The actinobacterium Tsukamurella paurometabola has a functionally divergent arylamine N-acetyltransferase (NAT) homolog
Actinobacteria in the Tsukamurella genus are aerobic, high-GC, Gram-positive mycolata, considered as opportunistic pathogens and isolated from various environmental sources, including sites contaminated with oil, urban or industrial waste and pesticides. Although studies look into xenobiotic biotransformation by Tsukamurella isolates, the relevant enzymes remain uncharacterized. We investigated the arylamine N-acetyltransferase (NAT) enzyme family, known for its role in the xenobiotic metabolism of prokaryotes and eukaryotes. Xenobiotic sensitivity of Tsukamurella paurometabola type strain DSM 20162T was assessed, followed by cloning, recombinant expression and functional characterization of its single NAT homolog (TSUPD)NAT1. The bacterium appeared quite robust against chloroanilines, but more sensitive to 4-anisidine and 2-aminophenol. However, metabolic activity was not evident towards those compounds, presumably due to mechanisms protecting cells from xenobiotic entry. Of the pharmaceutical arylhydrazines tested, hydralazine was toxic, but the bacterium was less sensitive to isoniazid, a drug targeting mycolic acid biosynthesis in mycobacteria. Although (TSUPD)NAT1 protein has an atypical Cys-His-Glu (instead of the expected Cys-His-Asp) catalytic triad, it is enzymatically active, suggesting that this deviation is likely due to evolutionary adaptation potentially serving a different function. The protein was indeed found to use malonyl-CoA, instead of the archetypal acetyl-CoA, as its preferred donor substrate. Malonyl-CoA is important for microbial biosynthesis of fatty acids (including mycolic acids) and polyketide chains, and the corresponding enzymatic systems have common evolutionary histories, also linked to xenobiotic metabolism. This study adds to accummulating evidence suggesting broad phylogenetic and functional divergence of microbial NAT enzymes that goes beyond xenobiotic metabolism and merits investigation.
KeywordsAcylated dichloroaniline derivatives Malonyl-coenzyme A N-acetyltransferase NAT enzyme family N-malonyltransferase Tsukamurella paurometabola Xenobiotics
This research was partly funded by a Joint Research & Technology 2009 Programme between Greece and Hungary (Grant No. HUN40), co-financed by Greece, Hungary and the European Union (European Regional Development Fund-ERDF) through Operational Program “Competitiveness and Entrepreneurship” in the context of project “Bilateral, Multilateral and Regional R&T Cooperation” implemented by the Greek General Secretariat for Research and Technology (GSRT) and the Hungarian National Office for Research and Technology (NKTH), and conducted by the two partners in 2012–2014. E.K. is recipient of a Ph.D. scholarship (2016–2019) and her research is co-financed by Greece and the European Union (European Social Fund-ESF) through Operational Program “Human Resources Development, Education and Lifelong Learning” in the context of project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432), implemented by the State Scholarships Foundation (ΙΚΥ). None of the funding bodies had any involvement in the study design, in the collection, analysis or interpretation of data, in the writing of the report or the decision to submit the article for publication.
VG performed the bulk of experimental procedures and data analyses; EK contributed to xenobiotic sensitivity tests and TLC assays; CI contributed to enzyme kinetic assays; OS, CVP and MGP contributed to DSF assays; IO and DP contributed to computational work; KF synthesized and characterised chemical compounds; TF, KM, GF and SB generated the biological material used and provided expert scientific input; KM, GF and SB conceived of, designed and pursued funding for the study; SB supervised the project, analysed data and wrote the manuscript. The materials and datasets generated during the study are available from SB on reasonable request. All authors reviewed the manuscript.
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Conflict of interest
The authors declare that they have no conflict of interest.
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