p-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA.

The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C8) acyl chains into structurally-opposed hydrophobic channels, denoted A and B, to carry out a non-decarboxylative Claisen condensation reaction and initiate the biosynthesis of membrane hydrocarbons and ß-lactone natural products. OleA has now been identified in hundreds of diverse bacteria via bioinformatics and high-throughput screening using p-nitrophenyl alkanoate esters as surrogate substrates. In the present study, p-nitrophenyl esters were used to probe the reaction mechanism of OleA and shown to be incorporated into Claisen condensation products for the first time. p-Nitrophenyl alkanoate substrates alone were shown not to undergo Claisen condensation, but co-incubation of p-nitrophenyl esters and CoA thioesters produced mixed Claisen products. Mixed product reactions were shown to initiate via acyl group transfer from a p-nitrophenyl carrier to the enzyme active site cysteine, C143. Acyl chains esterified to p-nitrophenol were synthesized and shown to undergo Claisen condensation with an acyl-CoA substrate, showing potential to greatly expand the range of possible Claisen products. Using p-nitrophenyl 1-13C-decanoate, the Channel A bound thioester chain was shown to act as the Claisen nucleophile, representing the first direct evidence for the directionality of the Claisen reaction in any OleA enzyme. These results both provide new insights into OleA catalysis and open a path for making unnatural hydrocarbon and ß-lactone natural products for biotechnological applications using cheap and easily synthesized p-nitrophenyl esters.

Development of the Organonitrogen Biodegradation Database: Teaching Bioinformatics and Collaborative Skills to Undergraduates during a Pandemic.

Physical distancing and inaccessibility to laboratory facilities created an opportunity to transition undergraduate research experiences to remote, digital platforms, adding another level of pedagogy to their training. Basic bioinformatics skills together with critical analysis of scientific literature are essential for addressing research questions in modern biology. The work presented here describes a fully online, collaborative research experience created to allow undergraduate students to learn those skills. The research experience was focused on the development and implementation of the Organonitrogen Biodegradation Database (ONDB, z.umn.edu/ondb). The ONDB was developed to catalog information about the cost, chemical properties, and biodegradation potential of commonly used organonitrogen compounds. A cross-institutional team of undergraduate researchers worked in collaboration with two faculty members and a postdoctoral fellow to develop the database. Students carried out extensive online literature searches and used a biodegradation prediction website to research and represent the microbial catabolism of different organonitrogen compounds. Participants employed computational tools such as R, Shiny, and flexdashboard to construct the database pages and interactive web interface for the ONDB. Worksheets and forms were created to encourage other students and researchers to gather information about organonitrogen compounds and expand the database. Student progress was evaluated through biweekly project meetings, presentations, and a final reflection. The ONDB undergraduate research experience provided a platform for students to learn bioinformatics skills while simultaneously developing a teaching and research tool for others.