Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage ...offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.
Widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems; thus, the enzymatic degradation of PET can be a promising solution. Although ...PETase from Ideonalla sakaiensis (IsPETase) has been reported to have the highest PET degradation activity under mild conditions of all PET-degrading enzymes reported to date, its low thermal stability limits its ability for efficient and practical enzymatic degradation of PET. Using the structural information on IsPETase, we developed a rational protein engineering strategy using several IsPETase variants that were screened for high thermal stability to improve PET degradation activity. In particular, the IsPETaseS121E/D186H/R280A variant, which was designed to have a stabilized β6-β7 connecting loop and extended subsite IIc, had a T m value that was increased by 8.81 °C and PET degradation activity was enhanced by 14-fold at 40 °C in comparison with IsPETaseWT. The designed structural modifications were further verified through structure determination of the variants, and high thermal stability was further confirmed by a heat-inactivation experiment. The proposed strategy and developed variants represent an important advancement for achieving the complete biodegradation of PET under mild conditions.
Amylomaltase is an essential enzyme in maltose utilization and maltodextrin metabolism, and it has been industrially used for the production of cyclodextrin and modification of starch. We determined ...the crystal structure of amylomaltase from Corynebacterium glutamicum (CgAM) at a resolution of 1.7 Å. Although CgAM forms a dimer without NaCl, it exists as a monomer in physiological concentration of NaCl. CgAM is composed of N- and C-terminal domains, which can be further divided into two and four subdomains, respectively. It exhibits a unique structural feature at the functionally unknown N-domain and also shows two striking differences at the C-domain compared to other amylomaltases. These differences at extended edge of the substrate-binding site might affect substrate specificity for large cyclodextrin formation. The bis-tris methane and sulfate molecules bound at the substrate-binding site of our current structure mimic the binding of the hydroxyl groups of glucose bound at subsites −1 and −2, respectively.
•To optimize the substrate binding site of IsPETase, we compared the residues constituting the substrate binding site with those from other PETase candidates.•IsPETaseS242T and IsPETaseN246D variants ...showed increased PET degradation activity.•We introduced S242 T and N246D to the IsPETaseS121E/D186H/R280A variant developed in the previous paper and generated the IsPETaseS121E/D186H/S242T/N246D variant with PET degradation activity increased by 58-fold compared to IsPETaseWT.
Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70 °C. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETaseS242T and IsPETaseN246D, with higher enzymatic activity at both 25 and 37 °C compared with IsPETaseWT. We also developed the IsPETaseS121E/D186H/S242T/N246D variant by integrating the S242 T and N246D mutations into the previously reported IsPETaseS121E/D186H/R208A variant. At the 37 °C incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETaseWT that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETaseWT.
Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET ...hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SPMalE:IsPETase and pET22b-SPLamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.
•PETase from Ideonella sakaiensis (IsPETase) was successfully produced using the protein secretory expression system.•Extracellular production of IsPETase is achieved by sec-dependent secretion system.•The extracellularly produced IsPETase shows a PET degradation activity.
Monohydroxyethyl terephthalate (MHET) hydrolase (MHETase) is an enzyme known to be involved in the final degradation step of poly(ethylene terephthalate) (PET) by hydrolyzing MHET into terephthalic ...acid and ethylene glycol in Ideonella sakaiensis. Here, we report the extracellular production of MHETase in an active form with a proper folding. Based on the structural observations and biochemical experiments, we reveal that MHETase also functions as exo-PETase by hydrolyzing the synthesized PET pentamer. We further present that MHETase has a hydrolysis activity against the termini-generated PET film, demonstrating the exo-PETase function of the enzyme. We also develop a MHETaseR411K/S416A/F424I variant with a higher BHET activity, and the variant exhibits an enhanced degradation activity against the PET film. Based on these results, we propose that MHETase plays several roles in the biodegradation of PET using the BHETase and exo-PETase activities as well as the MHET hydrolysis function.
Cysteine is a semiessential amino acid and plays an important role in metabolism and protein structure and has also been applied in various industrial fields, such as pharmaceutical, food, cosmetic, ...and animal feed industries. Metabolic engineering studies have been conducted for the cysteine production through bacterial fermentation, but studies on the cysteine biosynthetic pathway in microorganisms are limited. We report the biochemical characteristics of cystathionine γ-lyase from Bacillus cereus ATCC 14579 (BcCGL). We also determined the crystal structure of BcCGL in complex with the PLP cofactor and identified the substrate binding mode. We observed that the replacement of the conserved Glu321 residue to alanine showed increased activity by providing wider active site entrance and hydrophobic interaction for the substrate. We suggest that the structural differences of the α13−α14 region in CGL enzymes might determine the active site conformation.
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