Sequence annotations of plastics-active enzymes
Enzymes of different EC classes have been proposed to contribute to degradation of PET or PUR. α/β-hydrolases annotated as cutinases, esterases, or lipases were described to catalyze the hydrolysis of ester bonds and were collected in the PMBD 14, and peroxidases and laccases have been reported to enhance degradation of PUR 49. For most types of plastics, however, knowledge on enzymatic degradation is missing, although materials such as polypropylene (PP) are produced in large scales and contribute extensively to global plastics pollution. In this paper, we focus on PET- and PUR-degrading α/β-hydrolases due to the availability of sequence information and detailed experimental data from literature and public databases 14.
Many PETase homologues were found in the NCBI non-redundant protein database, due to the sequencing efforts of the cutinase-expressing bacterial phyla such as Actinobacteria and Proteobacteria. In contrast, sequences from fungal origin are unrepresented. The usage of metagenomics is expected to further broaden the scope of currently known PETases 50. The PETase sequence motif suggested herein (Table 2 ) is based on current knowledge from literature, such as the occurrence of an additional flexible loop region and amino acids that seem relevant for interaction with PET. The seven suggested PETase candidates can be used as starting points for wet-lab experiments, such as protein design experiments for improved PETase activity or thermostability.
Although PETases and PURases share the α/β-hydrolase fold as catalytic domain, their structure and their oxyanion hole types differ. All the 2930 PETase homologues belong to a large family (36936 sequences) of GX-types, 37 which consist of the core domain only (superfamily 1 in the Lipase Engineering Database,20), whereas PURases belong to either of three superfamilies: superfamily 1, superfamily 11, which consists of GX-types with two additional domains, an N-terminal mobile lid and a C-terminal β-sandwich domain (2054 sequences), or to superfamily 13 , which are carboxylesterases of the GGGX-type (44 sequences). Ample structural information is available for PET-degrading α/β-hydrolases, especially for LCC and Is PETase, whereas the structures of PUR-degrading α/β-hydrolases have not been resolved, yet. The former has also inspired the design of improved PETase variants, as recently demonstrated for variants based on LCC 40. Previously, conserved subsite I, subsite II, and an extended loop region were identified inIs PETase and used for a systematic comparison with its homologues39, which was confirmed by our conservation analysis of 2930 protein sequences. The profile HMM for PETases and the derived standard numbering scheme is available at the PAZy. It will help in the identification and comparison of amino acid positions reported in literature and will facilitate the design of new PETase variants. For PURases, homology models predict substrate binding regions44,45. Recently, a putative PURases was identified in the Proteobacterium Serratia liquefaciens 51. Similarly, most putative PURases in the PAZy are from Proteobacteria (mainly from Pseudomonas chlororaphis and Delftia acidovorans ).