Analysis of enzymatic transformation of trace contaminants through proteomics approaches

Microorganisms play a crucial role in environmental remediation by enzymatically transforming contaminants into less toxic or structurally simpler compounds. Understanding these metabolic pathways is essential for developing effective bioremediation strategies. Among multi-omics approaches, proteomics provides unique functional insights by directly identifying and characterizing catalytic proteins involved in contaminant transformation. This research applied proteomic approaches to investigate microbial transformation of emerging contaminants using bacterial strains. Bosea sp. 100-5 and Shinella sp. WSC3 were studied for degradation of the artificial sweetener acesulfame, while Nitratidesulfovibrio vulgaris Hildenborough (NvH) was investigated for transformation of the antibiotic sulfamethoxazole (SMX). In Bosea sp. 100-5 grown with acesulfame as the sole carbon source, proteomic analysis revealed high expression of a Mn²⁺-dependent metallo-β-lactamase-type sulfatase (BOSEA1005₄0015) and an amidase (BOSEA1005₄0030). Coupling proteomics with size-exclusion chromatography (SEC) and activity assays confirmed the sulfatase as indispensable for acesulfame hydrolysis. To extend the methods to investigate microbial contaminant transformation systems, limited proteolysis–mass spectrometry (LiP-MS) was adapted from pharmaceutical research to environmental systems. An optimized LiP-MS workflow incorporating multiple contaminant concentrations and data-independent acquisition (DIA) successfully identified both an acesulfame-specific transporter and the sulfatase in Bosea sp. 100-5, demonstrating the necessity of methodological optimization for environmental applications. In Shinella strains from wastewater treatment plants, a novel formylglycine-dependent sulfatase encoded on a plasmid was identified and functionally validated. Metagenomic screening of 73 terabases of wastewater data revealed distinct temporal and geographical patterns for Bosea and Shinella acesulfame sulfatase gene signatures. For NvH, multidimensional fractionation combined with activity assays indicated that SMX transformation likely involves multiple enzymatic systems linked to electron transport components, particularly the dissimilatory sulfite reductase complex. Overall, this work demonstrates that integrated proteomic and activity-based approaches provide a powerful framework for identifying microbial enzymes involved in contaminant biotransformation and for linking laboratory discoveries to ecosystem-scale processes.