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 ).