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.

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 CRISPRCas 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. Yan et al. recently published "Crystal structures of CRISPRassociated Csx3 reveal a manganese-dependent deadenylation exoribonuclease" in RNA Biology. 1 In this research article the authors present compelling evidence that Csx3 possesses manganese dependent RNase activity and also present crystal structures showing that the manganese and RNA binding sites (Fig. 1A) lie at opposite ends of the Csx3 homodimer. In all, their work represents a significant advance in our understanding of Csx3, providing critical new information relevant to its potential roles in CRISPR/Cas.They also describe the fold of Csx3 (Fig. 1B) as a "ferredoxin-like" fold. At first glance, it is not surprising that Csx3 might also harbor a ferredoxin-like fold. The ferredoxin-like fold (Fig. 1C) is commonly observed in proteins involved in CRISPR-Cas, where it may also be referred to as an RRM (RNA Recognition Motif) or RAMP (Repeat Associated Mysterious Protein) domain. RAMP domains are found in the Cas5, Cas6, Cas7 and Cmr3 families and RAMP-like domains are also found in Cas2 and Cas10. 2,3 However, the ferredoxin-like fold is defined in the SCOP database 4 (and elsewhere) as a babbab or (bab) 2 fold, resulting in a 4 stranded antiparallel b sheet arranged as b 2 b 3 b 1 b 4 . The two helices, present as righthanded crossovers between the b strands, lie on the same face of the b-sheet where they run antiparallel to each other (Fig. 1C). In this context, the Csx3 structure does indeed show a b-sheet flanked on one side by 2 a-helices. But in contrast to the ferredoxin-like fold, Csx3 lacks a 4-stranded antiparallel b sheet, the helices run antiparallel to each other, and the order and connectivity between the b-strands is substantially different (Fig. 1B).The Csx3 fold can, however, be described as a bbababbb(a) structure resulting in a 6-stranded mixed b-sheet in which the b-strands run sequentially across the sheet from b1-b6 (right to left in Fig. 1B). In addition, as a result of right-handed crossover connections provided by helices a1...