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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, pred...

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Autores principales: Prabhu, Dhamodharan, Rajamanikandan, Sundararaj, Amala, Mathimaran, Saritha, Poopandi, Jeyakanthan, Jeyaraman, Ramasamy, Palaniappan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9774460/
https://www.ncbi.nlm.nih.gov/pubmed/36551379
http://dx.doi.org/10.3390/antibiotics11121722
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author Prabhu, Dhamodharan
Rajamanikandan, Sundararaj
Amala, Mathimaran
Saritha, Poopandi
Jeyakanthan, Jeyaraman
Ramasamy, Palaniappan
author_facet Prabhu, Dhamodharan
Rajamanikandan, Sundararaj
Amala, Mathimaran
Saritha, Poopandi
Jeyakanthan, Jeyaraman
Ramasamy, Palaniappan
author_sort Prabhu, Dhamodharan
collection PubMed
description 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.
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spelling pubmed-97744602022-12-23 Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens Prabhu, Dhamodharan Rajamanikandan, Sundararaj Amala, Mathimaran Saritha, Poopandi Jeyakanthan, Jeyaraman Ramasamy, Palaniappan Antibiotics (Basel) Article 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. MDPI 2022-11-30 /pmc/articles/PMC9774460/ /pubmed/36551379 http://dx.doi.org/10.3390/antibiotics11121722 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Prabhu, Dhamodharan
Rajamanikandan, Sundararaj
Amala, Mathimaran
Saritha, Poopandi
Jeyakanthan, Jeyaraman
Ramasamy, Palaniappan
Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens
title Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens
title_full Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens
title_fullStr Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens
title_full_unstemmed Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens
title_short Functional Characterization, Mechanism, and Mode of Action of Putative Streptomycin Adenylyltransferase from Serratia marcescens
title_sort functional characterization, mechanism, and mode of action of putative streptomycin adenylyltransferase from serratia marcescens
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9774460/
https://www.ncbi.nlm.nih.gov/pubmed/36551379
http://dx.doi.org/10.3390/antibiotics11121722
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