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.
The development of a superb polyethylene terephthalate (PET) hydrolyzing enzyme requires an accurate understanding of the PET decomposition mechanism. However, studies on PET degrading enzymes, ...including the PET hydrolase from Ideonella sakaiensis (IsPETase), have not provided sufficient knowledge of the molecular mechanisms for the hardly accessible substrate. Here, we report a novel PET hydrolase from Rhizobacter gummiphilus (RgPETase), which has a hydrolyzing activity similar to IsPETase toward microcrystalline PET but distinct behavior toward low crystallinity PET film. Structural analysis of RgPETase reveals that the enzyme shares the key structural features of IsPETase for high PET hydrolysis activity but has distinguished structures at the surface-exposed regions. RgPETase shows a unique conformation of the wobbling tryptophan containing loop (WW-loop) and change of the electrostatic surface charge on the loop dramatically affects the PET-degrading activity. We further show that effect of the electrostatic surface charge to the activity varies depending on locations. This work provides valuable information underlying the uncovered PET decomposition mechanism.
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•Identification of a mesophilic PET hydrolase from Rhizobacter gummiphilus.•Report on distinct behavior of homologous PET hydrolases toward different PET samples.•Structural comparison of the mesophilic PET hydrolases implying uncovered mechanism.
•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.
Structural Insights into Polyhydroxyalkanoates Biosynthesis Sagong, Hye-Young; Son, Hyeoncheol Francis; Choi, So Young ...
Trends in biochemical sciences (Amsterdam. Regular ed.),
October 2018, 2018-10-00, 20181001, Letnik:
43, Številka:
10
Journal Article
Recenzirano
Polyhydroxyalkanoates (PHAs) are diverse biopolyesters produced by numerous microorganisms and have attracted much attention as a substitute for petroleum-based polymers. Despite several decades of ...study, the detailed molecular mechanisms of PHA biosynthesis have remained unknown due to the lack of structural information on the key PHA biosynthetic enzyme PHA synthase. The recently determined crystal structure of PHA synthase, together with the structures of acetyl-coenzyme A (CoA) acetyltransferase and reductase, have changed this situation. Structural and biochemical studies provided important clues for the molecular mechanisms of each enzyme as well as the overall mechanism of PHA biosynthesis from acetyl-CoA. This new information and knowledge is expected to facilitate production of designed novel PHAs and also enhanced production of PHAs.
Polyhydroxyalkanoates (PHAs) are natural polyesters consisting of various hydroxyalkanoates (HAs) and have attracted much attention as a feasible substitute to conventional petroleum-based plastics. The PHA biosynthetic pathway consists of three enzymes: acetyl-CoA acetyltransferase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC).
PhaA catalyzes the two-step condensation reaction of acetyl-CoA to acetoacetyl-CoA, and the size of the binding pocket for acyl-S-enzyme intermediate determines the substrate specificity of the enzyme. Acetoacetyl-CoA is reduced to (R)-3-hydroxybutyryl-CoA by PhaB using NADPH as a cofactor.
PHA polymerization reaction takes place at the C-terminal domain of PhaC via non-processive ping-pong mechanism, and the N-terminal domain of PhaC plays an important role in the binding and localization of PhaC to the PHA granule.
PhaM binds to the N-terminal domain of PhaC and activates PhaC by reinforcing the binding capacity of PhaC to the growing PHB polymer. The amphipathic surfactant properties of the PhaP are crucial for its interactions with polymer and the other granule-associated proteins PhaR and PhaZ.
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.
Biodegradation of polyethylene terephthalate (PET) is one of fundamental ways to solve plastic pollution. As various microbial hydrolases have an extra domain unlike PETase from Ideonella sakaiensis ...(IsPETase), research on the role of these extra domain in PET hydrolysis is crucial for the identification and selection of a novel PET hydrolase. Here, we report that a PET hydrolase from Burkholderiales bacterium RIFCSPLOWO2_02_FULL_57_36 (BbPETase) with an additional N-terminal domain (BbPETaseAND) shows a similar hydrolysis activity toward microcrystalline PET and a higher thermal stability than IsPETase. Based on detailed structural comparisons between BbPETase and IsPETase, we generated the BbPETaseS335N/T338I/M363I/N365G variant with an enhanced PET-degrading activity and thermal stability. We further revealed that BbPETaseAND contributes to the thermal stability of the enzyme through close contact with the core domain, but the domain might hinder the adhesion of enzyme to PET substrate. We suggest that BbPETase is an enzyme in the evolution of efficient PET degradation and molecular insight into a novel PET hydrolase provides a novel strategy for the development of biodegradation of PET.
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•Investigation of an auxiliary domain-containing PET hydrolase.•Development of a PET hydrolase with enhanced enzyme activity and thermal stability.•Structural and functional roles of auxiliary domain in PET decomposition.
Methionine is an essential amino acid in all living organisms and has been used in various industrial applications such as food and feed additives. However, inhibition of enzymes involved in ...methionine biosynthesis is considered to be a crucial bottleneck for an efficient bio-based methionine production process. Homoserine O-succinyltransferase fromEscherichia coli (EcHST) has been reported to be feedback inhibited by the final product methionine. To understand the regulation mechanism of the enzyme and generate a feedback-resistant mutant, we determined the crystal structure of EcHST and elucidated the binding site of homoserine and succinyl-CoA. The enzyme kinetic experiments of EcHST revealed that the enzyme is noncompetitively inhibited by methionine with a Ki value of 2.44 mM, and we also identified a putative inhibitor binding site located in the vicinity of the substrate binding site. We then generated the EcHSTT242A variant with reduced feedback inhibition with a Ki value of 17.40 mM.
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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