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We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions.As of September 19, 2020, about 30 million people have been infected with the novel corona virus disease 2019 (COVID-19) globally, and the