Characterization and engineering of a two-enzyme system for plastics depolymerization.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Recognition of a pseudo-symmetric RNA tetranucleotide by Csx3, a new member of the CRISPR associated Rossmann fold superfamily.

The CRISPR/Cas adaptive immune system shows extreme diversity in the number of CRISPR/Cas types and subtypes, and in the multitude of CRISPR associated protein families of which they are composed. Despite this diversity, the roles of many Cas protein families are now defined with regard to spacer acquisition, crRNA biogenesis, and DNA or RNA surveillance and targeting. However, a number of unclassified CRISPR-Cas proteins remain. Such proteins have traditionally been designated as CRISPR subtype x (Csx). Here we revisit the structural analysis of one such protein, Csx3, and show that this homodimeric protein utilizes a Rossmann fold for the recognition of an RNA tetranucleotide. Tertiary and quaternary structural similarities of Csx3 to CRISPR/Cas proteins Csx1 and Csa3 are identified and suggest Csx3 is a new member of the CRISPR Associated Rossmann Fold (CARF) superfamily. The structure of the Csx3/RNA complex illustrates one way CARF domain proteins may recognize pseudo-symmetric polynucleotides.