Camphor pathway redux: functional recombinant expression of 2,5- and 3,6-diketocamphane monooxygenases of Pseudomonas putida ATCC 17453 with their cognate flavin reductase catalyzing Baeyer-Villiger reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases is hitherto ill-defined. In this study, a 37-kDa Fred was purified from camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular mass of 18-kDa that uses NADH as electron donor (Km = 32 μM) and it catalyzes the reduction of FMN (Km = 3.6 μM; kcat = 283 s-1) in preference to FAD (Km = 19 μM; kcat = 128 s-1). Sequence determination of ∼40-kb of the camphor (CAM) degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1, and camE25-2 for 2,5-DKCMO-2) as well as that of 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533-kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO and 3,6-DKCMO encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates

Haploinsufficiency of PRR12 causes a spectrum of neurodevelopmental, eye, and multisystem abnormalities

PURPOSE: Proline Rich 12 (PRR12) is a gene of unknown function with suspected DNA-binding activity, expressed in developing mice and human brains. Predicted loss-of-function variants in this gene are extremely rare, indicating high intolerance of haploinsufficiency. METHODS: Three individuals with intellectual disability and iris anomalies and truncating de novo PRR12 variants were described previously. We add 21 individuals with similar PRR12 variants identified via matchmaking platforms, bringing the total number to 24. RESULTS: We observed 12 frameshift, 6 nonsense, 1 splice-site, and 2 missense variants and one patient with a gross deletion involving PRR12. Three individuals had additional genetic findings, possibly confounding the phenotype. All patients had developmental impairment. Variable structural eye defects were observed in 12/24 individuals (50%) including anophthalmia, microphthalmia, colobomas, optic nerve and iris abnormalities. Additional common features included hypotonia (61%), heart defects (52%), growth failure (54%), and kidney anomalies (35%). PrediXcan analysis showed that phecodes most strongly associated with reduced predicted PRR12 expression were enriched for eye- (7/30) and kidney- (4/30) phenotypes, such as wet macular degeneration and chronic kidney disease. CONCLUSION: These findings support PRR12 haploinsufficiency as a cause for a novel disorder with a wide clinical spectrum marked chiefly by neurodevelopmental and eye abnormalities

Focusing mutagenesis into the active site to improve hydrolase selectivity

Occasionally, researchers need to modify enzymes through amino acid substitutions to make them more efficient catalysts for organic synthesis. There is still debate over the best protein engineering strategy for improving enzyme enantioselectivity: rational design or directed evolution. Rational design experiments focus mutations close to the active site, while directed evolution experiments often find mutations far from the active site.In this thesis, a combination of the two strategies improved Pseudomonas fluorescens esterase (PFE) for production of a useful synthetic building block for organic synthesis. Random mutagenesis within the active site increased enantioselectivity more effectively (up to 5-fold reaching E = 61) than random mutagenesis of the entire protein (only 1.5-fold reaching E = 19). A general survey of previously published enzyme improvements showed that closer mutations were more effective than distant mutations for improving enantioselectivity. On this basis, we proposed that random mutagenesis focused in the active site may dramatically increase the success rate in future directed evolution experiments. The X-ray crystal structures of three improved PFE mutants showed that mutations directly in the active site can increase enantioselectivity without significantly altering the shape of the binding pocket. For rationalizing the improved enantioselectivity, a crystal structure of a transition state analogue-complex provided the conformation of the fast reacting enantiomer and computer modeling determined the conformation of the slow reacting enantiomer.When novel esterases are discovered from directed evolution experiments, they are screened with libraries of esters to identify their preferred substrates. A convenient method for the parallel synthesis of esters was developed by using solid-supported reagents to eliminate traditional purification.Acetyl xylan esterase (AxeA) was examined as a potential catalyst for the production of chitosan, a biopolymer with many commercial applications. Screening for chitin deacetylase activity showed that AxeA preferentially deacetylates chitosan oligosaccharides over alkali-treated chitin and crystalline chitin

Baeyer-Villiger monooxygenases : more than just green chemistry

What is life without oxygen is a rhetorical question. On the other hand, unraveling the intricacies and understanding the various mechanisms underpinning the biological processes beg many answers. The activation of hydrocarbon C-H bonds by oxygenases exemplifies an important biological process and prerequisite to the eventual transformation of these raw materials into value-added chemicals or bioproducts. Oxygenases come in two forms: those that introduce one atom of molecular oxygen into an organic substrate, called monooxygenases (also referred to as mixed function oxygenases) and those that insert both oxygen atoms into a substrate, namely, dioxygenases. For a historical account on the discovery of oxygenases see a review by Hayaishi.(1) In monooxygenase-catalyzed reactions, the other oxygen atom undergoes reduction to water. Hence, in a biotransformation or biocatalysis setting, having water as a byproduct cannot be greener. This review focuses on the monooxygenase-catalyzed Baeyer-Villiger oxidation of linear or cyclic ketones as a green chemistry tool to address environmental sustainability, a system to study its molecular diversity and catalytic mechanism, industrial-scale bioprocess development, and a challenging model for protein engineering to evolve new biotechnological applications.Biocatalysis at large is poised to play an ever increasingly important role in meeting the needs of industrial and environmental sustainability as manufacturers and industries are striving to improve efficiency and implement cleaner processes.(2) In the context of the three pillars of sustainable development, the use of biocatalysts, as opposed to strictly harsh chemical methods, is meeting the needs of environmental care and social responsibility, although rapid economic progress requires serious financial investment.Erratum published in Volume 113, Issue 7, page 5700, June 2013. DOI: 10.1021/cr400263mPeer reviewed: YesNRC publication: Ye

Physician Wellness During a Pandemic

Introduction: We are currently in the midst of the coronavirus disease 2019 (COVID-19) pandemic. Research into previous infectious disease outbreaks has shown that healthcare workers are at increased risk for burnout during these dire times, with those on the front lines at greatest risk. The purpose of this prospective study was to determine the effect that the COVID-19 pandemic has had on the wellness of emergency physicians (EP).Methods: A survey was sent to 137 EPs in a multi-hospital network in eastern Pennsylvania. We compared 10 primary and two supplemental questions based on how the physicians had been feeling in the prior 2-3 weeks (COVID-19 period) to the same questions based on how they were feeling in the prior 4-6 months (pre-COVID-19 period).Results: We received 55 responses to the survey (40.1% response rate). The study found that during the pandemic, EPs felt less in control (p-value = 0.001); felt decreased happiness while at work (p-value 0.001); had more trouble falling asleep (p-value = 0.001); had an increased sense of dread when thinking of work needing to be done (p-value = 0.04); felt more stress on days not at work (p-value <0.0001); and were more concerned about their own health (p-value <0.0001) and the health of their families and loved ones (p-value <0.0001).Conclusion: This study showed a statistically significant decrease in EP wellness during the COVID-19 pandemic when compared to the pre-pandemic period. We need to be aware of evidence-based recommendations to help mitigate the risks and prevent physician burnout

Physician Wellness During a Pandemic

Introduction: We are currently in the midst of the coronavirus disease 2019 (COVID-19) pandemic. Research into previous infectious disease outbreaks has shown that healthcare workers are at increased risk for burnout during these dire times, with those on the front lines at greatest risk. The purpose of this prospective study was to determine the effect that the COVID-19 pandemic has had on the wellness of emergency physicians (EP).Methods: A survey was sent to 137 EPs in a multi-hospital network in eastern Pennsylvania. We compared 10 primary and two supplemental questions based on how the physicians had been feeling in the prior 2-3 weeks (COVID-19 period) to the same questions based on how they were feeling in the prior 4-6 months (pre-COVID-19 period).Results: We received 55 responses to the survey (40.1% response rate). The study found that during the pandemic, EPs felt less in control (p-value = 0.001); felt decreased happiness while at work (p-value 0.001); had more trouble falling asleep (p-value = 0.001); had an increased sense of dread when thinking of work needing to be done (p-value = 0.04); felt more stress on days not at work (p-value <0.0001); and were more concerned about their own health (p-value <0.0001) and the health of their families and loved ones (p-value <0.0001).Conclusion: This study showed a statistically significant decrease in EP wellness during the COVID-19 pandemic when compared to the pre-pandemic period. We need to be aware of evidence-based recommendations to help mitigate the risks and prevent physician burnout

Focusing Mutations into the P. fluorescens Esterase Binding Site Increases Enantioselectivity More Effectively than Distant Mutations

SummaryRational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach—random mutagenesis within the substrate binding site—is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme

Camphor pathway redux: functional recombinant expression of 2,5-and 3,6-diketocamphane monooxygenases of Pseudomonas putida ATCC 17453 with their cognate flavin reductase catalyzing Baeyer-Villiger reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km(32 \u3bcM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km=3.6 \u3bcM; kcat=283 s-\ub9) in preference to flavin adenine dinucleotide (FAD) (Km(19 \u3bcM; kcat(128 s-\ub9). Sequence determination of~40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and~533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.Peer reviewed: YesNRC publication: Ye