extracted ion chromatograms for the peptides RLDAVNAIASNASC48MVSDAVAGMIC59ENQGLIQAGGNCYPNR at m/z 1200. 06 ((M + 4H)4+, PEB-modified; blue line) and m/z 1404.32 ((M + 3H)3+, unmodified (unmod) + one BME; red line) of CpeB isolated from ΔcpeF cells grown in green light. Numbers 1–3 indicate three versions of the unmodified peptide observed (one BME on Cys-48, -59, or -71). B, extracted ion chromatograms for the peptides MAAC80LR at m/z 625.81 ((M + 2H)2+, bilin-modified; blue line) and m/z 332.67 ((M + 2H)2+, unmodified; red line and asterisk) of CpeB isolated from ΔcpeF cells grown in green light. Numbers 1–5 indicate the MAAC80LR peptides that have a bilin bound. C, MS of unmodified MAAC80LR. D–H, MS for the five modified versions of the peptide MAAC80LR containing Cys-80 as specified in B. Inset graphs are the UV-visible absorbance spectra for the peaks at the retention times that are numbered with a + charge. The type of bilin bound to Cys-80 is indicated per panel. These results are representative of two independent replicates.
Figure 4.
Figure 4.
Recombinant CpeF coexpressions with HT-CpeB. A, fluorescence emission (excitation set at 490 nm) spectra of purified HT-CpeB obtained from E. coli cells expressing pPebS in addition to pCpeB2 and pCpeS (B+S+PEB; blue); pCpeB2 and pHT-CpeF2 (B+F+PEB; gray); pCpeB2, pCpeS, and pHT-CpeF2 (B+S+F+PEB; orange); pCpeBZ and pCpeS (BZ+S+PEB; cyan); pCpeBZ and pHT-CpeF2 (BZ+F+PEB; magenta); or pCpeBZ, pCpeS, and pHT-CpeF2 (BZ+S+F+PEB; red). B, the Coomassie-stained SDS-polyacrylamide gel for purified HT-CpeB samples from A. Lane Std indicates the molecular mass standard. C, the Zn-enhanced fluorescence of the gel in B excited at 532 nm. These results are representative of three independent replicates.
Figure 5.
Figure 5.
MALDI-MS spectrum of recombinant HT-CpeB peptides. The MALDI-MS spectrum of the peptide mixture resulting from the trypsin digestion of HT-CpeB purified from E. coli cells expressing pCpeBZ, pCpeS, pHT-CpeF2, and pPebS (BZ+S+F+PEB sample) is shown. Arrows bearing numbers indicate peaks that were identified as tryptic peptides containing a Cys residue without PEB bound: Cys-80 (arrow 1, m/z 664.41+), Cys-165 (arrow 2, m/z 1518.81+), and Cys-48/Cys-59 (arrow 3, m/z 40401+). Arrows bearing numbers with asterisks indicate peaks that were identified as tryptic peptides containing a PEB chromophore. Attachment was found to occur at Cys-80 (arrow 1*, m/z 1250.71+) and double attachment at Cys-48/Cys-59 (arrow 3*, m/z 4627.41+). No attachment to Cys-165 was detected. The inset graph is a close-up view of the m/z 4500–4900 range. These results are representative of two independent replicates.
Figure 6.
Figure 6.
Extracted ion chromatograms for trypsin-digested peptides from HT-CpeBA coexpressions. Combined extracted ion chromatograms for unmodified Cys-48/Cys-59 peptides (red) and PEB-cross-linked versions of Cys-48/Cys-59 peptides (blue and indicated with arrows) from purified HT-CpeBA obtained from E. coli cells expressing pCpeBA and pNT-PebS in addition to pCpeS (BA+S+PEB) (A), pCpeF2 and pNT-CpeZ3 (BA+F+Z+PEB) (B), pCpeSF2 and pNT-CpeZ3 (BA+SF+Z+PEB) (C), or pCpeSF2 and pNT-CpeYZ (BA+SF+YZ+PEB) (D) are shown. These results are representative of two independent replicates.
Figure 7.
Figure 7.
Recombinant CpeF coexpressions with HT-CpeB variants show the order of PEB ring A versus ring D ligation. A, fluorescence emission (excitation set at 490 nm) spectra of purified HT-CpeBA variants obtained from E. coli cells expressing pCpeF2, pNT-CpeZ3, and pNT-PebS in addition to pCpeBA (BA+F+Z+PEB; magenta); pCpeB(C48S)CpeA (B(C48S)A+F+Z+PEB; purple); or pCpeB(C59S)CpeA (B(C59S)A+F+Z+PEB; cyan). All protein samples were diluted to 0.75 μg μl−1 prior to obtaining the fluorescence spectra. B, the Zn-stained SDS-polyacrylamide gel for purified HT-CpeBA variant samples from A. Lane Std indicates the molecular mass standard with size markers shown at left. The gel was loaded with 2 μg of total protein per lane. C, Western blot analysis of purified HT-CpeBA variant samples using anti-CpeB antibodies. The gel was loaded with 2 μg of total protein per sample. The asterisk indicates degraded CpeB; this degradation product was not observed in B because it did not have PEB covalently attached. D, scatter plot representing the normalized ratio of Zn fluorescence from PEB (B):CpeB intensities as obtained from the Western blotting in C for the HT-CpeB samples. Error bars represent the S.D. of four independent replicates (mean values are indicated with a horizontal line for each sample set). The average BA+F+Z+PEB ratio was set to 1, and the mutants are represented relative to this value in D. Normalized fluorescence data were analyzed by a two-way analysis of variance (α = 0.05) with experimental replicate and strain (or mutant) as factors. Fluorescence of the strains was significantly different (p = 0.0292). Of note, B(C48S)A+F+Z+PEB showed significantly lower fluorescence compared with BA+F+Z+PEB (Tukey's honestly, p = 0.0277, noted with asterisk). Strain B(C59S)A+F+Z+PEB was intermediate between B(C48S)A+F+Z+PEB and BA+F+Z+PEB (p = 0.0941 versus B(C48S)A+F+Z+PEB and p = 0.6054 versus BA+F+Z+PEB).

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