Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystalstructure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded beta barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced

Use of Prostate-specific Antigen Testing in Medicare Beneficiaries: Association with Previous Evaluation

Objective: Determine uptake of prostate-specific antigen (PSA) testing in Medicare beneficiaries according to previous receipt of PSA testing. Methods: A 5% random sample of men aged 67 years or older without a previous diagnosis of prostate cancer was identified through 2009-2012 Medicare claims. We measured the annualized frequency of PSA screening among men due for PSA testing, stratified by PSA testing use in the previous 2 years, and clustered by ordering provider. Results: Throughout the study period, PSA testing use was consistently higher for men with previous screening than for men without previous screening. For men without previous screening, there was a decline in testing that was most pronounced in 2012. Compared with 2009, the corresponding odds ratios were 0.98 [95% confidence interval (CI) (0.96-1.00)] in 2010, 0.94 [95% CI (0.92-0.95)] in 2011, and 0.66 [95% CI (0.65-0.68)] in 2012. In contrast, for men with previous screening, PSA testing frequency was stable from 2009 to 2011, and declined to a lesser extent in 2012 [odds ratio 0.80, 95% CI (0.79-0.81)]. Conclusion: Receipt of PSA testing is highly dependent on whether an individual was tested in the recent past. In previously unscreened men, the largest decrease occurred in 2012, which may reflect in part the publication of US Preventive Services Task Force guidelines, but there was much less impact among men already being screened. © 2017 Family Medicine and Community Health

The Roles of the Chaperone-like Protein CpeZ and the Phycoerythrobilin Lyase CpeY in Phycoerythrin Biogenesis

Phycoerythrin (PE) present in the distal ends of light-harvesting phycobilisome rods in Fremyella diplosiphon (Tolypothrix sp. PCC 7601) contains five phycoerythrobilin (PEB) chromophores attached to six cysteine residues for efficient green light capture for photosynthesis. Chromophore ligation on PE subunits occurs through bilin lyase catalyzed reactions, but the characterization of the roles of all bilin lyases for phycoerythrin is not yet complete. To gain a more complete understanding about the individual functions of CpeZ and CpeY in PE biogenesis in cyanobacteria, we examined PE and phycobilisomes purified from wild type F. diplosiphon, cpeZ and cpeY knockout mutants. We find that the cpeZ and cpeY mutants accumulate less PE than wild type cells. We show that in the cpeZ mutant, chromophorylation of both PE subunits is affected, especially the Cys-80 and Cys-48/Cys-59 sites of CpeB, the beta-subunit of PE. The cpeY mutant showed reduced chromophorylation at Cys-82 of CpeA. We also show that, in vitro, CpeZ stabilizes PE subunits and assists in refolding of CpeB after denaturation. Taken together, we conclude that CpeZ acts as a chaperone-like protein, assisting in the folding/stability of PE subunits, allowing bilin lyases such as CpeY and CpeS to attach PEB to their PE subunit

CpeT is the Phycoerythrobilin Lyase for Cys-165 on Beta-Phycoerythrin from Fremyella Diplosiphon and the Chaperone-like Protein CpeZ Greatly Improves its Activity.

Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae. The lyases responsible for chromophorylating the light harvesting protein phycoerythrin (PE) have not been fully characterized. In this study, we explore the role of CpeT, a putative bilin lyase, in the biosynthesis of PE in the cyanobacterium Fremyella diplosiphon. Recombinant protein studies show that CpeT alone can bind phycoerythrobilin (PEB), but CpeZ, a chaperone-like protein, is needed in order to correctly and efficiently attach PEB to the beta-subunit of PE. MS analyses of the recombinant beta-subunit of PE coexpressed with CpeT and CpeZ show that PEB is attached at Cys-165. Purified phycobilisomes from a cpeT knockout mutant and wild type (WT) samples from F. diplosiphon were analyzed and compared. The cpeT mutant contained much less PE and more phycocyanin than WT cells grown under green light, conditions which should maximize the production of PE. In addition, Northern blot analyses showed that the cpeCDESTR operon mRNAs were upregulated while the cpeBcpeA mRNAs were downregulated in the cpeT mutant strain when compared with WT, suggesting that CpeT may also play a direct or indirect regulatory role in transcription of these operons or their mRNA stability, in addition to its role as a PEB lyase for Cys-165 on beta-PE

Adaptation to Blue Light in Marine Synechococcus Requires MpeU, an Enzyme with Similarity to Phycoerythrobilin Lyase Isomerases

Marine Synechococcus has successfully adapted to environments with different light colors, which likely contributes to this genus being the second most abundant group of microorganisms worldwide. Populations of Synechococcus that grow in deep, blue ocean waters contain large amounts of the blue-light absorbing chromophore phycourobilin (PUB) in their light harvesting complexes (phycobilisomes). Here, we show that all Synechococcus strains adapted to blue light possess a gene called mpeU. MpeU is structurally similar to phycobilin lyases, enzymes that ligate chromophores to phycobiliproteins. Interruption of mpeU caused a reduction in PUB content, impaired phycobilisome assembly and reduced growth rate more strongly in blue than green light. When mpeU was reintroduced in the mpeU mutant background, the mpeU-less phenotype was complemented in terms of PUB content and phycobilisome content. Fluorescence spectra of mpeU mutant cells and purified phycobilisomes revealed red-shifted phycoerythrin emission peaks, likely indicating a defect in chromophore ligation to phycoerythrin-I (PE-I) or phycoerythrin-II (PE-II). Our results suggest that MpeU is a lyase-isomerase that attaches a phycoerythrobilin to a PEI or PEII subunit and isomerizes it to PUB. MpeU is therefore an important determinant in adaptation of Synechococcus spp. to capture photons in blue light environments throughout the world\u27s oceans

CpeF is the Bilin Lyase that Ligates the Doubly Linked Phycoerythrobilin on Phycoerythrin in the Cyanobacterium Fremyella Diplosiphon

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