Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens .

Nosocomial infections are serious threats to the entire world in healthcare settings. The major causative agents of nosocomial infections are bacterial pathogens, among which Enterobacteriaceae family member Serratia marcescens plays a crucial role. It is a gram-negative opportunistic pathogen, predominantly affecting patients in intensive-care units. The presence of intrinsic genes in S. marcescens led to the development of resistance to antibiotics for survival. Complete scanning of the proteome, including hypothetical and partially annotated proteins, paves the way for a better understanding of potential drug targets. The targeted protein expressed in E. coli BL21 (DE3) pLysS cells has shown complete resistance to aminoglycoside antibiotic streptomycin (>256 MCG). The recombinant protein was purified using affinity and size-exclusion chromatography and characterized using SDS-PAGE, western blotting, and MALDI-TOF analysis. Free phosphate bound to malachite green was detected at 620 nm, evident of the conversion of adenosine triphosphate to adenosine monophosphate during the adenylation process. Similarly, in the chromatographic assay, adenylated streptomycin absorbed at 260 nm in AKTA (FPLC), confirming the enzyme-catalyzed adenylation of streptomycin. Further, the adenylated product of streptomycin was confirmed through HPLC and mass spectrometry analysis. In conclusion, our characterization studies identified the partially annotated hypothetical protein as streptomycin adenylyltransferase.