The N121 site on the spike protein of SARS-CoV-2 is associated with heme and its metabolite, biliverdin, which can affect antibody binding. Both N121T and N121S substitutions have been observed in natural conditions and in a hamster model of dual infection with SARS-CoV-2 and Influenza A virus. Serum pseudotype neutralization assays against HIV-1 particles carrying wild-type, N121T, and N121S spikes with immune mouse and human sera revealed that N121T and N121S mutations had a greater impact on serum neutralization than biliverdin treatment. Although N121T and N121S substitutions are not currently major SARS-CoV-2 variants of concern, this study could provide fundamental information to prepare for potential future mutations at the N121 site of SARS-CoV-2.

Renoliths were removed at necropsy from dogs that had died from acute kidney injury in Asia in 2004 and submitted to our laboratories for analysis including elemental composition, mass spectrometry, and nuclear magnetic resonance spectroscopy. The presence of a mixed s-triazine matrix comprising melamine, cyanuric acid, and ammelide, but no detectable ammeline, was found in the stone samples we analyzed. The unusual and unique green coloration of these stones was determined to be due to the presence of biliverdin. The occurrence of these green stones distinguished the 2004 incident from another incident in 2007 in the USA and other reported cases. The presence of crystals was reported in renal tubules and collecting ducts in both outbreaks, but no stones were reported in the 2007 incident. This difference suggested a variation in the disease process caused by mixed s-triazine ingestion. Careful monitoring of food additives is warranted to prevent future problems in animals and humans.

Biliverdin, a bile pigment hydrolyzed from heme by heme oxygenase (HO), serves multiple functions in the human body, including antioxidant, anti-inflammatory, and immune response inhibitory activities. Biliverdin has great potential as a clinical drug; however, no economic and efficient production method is available currently. Therefore, the production of biliverdin by the biotransformation of exogenous heme using recombinant HO-expressing yeast cells was studied in this research. First, the heme oxygenase-1 gene (HO1) encoding the inducible plastidic isozyme from Arabidopsis thaliana, with the plastid transport peptide sequence removed, was recombined into Pichia pastoris GS115 cells. This resulted in the construction of a recombinant P. pastoris GS115-HO1 strain that expressed active HO1 in the cytoplasm. After that, the concentration of the inducer methanol, the induction culture time, the pH of the medium, and the concentration of sorbitol supplied in the medium were optimized, resulting in a significant improvement in the yield of HO1. Subsequently, the whole cells of GS115-HO1 were employed as catalysts to convert heme chloride (hemin) into biliverdin. The results showed that the yield of biliverdin was 132 mg/L when hemin was added to the culture of GS115-HO1 and incubated for 4 h at 30 °C. The findings of this study have laid a good foundation for future applications of this method for the economical production of biliverdin.

The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB\'s antioxidation, anti-inflammatory and anti-cancer properties, it has been utilized in the food, pharmaceutical and cosmetic industries. Currently, the extraction of PCB from Spirulina involves complex processes, which has led to increasing interest in the biosynthesis of PCB in Escherichia coli. However, the PCB titer remains low because of the poor activity of key enzymes and the insufficient precursor supply. Here, the synthesis of PCB was firstly improved by screening the optimal heme oxygenase (HO) from Thermosynechococcus elongatus BP-1(HOT) and PCB: ferredoxin oxidoreductase from Synechocystis sp. PCC6803 (PcyAS). In addition, based on a rational design and the infrared fluorescence method for high-throughput screening, the mutants of HOT(F29W/K166D) and PcyAS(D220G/H74M) with significantly higher activities were obtained. Furthermore, a DNA scaffold was applied in the assembly of HOT and PcyAS mutants to reduce the spatial barriers, and the heme supply was enhanced via the moderate overexpression of hemB and hemH, resulting in the highest PCB titer (184.20 mg/L) obtained in a 5 L fermenter. The strategies applied in this study lay the foundation for the industrial production of PCB and its heme derivatives.

Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IXα chromophore. In contrast to prototypical phytochromes that adopt a red-light-absorbing Pr ground state, the far-red light-absorbing Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 s to 50 min half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.

Equid alphaherpesvirus 8 (EqHV-8) is one of the most economically important viruses that is known to cause severe respiratory disease, abortion, and neurological syndromes in equines. However, no effective vaccines or therapeutic agents are available to control EqHV-8 infection. Heme oxygenase-1 (HO-1) is an antioxidant defense enzyme that displays significant cytoprotective effects against different viral infections. However, the literature on the function of HO-1 during EqHV-8 infection is little. We explored the effects of HO-1 on EqHV-8 infection and revealed its potential mechanisms. Our results demonstrated that HO-1 induced by cobalt-protoporphyrin (CoPP) or HO-1 overexpression inhibited EqHV-8 replication in susceptible cells. In contrast, HO-1 inhibitor (zinc protoporphyria) or siRNA targeting HO-1 reversed the anti-EqHV-8 activity. Furthermore, biliverdin, a metabolic product of HO-1, mediated the anti-EqHV-8 effect of HO-1 via both the protein kinase C (PKC)β/extracellular signal-regulated kinase (ERK)1/ERK2 and nitric oxide (NO)-dependent cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathways. In addition, CoPP protected the mice by reducing the EqHV-8 infection in the lungs. Altogether, these results indicated that HO-1 can be developed as a promising therapeutic strategy to control EqHV-8 infection.IMPORTANCEEqHV-8 infections have threatened continuously donkey and horse industry worldwide, which induces huge economic losses every year. However, no effective vaccination strategies or drug against EqHV-8 infection until now. Our present study found that one host protien HO-1 restrict EqHV-8 replication in vitro and in vivo. Furthermore, we demonstrate that HO-1 and its metabolite biliverdin suppress EqHV-8 relication via the PKCβ/ERK1/ERK2 and NO/cGMP/PKG pathways. Hence, we believe that HO-1 can be developed as a promising therapeutic strategy to control EqHV-8 infection.

Pseudomonas aeruginosa is a versatile opportunistic pathogen requiring iron for its survival and virulence within the host. The ability to switch to heme as an iron source and away from siderophore uptake provides an advantage in chronic infection. We have recently shown the extracellular heme metabolites biliverdin IXβ (BVIXβ) and BVIXδ positively regulate the heme-dependent cell surface signaling cascade. We further investigated the role of BVIXβ and BVIXδ in cell signaling utilizing allelic strains lacking a functional heme oxygenase (hemOin) or one reengineered to produce BVIXα (hemOα). Compared to PAO1, both strains show a heme-dependent growth defect, decreased swarming and twitching, and less robust biofilm formation. Interestingly, the motility and biofilm defects were partially rescued on addition of exogenous BVIXβ and BVIXδ. Utilizing liquid chromatography-tandem mass spectrometry, we performed a comparative proteomics and metabolomics analysis of PAO1 versus the allelic strains in shaking and static conditions. In shaking conditions, the hemO allelic strains showed a significant increase in proteins involved in quorum sensing, phenazine production, and chemotaxis. Metabolite profiling further revealed increased levels of Pseudomonas quinolone signal and phenazine metabolites. In static conditions, we observed a significant repression of chemosensory pathways and type IV pili biogenesis proteins as well as several phosphodiesterases associated with biofilm dispersal. We propose BVIX metabolites function as signaling and chemotactic molecules integrating heme utilization as an iron source into the adaptation of P. aeruginosa from a planktonic to sessile lifestyle.
OBJECTIVE: The opportunistic pathogen Pseudomonas aeruginosa causes long-term chronic infection in the airways of cystic fibrosis patients. The ability to scavenge iron and to establish chronic infection within this environment coincides with a switch to utilize heme as the primary iron source. Herein, we show the heme metabolites biliverdin beta and delta are themselves important signaling molecules integrating the switch in iron acquisition systems with cooperative behaviors such as motility and biofilm formation that are essential for long-term chronic infection. These significant findings will enhance the development of viable multi-targeted therapeutics effective against both heme utilization and cooperative behaviors essential for survival and persistence within the host.

The eggshell color of avian species is an important trait that is predominantly determined by the pigments biliverdin and protoporphyrin. Various factors affect eggshell pigment deposition and coloration; however, the underlying mechanisms remain unclear. We analyzed the hepatic transcriptomes and metabolomes of Changshun green-shell hens laying dark green and light green eggs to investigate the potential role of the liver in regulating the intensity of the green eggshell color. In total, 350 differentially expressed genes and 211 differentially altered metabolites were identified. Gene set enrichment analysis revealed that the enriched pathways and Gene Ontology (GO) terms were mainly associated with energy, immunity, and nutrient metabolism. Metabolite set enrichment analysis revealed that the enriched pathways were mainly associated with amino acid, vitamin, bile acid, and lipid metabolism. Moreover, gene-metabolite interaction network analysis revealed 1 subnetwork. Most genes and metabolites in this subnetwork were determined to be related to melanin metabolism and transport. In conclusion, our results suggest that hepatic melanin metabolism and transport are critical for eggshell coloration. Six candidate genes (CDKN2B, DDC, PYCR1, ABCG5, SLC3A1, and P2RX2) and 7 candidate metabolites (serotonin, 5-hydroxyindoleacetic acid, ornithine, acetylcholine, L-tryptophan, D-ornithine, and ADP) were suggested to play important roles in this process. Meanwhile, this study suggests that changes in hepatic energy metabolism, immune status, antioxidation activity, nutrient availability, and bile acid synthesis can impair eggshell coloration.

Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of μJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.