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

Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases. Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases. 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Although PEB is readily produced in cyanobacteria as a free precursor molecule (41Frankenberg N. Mukougawa K.K. Lagarias J.C. Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms.Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (193) Google Scholar), the bilin PUB is produced through the activity of specialized bilin lyase isomerases that attach PEB while simultaneously isomerizing it to PUB (Fig. 2) (31Shukla A. Biswas A. Blot N. Partensky F. Karty J.A. Hammad L.A. Garczarek L. Gutu A. Schluchter W.M. Kehoe D.M. Phycoerythrin-specific biln lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 20136-20141Crossref PubMed Scopus (57) Google Scholar, 42Blot N. Wu X.J. Thomas J.C. Zhang J. Garczarek L. Bohm S. Tu J.M. Zhou M. Ploscher M. Eichacker L. Partensky F. Scheer H. Zhao K.H. Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyase-isomerase.J. Biol. Chem. 2009; 284: 9290-9298Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Increased PUB content in PBS allows these organisms to better absorb the blue light available in deeper water (43Mahmoud R.M. Sanfilippo J.E. Nguyen A.A. Strnat J.A. Partensky F. Garczarek L. El-Kassem N.A. Kehoe D.M. Schluchter W.M. Adaptation to blue light in marine Synechoccous requires MpeU, an enzyme with similarity to phycoerythrobilin lyase isomerases.Front. Microbiol. 2017; 8: 243Crossref PubMed Scopus (19) Google Scholar). In RS9916, there are five possible sites for bilin attachment on PEI, two on the α-subunit (CpeA) and three on the β-subunit (CpeB), and six possible sites on PEII, three each on the α-subunit (MpeA) and β-subunit (MpeB) (31Shukla A. Biswas A. Blot N. Partensky F. Karty J.A. Hammad L.A. Garczarek L. Gutu A. Schluchter W.M. Kehoe D.M. Phycoerythrin-specific biln lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 20136-20141Crossref PubMed Scopus (57) Google Scholar). It is hypothesized that a bilin lyase is responsible for ligation of a given bilin at each individual site. Thus far, only four PE lyases (all members of the CpcE/CpcF clan) have been characterized for RS9916 in the literature (31Shukla A. Biswas A. Blot N. Partensky F. Karty J.A. Hammad L.A. Garczarek L. Gutu A. Schluchter W.M. Kehoe D.M. Phycoerythrin-specific biln lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 20136-20141Crossref PubMed Scopus (57) Google Scholar, 38Sanfilippo J.E. Nguyen A.A. Garczarek L. Karty J.A. Pokhrel S. Strnat J.A. Partensky F. Schluchter W.M. Kehoe D.M. Interplay between differentially expressed enzymes contribures to light color acclimation in marine Synechococcus.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 6457-6462Crossref PubMed Scopus (21) Google Scholar, 43Mahmoud R.M. Sanfilippo J.E. Nguyen A.A. Strnat J.A. Partensky F. Garczarek L. El-Kassem N.A. Kehoe D.M. Schluchter W.M. Adaptation to blue light in marine Synechoccous requires MpeU, an enzyme with similarity to phycoerythrobilin lyase isomerases.Front. Microbiol. 2017; 8: 243Crossref PubMed Scopus (19) Google Scholar, 44Carrigee L. Mahmoud R.M. Sanfilippo J.E. Frick J.P. Strnat J.A. Karty J.A. Chen B. Kehoe D.M. Schluchter W.M. CpeY is a phycoerythrobilin lyase for cysteine 82 of the phycoerythrin I α-subunit in marine Synechococcus.Biochim. Biophys. Acta Bioenerg. 2020; 1861: 148215Crossref PubMed Scopus (3) Google Scholar). During CA4, MpeY (PEB lyase) and MpeZ (PUB lyase isomerase) chromophorylate the C83 position of MpeA in green or blue light, respectively (31Shukla A. Biswas A. Blot N. Partensky F. Karty J.A. Hammad L.A. Garczarek L. Gutu A. Schluchter W.M. Kehoe D.M. Phycoerythrin-specific biln lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 20136-20141Crossref PubMed Scopus (57) Google Scholar, 38Sanfilippo J.E. Nguyen A.A. Garczarek L. Karty J.A. Pokhrel S. Strnat J.A. Partensky F. Schluchter W.M. Kehoe D.M. Interplay between differentially expressed enzymes contribures to light color acclimation in marine Synechococcus.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 6457-6462Crossref PubMed Scopus (21) Google Scholar). CpeY adds PEB to the C82 position of CpeA, a site of constitutive attachment, not involved in any of the major CA4 changes that occur (44Carrigee L. Mahmoud R.M. Sanfilippo J.E. Frick J.P. Strnat J.A. Karty J.A. Chen B. Kehoe D.M. Schluchter W.M. CpeY is a phycoerythrobilin lyase for cysteine 82 of the phycoerythrin I α-subunit in marine Synechococcus.Biochim. Biophys. Acta Bioenerg. 2020; 1861: 148215Crossref PubMed Scopus (3) Google Scholar). MpeU is responsible for PUB attachment in BL (43Mahmoud R.M. Sanfilippo J.E. Nguyen A.A. Strnat J.A. Partensky F. Garczarek L. El-Kassem N.A. Kehoe D.M. Schluchter W.M. Adaptation to blue light in marine Synechoccous requires MpeU, an enzyme with similarity to phycoerythrobilin lyase isomerases.Front. Microbiol. 2017; 8: 243Crossref PubMed Scopus (19) Google Scholar, 45Nguyen A. Characterization of Genes Involved in the Biosynthesis of Phycoerythrin I and II in Cyanobacteria. University of New Orleans, 2018Google Scholar); 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 d