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Appl Environ Microbiol. 2019 Jan 18. pii: AEM.03029-18. doi: 10.1128/AEM.03029-18. [Epub ahead of print]

Insights into the function of the N-acetyltransferase SatA that detoxifies streptothricin in Bacillus subtilis and Bacillus anthracis.

Author information

1
Department of Microbiology, University of Georgia, Athens.
2
Department of Microbiology, University of Georgia, Athens jcescala@uga.edu.

Abstract

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the Gcn5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactive streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA, a mutational screen was performed, highlighting the importance of a conserved area near the C-terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (PDB 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region could contain the streptothricin-binding site. Chemical and site-directed mutagenesis were used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry (ITC) experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Result of size-exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic, and probes the contributions of substituting side chains to those in the native protein, providing information regarding hydrophobicity, size and flexibility of the antibiotic binding site.

PMID:
30658980
DOI:
10.1128/AEM.03029-18

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