(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > unclassified sequences: NE > environmental samples: NE > uncultured organism: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MKQVAWSKSRRPKSKPIFYKNLLIMMAIAVLGIAMVPVTGQAVSRVPLNA SGIYSATADDGSKIFLYRYAPYTTGTPKFNTSATPVLIFTGITMNMNQYL SCTPPDKKDVYSSVYVPPVDQAPEWALNEDKTDYEPYIKADKMRYYSLAH YLWLKGYDPWFVNYRGTGRGEVRSDGTNANSLTTLDTWATQDAPAAIAKV KSVTGKRMFIGGHSTGGLVSYDYLQGAYMESGIASSAWAKKAYYKLCYSL GYMPHVRASADLAKKRNEDVKGFIGIDPAGVPSLPNLLDTPLFWTIVGSR LYLPLDYLSDNLIQVLPSKPLVGLLEGMFGLLNKAAAGDSAISDFFDYLN FWVVEDMDPCLEDWTVRYAVGSAAIRGFGHYMDMGLNNTLREHYLNGKEN YLSSKLVKGVSAPNPGRDGYYYYSENMARMTVPMIVFSSSTGSLVSPQAT YDFIISKKTPTAYDEWYVLDGTGHFDLAMGKKMPTEMFPRLGAWLDTVDA L
The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 A resolution and revealed a modified alpha/beta hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.
Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
