Morphology, behavior, and phylogenomics of Oxytoxum lohmannii, Dinoflagellata.

Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology.

Recent innovations in long-term care coverage and financing: a rapid scoping review.

OBJECTIVES: To identify, chart and analyse the literature on recent initiatives to improve long-term care (LTC) coverage, financial protection and financial sustainability for persons aged 60 and older. DESIGN: Rapid scoping review. DATA SOURCES: Four databases and four sources of grey literature were searched for reports published between 2017 and 2022. After using a supervised machine learning tool to rank titles and abstracts, two reviewers independently screened sources against inclusion criteria. ELIGIBILITY CRITERIA: Studies published from 2017-2022 in any language that captured recent LTC initiatives for people aged 60 and older, involved evaluation and directly addressed financing were included. DATA EXTRACTION AND ANALYSIS: Data were extracted using a form designed to answer the review questions and analysed using descriptive qualitative content analysis, with data categorised according to a prespecified framework to capture the outcomes of interest. RESULTS: Of 24 reports, 22 were published in peer-reviewed journals, and two were grey literature sources. Study designs included quasi-experimental study, policy analysis or comparison, qualitative description, comparative case study, cross-sectional study, systematic literature review, economic evaluation and survey. Studies addressed coverage based on the level of disability, income, rural/urban residence, employment and citizenship. Studies also addressed financial protection, including out-of-pocket (OOP) expenditures, copayments and risk of poverty related to costs of care. The reports addressed challenges to financial sustainability such as lack of service coordination and system integration, insufficient economic development and inadequate funding models. CONCLUSIONS: Initiatives where LTC insurance is mandatory and accompanied by commensurate funding are situated to facilitate ageing in place. Efforts to expand population coverage are common across the initiatives, with the potential for wider economic benefits. Initiatives that enable older people to access the services needed while avoiding OOP-induced poverty contribute to improved health and well-being. Preserving health in older people longer may alleviate downstream costs and contribute to financial sustainability.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Masked cerulenin enables a dual-site selective protein crosslink.

Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases. We also describe two new classes of silylcyanohydrin and silylhemiaminal masked crosslinking probes that serve as new tools for activity and structure studies of these biosynthetic pathways.

Biochemistry and Protein Interactions of the CYREN Microprotein.

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

Nutrition in decompensated liver disease: A regional trainee-led audit investigating patient nutrition and outcomes following admission with decompensated chronic liver disease.

OBJECTIVE: To conduct a regional audit assessing the prevalence and management of malnutrition in decompensated liver disease. METHOD: All adults admitted with decompensated cirrhosis over one-month period to participating trusts were included. Malnutrition was identified using MUST and Royal Free Hospital-Nutritional Prioritisation Tool (RFH-NPT). RESULTS: 47 patients were identified. The prevalence of malnutrition was 76.6%. This was independent of age (<65 versus ≥65; p = 1) or aetiology of liver disease (alcohol-related versus not; p = 0.55). Screening was significantly higher on Gastroenterology wards than other wards (77% versus 23%; p = 0.012). RFH-NPT identified 76.6% of patients as malnourished whereas MUST identified 55.3%. Supplementation was prescribed to 83% of eligible patients. 80% was oral supplementation and 20% received NG feeding. Median length of stay (9 (2-62) days) was higher in those prescribed supplements (11 vs 7 days, p = 0.041). Readmission rates were similar regardless of supplementation. Mortality was higher in malnourished patients (p = 0.03) and in those prescribed nutritional supplements at 1, 3 and 6 months (p = 0.026, p = 0.026 and p = 0.008) respectively, who were more likely to have severe liver disease. CONCLUSION: Prevalence of malnutrition is high in patients with decompensated cirrhosis but independent of age and aetiology and associated with higher Child-Pugh scores. The RFH-NPT was a more sensitive screening tool than MUST. Increased nutritional screening was noted on gastroenterology wards with more intervention in those with severe liver disease. Despite the study's limitations, once malnourished, nutritional intervention did not appear to impact on patient readmission or mortality rates therefore, we propose addressing malnutrition by utilising specialty dietician involvement at an earlier stage.

Comparative-effectiveness research of COVID-19 treatment: a rapid scoping review.

OBJECTIVES: The COVID-19 pandemic has stimulated growing research on treatment options. We aim to provide an overview of the characteristics of studies evaluating COVID-19 treatment. DESIGN: Rapid scoping review DATA SOURCES: Medline, Embase and biorxiv/medrxiv from inception to 15 May 2021. SETTING: Hospital and community care. PARTICIPANTS: COVID-19 patients of all ages. INTERVENTIONS: COVID-19 treatment. RESULTS: The literature search identified 616 relevant primary studies of which 188 were randomised controlled trials and 299 relevant evidence syntheses. The studies and evidence syntheses were conducted in 51 and 39 countries, respectively.Most studies enrolled patients admitted to acute care hospitals (84%), included on average 169 participants, with an average age of 60 years, study duration of 28 days, number of effect outcomes of four and number of harm outcomes of one. The most common primary outcome was death (32%).The included studies evaluated 214 treatment options. The most common treatments were tocilizumab (11%), hydroxychloroquine (9%) and convalescent plasma (7%). The most common therapeutic categories were non-steroidal immunosuppressants (18%), steroids (15%) and antivirals (14%). The most common therapeutic categories involving multiple drugs were antimalarials/antibiotics (16%), steroids/non-steroidal immunosuppressants (9%) and antimalarials/antivirals/antivirals (7%). The most common treatments evaluated in systematic reviews were hydroxychloroquine (11%), remdesivir (8%), tocilizumab (7%) and steroids (7%).The evaluated treatment was in favour 50% and 36% of the evaluations, according to the conclusion of the authors of primary studies and evidence syntheses, respectively. CONCLUSIONS: This rapid scoping review characterised a growing body of comparative-effectiveness primary studies and evidence syntheses. The results suggest future studies should focus on children, elderly ≥65 years of age, patients with mild symptoms, outpatient treatment, multimechanism therapies, harms and active comparators. The results also suggest that future living evidence synthesis and network meta-analysis would provide additional information for decision-makers on managing COVID-19.

Efficacy of EUS-guided needle-based confocal laser endomicroscopy in the diagnosis of pancreatic lesions: A systematic review and meta-analysis.

Background and Objectives: Needle-based confocal laser endomicroscopy (nCLE) is a procedure in which an AQ-Flex nCLE mini-probe is passed through an EUS-FNA needle into a pancreatic lesion to enable subsurface in vivo tissue analysis. In this study, we conducted a systematic review and meta-analysis of nCLE for the diagnosis of pancreatic lesions. Materials and Methods: We conducted a comprehensive search of several databases and conference proceedings, including PubMed, EMBASE, Google-Scholar, MEDLINE, SCOPUS, and Web of Science databases (earliest inception to March 2020). The primary outcomes assessed the pooled rate of diagnostic accuracy for nCLE and the secondary outcomes assessed the pooled rate of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and adverse events (AE) of nCLE to diagnose premalignant/malignant pancreatic lesions. Results: Eleven studies on 443 patients were included in our analysis. The pooled rate of diagnostic accuracy of EUS nCLE was 83% (95 confidence interval [CI] = 79-87; I 2 = 0). The pooled rate of sensitivity, specificity, PPV and NPV of EUS nCLE was 85.29% (95% CI = 76.9-93.68; I 2 = 85%), 90.49% (95% CI = 82.24-98.74; I 2 = 64%), 94.15% (95% CI = 88.55-99.76; I 2 = 68%), and 73.44% (95% CI = 60.16-86.72; I 2 = 93%), respectively. The total AE rate was 5.41% (±5.92) with postprocedure pancreatitis being the most common AE at 2.28% (±3.73). Conclusion: In summary, this study highlights the rate of diagnostic accuracy, sensitivity, specificity, and PPV for distinguishing premalignant/malignant lesions. Pancreatic lesions need to be further defined with more validation studies to characterize CLE diagnosis criteria and to evaluate its use as an adjunct to EUS-FNA.

Preventing the transmission of COVID-19 and other coronaviruses in older adults aged 60 years and above living in long-term care: a rapid review.

BACKGROUND: The objective of this review was to examine the current guidelines for infection prevention and control (IPAC) of coronavirus disease-19 (COVID-19) or other coronaviruses in adults 60 years or older living in long-term care facilities (LTCF). METHODS: EMBASE, MEDLINE, Cochrane library, pre-print servers, clinical trial registries, and relevant grey literature sources were searched until July 31, 2020, using database searching and an automated method called Continuous Active Learning® (CAL®). All search results were processed using CAL® to identify the most likely relevant citations that were then screened by a single human reviewer. Full-text screening, data abstraction, and quality appraisal were completed by a single reviewer and verified by a second. RESULTS: Nine clinical practice guidelines (CPGs) were included. The most common recommendation in the CPGs was establishing surveillance and monitoring systems followed by mandating the use of PPE; physically distancing or cohorting residents; environmental cleaning and disinfection; promoting hand and respiratory hygiene among residents, staff, and visitors; and providing sick leave compensation for staff. CONCLUSIONS: Current evidence suggests robust surveillance and monitoring along with support for IPAC initiatives are key to preventing the spread of COVID-19 in LTCF. However, there are significant gaps in the current recommendations especially with regard to the movement of staff between LTCF and their role as possible transmission vectors. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020181993.

Substrate Specificity and Engineering of Mevalonate 5-Phosphate Decarboxylase.

A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)-mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)-MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomic-resolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)-MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically, plants, which universally encode a structural and functional homologue of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway.

Autoimmune Hepatitis A Case Report and Literature Review.

INTRODUCTION: Autoimmune hepatitis (AIH) is a cause of chronic liver disease. It is usually suspected based on clinical presentation and laboratory findings, but the diagnosis relies on the presence of specific autoantibodies and characteristic histology. Other unexplained findings should always prompt investigation for coexisting syndromes. CASE PRESENTATION: The patient is a 60-year-old Hispanic female with a history of mild asthma presented with exertional and pleuritic chest pain with weight loss, arthralgia, subjective fever, and night sweats for the last 3 months. Given the nonspecific nature of the presentation, further workup was pursued. Laboratory results indicated pancytopenia, elevated INR, and positive autoimmune panel including ANA, anti-chromatin, anti-histone, and rheumatoid factor as well as abnormal C3 and C4. Subsequent liver biopsy with interface hepatitis lead to a diagnosis of AIH with concurrent systemic lupus erythematosus suspected. CONCLUSION: The diagnostic work up for AIH is multimodal and aims to differentiate other etiologies such as congestive hepatopathy, iron overload, viral hepatitis, and other autoimmune liver diseases. In this particular case, unusual clinical and laboratory findings led to diagnosis of the overlap syndrome. Treatment for both was necessary to prevent further progression of disease.

Report from the 20th annual Western Canadian Gastrointestinal Cancer Consensus Conference; Saskatoon, Saskatchewan; 28-29 September 2018.

The 20th annual Western Canadian Gastrointestinal Cancer Consensus Conference was held in Saskatoon, Saskatchewan, 28-29 September 2018. This interactive multidisciplinary conference is attended by health care professionals from across Western Canada (British Columbia, Alberta, Saskatchewan, and Manitoba) who are involved in the care of patients with gastrointestinal cancers. In addition, invited speakers from other provinces participate. Surgical, medical, and radiation oncologists, and allied health care professionals participated in presentations and discussion sessions for the purpose of developing the recommendations presented here. This consensus statement addresses current issues in the management of colorectal cancers.

Report from the 19th annual Western Canadian Gastrointestinal Cancer Consensus Conference; Winnipeg, Manitoba; 29-30 September 2017.

The 19th annual Western Canadian Gastrointestinal Cancer Consensus Conference (wcgccc) was held in Winnipeg, Manitoba, 29-30 September 2017. The wcgccc is an interactive multidisciplinary conference attended by health care professionals from across Western Canada (British Columbia, Alberta, Saskatchewan, and Manitoba) who are involved in the care of patients with gastrointestinal cancer. Surgical, medical, and radiation oncologists; pathologists; radiologists; and allied health care professionals participated in presentation and discussion sessions for the purpose of developing the recommendations presented here. This consensus statement addresses current issues in the management of colorectal cancer.

Comparing assessment tools for detecting undernutrition in patients with liver cirrhosis.

BACKGROUND AND AIMS: Undernutrition in cirrhotic patients is often poorly recognised until late-stages. The current UK screening tool, the Malnutrition Universal Screening Tool, can miss undernutrition in patients with ascites/fluid retention. A 6-question Liver Disease Undernutrition Screening Tool (LDUST) has been developed in America. METHODS: We sought to compare LDUST with MUST in the detection of undernutrition in 50 inpatients and 50 outpatients with liver cirrhosis in a secondary care setting. This was then validated by a dietitian assessment. RESULTS: Similar patient demographics and liver disease aetiologies were found in the two cohorts. Mean Child-Pugh scores were higher for inpatients, 8.3 (SD 1.9) vs 5.9 (SD 1.2). LDUST detected undernutrition in 45/50 inpatients (90%) and 34/50 outpatients (68%). MUST scores ≥2 were present in 19/50 (38%) inpatients and 9/50 (18%) outpatients. In those with a MUST score <2, LDUST detected undernutrition in 26/31 (84%) inpatients and 27/41 (66%) outpatients. 26 inpatients had undernutrition using LDUST but had a MUST score <2, 20 (76%) of these were deemed to be undernourished by dietetics assessment. LDUST was mostly completed independently or with minimal assistance (80% inpatients, 100% outpatients), with mean completion times of 4 and 3 min for in- and outpatients respectively. CONCLUSION: LDUST is a quick and easy screening tool, which appears better able than MUST to detect undernutrition in cirrhotic patients, including undernutrition missed by MUST. Importantly the tool was validated against dietitian assessments. The high rates of undernutrition among cirrhotic inpatients suggest that screening this cohort is unnecessary, and instead all should undergo dietitian review, with LDUST utilised in an outpatient setting.

Post-admission outcomes of participants in the PARAMEDIC trial: A cluster randomised trial of mechanical or manual chest compressions.

BACKGROUND: The PARAMEDIC cluster randomised trial evaluated the LUCAS mechanical chest compression device, and did not find evidence that use of mechanical chest compression led to an improvement in survival at 30 days. This paper reports patient outcomes from admission to hospital to 12 months after randomisation. METHODS: Information about hospital length of stay and intensive care management was obtained through linkage with Hospital Episode Statistics and the Intensive Care National Audit and Research Centre. Patients surviving to hospital discharge were approached to complete questionnaires (SF-12v2, EQ-5D, MMSE, HADS and PTSD-CL) at 90days and 12 months. The study is registered with Current Controlled Trials, number ISRCTN08233942. RESULTS: 377 patients in the LUCAS arm and 658 patients in the manual chest compression were admitted to hospital. Hospital and intensive care length of stay were similar. Long term follow-up assessments were limited by poor response rates (53.7% at 3 months and 55.6% at 12 months). Follow-up rates were lower in those with worse neurological function. Among respondents, long term health related quality of life outcomes and emotional well-being was similar between groups. Cognitive function, measured by MMSE, was marginally lower in the LUCAS arm mean 26.9 (SD 3.7) compared to control mean 28.0 (SD 2.3), adjusted mean difference -1.5 (95% CI -2.6 to -0.4). CONCLUSION: There were no clinically important differences identified in outcomes at long term follow-up between those allocated to the mechanical chest compression compared to those receiving manual chest compression.

Doppler Endoscopic Probe Monitoring of Blood Flow Improves Risk Stratification and Outcomes of Patients With Severe Nonvariceal Upper Gastrointestinal Hemorrhage.

BACKGROUND & AIMS: For 4 decades, stigmata of recent hemorrhage in patients with nonvariceal lesions have been used for risk stratification and endoscopic hemostasis. The arterial blood flow that underlies the stigmata rarely is monitored, but can be used to determine risk for rebleeding. We performed a randomized controlled trial to determine whether Doppler endoscopic probe monitoring of blood flow improves risk stratification and outcomes in patients with severe nonvariceal upper gastrointestinal hemorrhage. METHODS: In a single-blind study performed at 2 referral centers we assigned 148 patients with severe nonvariceal upper gastrointestinal bleeding (125 with ulcers, 19 with Dieulafoy's lesions, and 4 with Mallory Weiss tears) to groups that underwent standard, visually guided endoscopic hemostasis (control, n = 76), or endoscopic hemostasis assisted by Doppler monitoring of blood flow under the stigmata (n = 72). The primary outcome was the rate of rebleeding after 30 days; secondary outcomes were complications, death, and need for transfusions, surgery, or angiography. RESULTS: There was a significant difference in the rates of lesion rebleeding within 30 days of endoscopic hemostasis in the control group (26.3%) vs the Doppler group (11.1%) (P = .0214). The odds ratio for rebleeding with Doppler monitoring was 0.35 (95% confidence interval, 0.143-0.8565) and the number needed to treat was 7. CONCLUSIONS: In a randomized controlled trial of patients with severe upper gastrointestinal hemorrhage from ulcers or other lesions, Doppler probe guided endoscopic hemostasis significantly reduced 30-day rates of rebleeding compared with standard, visually guided hemostasis. Guidelines for nonvariceal gastrointestinal bleeding should incorporate these results. ClinicalTrials.gov no: NCT00732212 (CLIN-013-07F).

Origin, Clinical Characteristics and 30-Day Outcomes of Severe Hematochezia in Cirrhotics and Non-cirrhotics.

BACKGROUND: The sites of origin, causes and outcomes of severe hematochezia have not been compared between cirrhotics and non-cirrhotics. In cirrhotics versus non-cirrhotics presenting with severe hematochezia, we aimed at (1) identifying the site and etiology of gastro-intestinal bleeding and independent predictors of bleeding from the upper gastrointestinal tract versus small bowel or the colon, (2) comparing 30-day clinical outcomes, and (3) proposing an algorithm for management of severe hematochezia. METHODS: In this cohort study from two university-based medical centers, 860 consecutive patients with severe hematochezia admitted from 1995 to 2011 were prospectively enrolled with 160 (18.6 %) cirrhotics. We studied (a) general clinical and laboratory characteristics of cirrhotics versus non-cirrhotics, (b) predictors of bleeding sites in each patient group by multiple variable regression analysis, and compared (c) 30-day outcomes, including rebleeding, surgery and deaths. RESULTS: Cirrhosis independently predicted an upper gastrointestinal source of bleeding (OR 3.47; 95 % CI 2.01-5.96) as well as history of hematemesis, melena in the past 30 days, positive nasogastric aspirate, prior upper gastrointestinal bleeding or use of aspirin or non-steroidal anti-inflammatory. The most prevalent diagnoses were esophageal varices (20 %) in cirrhotics and colon diverticular bleeding (27.1 %) in non-cirrhotics. Thirty-day rates of rebleeding, surgical interventions and deaths were 23.1 versus 15 % (P = 0.01), 14.4 versus 6.4 % (P < 0.001), and 17.5 versus 4.1 % (P < 0.001), in cirrhotics versus non-cirrhotics, respectively. CONCLUSIONS: Cirrhosis predicted an upper gastrointestinal site of bleeding in patients presenting with severe hematochezia. The 30-day rates of rebleeding, surgery, and death were significantly higher in cirrhotics than in non-cirrhotics.

Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants.

The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair.

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.