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The FEBS Journal Oct 2018Poly(ethylene terephthalate) (PET) is a class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems.... (Review)
Review
Poly(ethylene terephthalate) (PET) is a class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems. Obtained from a PET-consuming bacterium Ideonella sakaiensis, the enzyme PETase exhibits superb hydrolytic activity and substrate preference toward PET. Here, we summarize some recent advances in the crystallographic analysis of PETase. These reports uncover structural features of PETase that are involved in its catalytic activity. In comparison to homologous enzymes, PETase contains an additional disulfide bond as well as an extended β8-α6 loop. More importantly, the crystal structures of PETase in complex with substrate and product analogs provide critical information for understanding the mechanism of action of PETase. In particular, the wobbling conformation of W156 is closely related to the binding of substrate and product. These new findings are of great importance for further in-depth research and engineering development of PETase, and should advance the implementation of plastic biodegradation strategy.
Topics: Bacterial Proteins; Biocatalysis; Biodegradation, Environmental; Catalytic Domain; Hydrolases; Polyethylene Terephthalates; Protein Conformation; Substrate Specificity
PubMed: 30048043
DOI: 10.1111/febs.14612 -
Critical Reviews in Biochemistry and... Feb 2018Protein depalmitoylation describes the removal of thioester-linked long chain fatty acids from cysteine residues in proteins. For many S-palmitoylated proteins, this... (Review)
Review
Protein depalmitoylation describes the removal of thioester-linked long chain fatty acids from cysteine residues in proteins. For many S-palmitoylated proteins, this process is promoted by acyl protein thioesterase enzymes, which catalyze thioester hydrolysis to solubilize and displace substrate proteins from membranes. The closely related enzymes acyl protein thioesterase 1 (APT1; LYPLA1) and acyl protein thioesterase 2 (APT2; LYPLA2) were initially identified from biochemical assays as G protein depalmitoylases, yet later were shown to accept a number of S-palmitoylated protein and phospholipid substrates. Leveraging the development of isoform-selective APT inhibitors, several studies report distinct roles for APT enzymes in growth factor and hormonal signaling. Recent crystal structures of APT1 and APT2 reveal convergent acyl binding channels, suggesting additional factors beyond acyl chain recognition mediate substrate selection. In addition to APT enzymes, the ABHD17 family of hydrolases contributes to the depalmitoylation of Ras-family GTPases and synaptic proteins. Overall, enzymatic depalmitoylation ensures efficient membrane targeting by balancing the palmitoylation cycle, and may play additional roles in signaling, growth, and cell organization. In this review, we provide a perspective on the biochemical, structural, and cellular analysis of protein depalmitoylases, and outline opportunities for future studies of systems-wide analysis of protein depalmitoylation.
Topics: Animals; Humans; Lipoylation; Models, Molecular; Monoacylglycerol Lipases; Protein Processing, Post-Translational; Proteins; Serine Proteases; Thiolester Hydrolases
PubMed: 29239216
DOI: 10.1080/10409238.2017.1409191 -
Applied and Environmental Microbiology Aug 2021Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a...
Development of a Targeted Gene Disruption System in the Poly(Ethylene Terephthalate)-Degrading Bacterium Ideonella sakaiensis and Its Applications to PETase and MHETase Genes.
Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes , we have developed a gene disruption system in . The pT18-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene () from the genome of the wild-type strain, producing the Δ strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ strain as a parent strain and as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ and Δ strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δ strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for metabolism of PET. The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many studies, including biochemical and crystal structure analyses, few studies have been reported. Here, we developed a targeted gene disruption system in , which was then applied for constructing Δ and Δ strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in , while PETase and MHETase play essential roles in its PET assimilation.
Topics: Bacterial Proteins; Burkholderiales; Ethylene Glycol; Genes, Bacterial; Hydrolases; Hydrolysis; Metabolic Engineering; Phthalic Acids; Polyethylene Terephthalates; Recycling
PubMed: 34260304
DOI: 10.1128/AEM.00020-21 -
International Journal of Molecular... Apr 2023Epoxide hydrolases are attractive and industrially important biocatalysts. They can catalyze the enantioselective hydrolysis of epoxides to the corresponding diols as... (Review)
Review
Epoxide hydrolases are attractive and industrially important biocatalysts. They can catalyze the enantioselective hydrolysis of epoxides to the corresponding diols as chiral building blocks for bioactive compounds and drugs. In this review article, we discuss the state of the art and development potential of epoxide hydrolases as biocatalysts based on the most recent approaches and techniques. The review covers new approaches to discover epoxide hydrolases using genome mining and enzyme metagenomics, as well as improving enzyme activity, enantioselectivity, enantioconvergence, and thermostability by directed evolution and a rational design. Further improvements in operational and storage stabilization, reusability, pH stabilization, and thermal stabilization by immobilization techniques are discussed in this study. New possibilities for expanding the synthetic capabilities of epoxide hydrolases by their involvement in non-natural enzyme cascade reactions are described.
Topics: Epoxide Hydrolases; Catalysis; Epoxy Compounds; Hydrolysis; Genetic Techniques; Stereoisomerism
PubMed: 37108499
DOI: 10.3390/ijms24087334 -
Glycobiology Sep 2022The substitution and de-substitution of carbohydrate materials are important steps in the biosynthesis and/or breakdown of a wide variety of biologically important... (Review)
Review
The substitution and de-substitution of carbohydrate materials are important steps in the biosynthesis and/or breakdown of a wide variety of biologically important polymers. The SGNH hydrolase superfamily is a group of related and well-studied proteins with a highly conserved catalytic fold and mechanism composed of 16 member families. SGNH hydrolases can be found in vertebrates, plants, fungi, bacteria, and archaea, and play a variety of important biological roles related to biomass conversion, pathogenesis, and cell signaling. The SGNH hydrolase superfamily is chiefly composed of a diverse range of carbohydrate-modifying enzymes, including but not limited to the carbohydrate esterase families 2, 3, 6, 12 and 17 under the carbohydrate-active enzyme classification system and database (CAZy.org). In this review, we summarize the structural and functional features that delineate these subfamilies of SGNH hydrolases, and which generate the wide variety of substrate preferences and enzymatic activities observed of these proteins to date.
Topics: Biopolymers; Carbohydrates; Esterases; Hydrolases; Protein Conformation
PubMed: 35871440
DOI: 10.1093/glycob/cwac045 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2023Plastics have brought invaluable convenience to human life since it was firstly synthesized in the last century. However, the stable polymer structure of plastics led to... (Review)
Review
Plastics have brought invaluable convenience to human life since it was firstly synthesized in the last century. However, the stable polymer structure of plastics led to the continuous accumulation of plastic wastes, which poses serious threats to the ecological environment and human health. Poly(ethylene terephthalate) (PET) is the most widely produced polyester plastics. Recent researches on PET hydrolases have shown great potential of enzymatic degradation and recycling of plastics. Meanwhile, the biodegradation pathway of PET has become a reference model for the biodegradation of other plastics. This review summarizes the sources of PET hydrolases and their degradation capacity, degradation mechanism of PET by the most representative PET hydrolase-PETase, and recently reported highly efficient degrading enzymes through enzyme engineering. The advances of PET hydrolases may facilitate the research on the degradation mechanism of PET and further exploration and engineering of efficient PET degradation enzymes.
Topics: Humans; Hydrolases; Polyethylene Terephthalates; Plastics; Ethylenes
PubMed: 37212227
DOI: 10.13345/j.cjb.220915 -
Biochimica Et Biophysica Acta Jun 2011The histidine triad (HIT) superfamily comprises proteins that share the histidine triad motif, His-ϕ-His-ϕ-His-ϕ-ϕ, where ϕ is a hydrophobic amino acid. HIT... (Review)
Review
The histidine triad (HIT) superfamily comprises proteins that share the histidine triad motif, His-ϕ-His-ϕ-His-ϕ-ϕ, where ϕ is a hydrophobic amino acid. HIT proteins are ubiquitous in prokaryotes and eukaryotes. HIT proteins bind nucleotides and exert dinucleotidyl hydrolase, nucleotidylyl transferase or phosphoramidate hydrolase enzymatic activity. In humans, 5 families of HIT proteins are recognized. The accumulated epidemiological and experimental evidence indicates that two branches of the superfamily, the HINT (Histidine Triad Nucleotide Binding) members and FHIT (Fragile Histidine Triad), have tumor suppressor properties but a conclusive physiological role can still not be assigned to these proteins. Aprataxin forms another discrete branch of the HIT superfamily, is implicated in DNA repair mechanisms and unlike the HINT and FHIT members, a defective protein can be conclusively linked to a disease, ataxia with oculomotor apraxia type 1. The scavenger mRNA decapping enzyme, DcpS, forms a fourth branch of the HIT superfamily. Finally, the GalT enzymes, which exert specific nucleoside monophosphate transferase activity, form a fifth branch that is not implicated in tumorigenesis. The molecular mechanisms by which the HINT and FHIT proteins participate in bioenergetics of cancer are just beginning to be unraveled. Their purported actions as tumor suppressors are highlighted in this review.
Topics: Amino Acid Sequence; Animals; Humans; Hydrolases; Mitochondria; Models, Biological; Molecular Sequence Data; Multigene Family; Neoplasms; Sequence Homology, Amino Acid
PubMed: 21316334
DOI: 10.1016/j.bbabio.2011.02.001 -
Biotechnology Advances 2023Endo-1,4-β-xylanases (EC 3.2.1.8) are O-glycoside hydrolases that cleave the internal β-1,4-D-xylosidic linkages of the complex plant polysaccharide xylan. They are... (Review)
Review
Endo-1,4-β-xylanases (EC 3.2.1.8) are O-glycoside hydrolases that cleave the internal β-1,4-D-xylosidic linkages of the complex plant polysaccharide xylan. They are produced by a vast array of organisms where they play critical roles in xylan saccharification and plant cell wall hydrolysis. They are also important industrial biocatalysts with widespread application. A large and ever growing number of xylanases with wildly different properties and functionalites are known and a better understanding of these would enable a more effective use in various applications. The Carbohydrate-Active enZYmes database (CAZy), which classifies evolutionarily related proteins into a glycoside hydrolase family-subfamily organisational scheme has proven powerful in understanding these enzymes. Nevertheless, ambiguity currently exists as to the number of glycoside hydrolase families and subfamilies harbouring catalytic domains with true endoxylanase activity and as to the specific characteristics of each of these families/subfamilies. This review seeks to clarify this, identifying 9 glycoside hydrolase families containing enzymes with endo-1,4-β-xylanase activity and discussing their properties, similarities, differences and biotechnological perspectives. In particular, substrate specificities and hydrolysis patterns and the structural determinants of these are detailed, with taxonomic aspects of source organisms being also presented. Shortcomings in current knowledge and research areas that require further clarification are highlighted and suggestions for future directions provided. This review seeks to motivate further research on these enzymes and especially of the lesser known endo-1,4-β-xylanase containing families. A better understanding of these enzymes will serve as a foundation for the knowledge-based development of process-fitted endo-1,4-β-xylanases and will accelerate their development for use with even the most recalcitrant of substrates in the biobased industries of the future.
Topics: Humans; Endo-1,4-beta Xylanases; Glycoside Hydrolases; Xylans; Substrate Specificity; Bacterial Proteins
PubMed: 37030552
DOI: 10.1016/j.biotechadv.2023.108148 -
Nature Communications Apr 2023The recently discovered metagenomic-derived polyester hydrolase PHL7 is able to efficiently degrade amorphous polyethylene terephthalate (PET) in post-consumer plastic...
The recently discovered metagenomic-derived polyester hydrolase PHL7 is able to efficiently degrade amorphous polyethylene terephthalate (PET) in post-consumer plastic waste. We present the cocrystal structure of this hydrolase with its hydrolysis product terephthalic acid and elucidate the influence of 17 single mutations on the PET-hydrolytic activity and thermal stability of PHL7. The substrate-binding mode of terephthalic acid is similar to that of the thermophilic polyester hydrolase LCC and deviates from the mesophilic IsPETase. The subsite I modifications L93F and Q95Y, derived from LCC, increased the thermal stability, while exchange of H185S, derived from IsPETase, reduced the stability of PHL7. The subsite II residue H130 is suggested to represent an adaptation for high thermal stability, whereas L210 emerged as the main contributor to the observed high PET-hydrolytic activity. Variant L210T showed significantly higher activity, achieving a degradation rate of 20 µm h with amorphous PET films.
Topics: Hydrolases; Plastics; Phthalic Acids; Polyethylene Terephthalates
PubMed: 37019924
DOI: 10.1038/s41467-023-37415-x -
Microbial Cell Factories Jun 2022Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET)....
BACKGROUND
Biocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.
RESULTS
P. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.
CONCLUSION
Our results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.
Topics: Biocatalysis; Hydrolases; Plastics; Polyethylene Terephthalates; Pseudomonas putida
PubMed: 35717313
DOI: 10.1186/s12934-022-01849-7