Identification and Quantification of Active Bacterial Monooxygenases in Environmental Samples Using Activity-Based Protein Profiling (ABPP)
EMSL Project ID
48372
Abstract
Bacterial monooxygenases are enzymes the initiate the aerobic biodegradation of a wide variety of environmentally important chemicals. These include key intermediates in the biogeochemical cycles of carbon (methane), nitrogen (ammonia), and sulfur (dimethylsulfide), as well as diverse environmental contaminants including petroleum hydrocarbons, chlorinated solvents and many ether-bonded compounds. The aim of this project is to develop an activity based protein profiling (ABPP) methodology that will enable the accurate identification and quantification of catalytically active forms of these enzymes in environmental samples such as soils and sediments. The ability to detect, identify and quantify active monooxygenases at the protein level would represent an important advance for microbial ecologists as current methodologies are largely restricted to detecting the potential for these activities through gene-based approaches. If successful, the proposed ABPP methodology has the potential to have widespread use in studies ranging from the role of microorganisms in trace gas and nutrient cycling to bioremediation of widespread organic pollutants. Using model organisms, we have obtained preliminary evidence that all of the major steps in the proposed methodology are feasible. The current project aims to expand these observations to develop a robust and validated method that can be applied to many environmental sample types. This project requests the combined use of facilities, analytical capabilities and collaborations that are uniquely available through EMSL and PNNL. Specific resources that we are requesting include 1) proteomics analysis using Orbitrap and Velos Orbitrap and 2) fluorescence-assisted cell sorting with Influx. The project involves collaboration between North Carolina State University, Los Alamos National Laboratory and Pacific Northwest National Laboratory and builds on respective expertise in microbial biochemistry, environmental microbiology and advanced methods for enzyme detection and identification.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2014-10-01
End Date
2016-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members
Related Publications
Bennett K, NC Sadler, AT Wright, C Yeager, and MR Hyman. 2016. "Activity-based protein profiling of ammonia monooxygenase in Nitrosomonas europaea." Applied and Environmental Microbiology 82(8):2270-2279. doi:10.1128/AEM.03556-15