BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF A DIENELACTONE HYDROLASE (DLH) FROM Hydrogenobacter thermophilus WITH ACTIVITY ON TEREPHTHALATE ESTERS

Dienolactone hydrolases (DLHs) are enzymes that catalyze the hydrolysis of dienelactone to maleylacetate. DLHs have significant potential in biotechnological applications due to their ability to degrade complex aromatic compounds into simpler and less toxic products. DLH can be useful in the bioremediation of environmental pollutants, such as petroleum-derived compounds and toxic organic solvents found in wastewater. A recent study has highlighted the discovery of a new bacterium, Ideonella sakaiensis 201-F6, which has a unique ability to utilize polyethylene terephthalate (PET) as its carbon source. This process occurs through the coordinated action of two enzymes, PETase and MHETase. Enzymatic depolymerization of PET presents a possible sustainable solution to the plastic accumulation problem. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67 Å, exhibits a canonical /-hydrolase fold formed by eight -sheet strands in the core, with one buried -helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 ºC. Furthermore, HtDLH maintains more than 50% of its activity even after incubation at 90 ºC for 16 h. Interestingly, HtDLH exhibits catalytic activity towards PET monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate (HMT), as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This study can provide novel insights for plastic recycling through protein engineering, which could lead to innovative solutions with biotechnological and environmental applications.