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Background   S -Formylglutathione is hydrolyzed to glutathione and formate by an  S -formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering.    Results  A novel cold-active  S -formylglutathione hydrolase ( Sf SFGH) from  Shewanella frigidimarina , composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of  Sf SFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that  Sf SFGH was active towards short-chain esters, such as  p -nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of  Sf SFGH, we determined the crystal structure of  Sf SFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to  Sf SFGH. Moreover,  Sf SFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea.    Conclusions  Mutation of Trp182 to Ala allowed  Sf SFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in  Sf SFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type  Sf SFGH. Taken together, this study provides useful structure–function data of a SFGH family member and may inform protein engineering strategies for industrial applications of  Sf SFGH.    Electronic supplementary material  The online version of this article (10.1186/s12934-019-1190-1) contains supplementary material, which is available to authorized users.

microbial cell factories

Structural and functional characterization of a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina, a psychrophilic bacterium