| Summary: | S-formylglutathione hydrolases (SFGHs) catalyze the hydrolysis of S-formylglutathione to formate and glutathione using the conserved serine hydrolase catalytic triad residues (Ser-His-Asp). SFGHs have broad substrate specificity, including, for example, ester bond-containing substrates. Here, we report the crystal structure of <i>Burkholderiaceae</i> sp. SFGH (<i>Bu</i>SFGH) at 1.73 Å resolution. Structural analysis showed that the overall structure of <i>Bu</i>SFGH has a typical α/β hydrolase fold, with a central β-sheet surrounded by α-helices. Analytical ultracentrifugation analysis showed that <i>Bu</i>SFGH formed a stable dimer in solution. The enzyme activity assay indicated that <i>Bu</i>SFGH has a high preference for short-chain <i>p</i>-nitrophenyl esters, such as <i>p</i>-nitrophenyl acetate. The activity of <i>Bu</i>SFGH toward <i>p</i>-nitrophenyl acetate was five times higher than that of <i>p</i>-nitrophenyl butylate. Molecular modeling studies on the <i>p</i>-nitrophenyl acetate-bound <i>Bu</i>SFGH structure indicate that Gly52, Leu53, Trp96, His147, Ser148, Trp182, Phe228, and His259 residues may be crucial for substrate binding. Collectively, these results are useful for understanding the substrate-binding mechanism and substrate specificity of <i>Bu</i>SFGH. They can also provide useful insights for designing modified <i>Bu</i>SFGHs with different substrate specificities.
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