Crystal structure and biochemical analysis of the polyester-degrading carboxylesterase SM0281 from the symbiotic legume-associated rhizobacterium Sinorhizobium (Ensifer) meliloti

The microbial degradation of synthetic polyesters such as polyethyleneterephthalate (PET) is mediated by diverse a/b-hydrolases, many of which remain poorly characterized. In this study, we investigated SM0281, an uncharacterized a/b-hydrolase from the symbiotic legume-associated rhizo-bacterium Sinorhizobium meliloti. Sequence analysis revealed that SM0281shares low similarity with known polyester-degrading enzymes. Biochemical characterization of purified SM0281 demonstrated that it is a carboxylesterase with preference for medium-chain length monoester substrates(C4–C8) and displays broad tolerance to pH, salts, glycerol, and organic solvents, with maximal activity at 30 °C and notable cold tolerance. In addition to monoesters, SM0281 hydrolyzed the PET model substratebis (benzoyloxyethyl) terephthalate (3PET), producing predominantly mono(2-hydroxyethyl) terephthalate (MHET), and showed detectable activity toward emulsified PET, polycaprolactone (PCL), and poly(D,L-lactide)(PLA). The crystal structure of SM0281 was determined at 2.46 A resolution and revealed a classical a/b-hydrolase core domain associated with a small lid domain positioned above the catalytic Ser121. Three polyethyleneglycol (PEG) molecules were observed bound to the lid domain, with one molecule (PEG1) occupying a tunnel-like cavity connecting the protein sur-face to the active site and suggesting a potential route for polyester binding. Structure-guided mutational analysis identified several residues from both the core domain (Tyr53, Arg54, Asp230) and the lid domain (Phe160,Leu164, Ile165, Phe183, Phe196) that contribute to catalytic activity toward monoester and polyester substrates. Together, these results expand the diversity of structurally characterized polyester-active a/b-hydrolases containing lid domains and provide insights into the molecular determinants of substrate recognition and hydrolysis in these enzymes.