Palm GJ

References (10)

Title : Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization - von Haugwitz_2022_ACS.Catal_12_15259
Author(s) : von Haugwitz G , Han X , Pfaff L , Li Q , Wei H , Gao J , Methling K , Ao Y , Brack Y , Jan Mican J , Feiler CG , Weiss MS , Bednar D , Palm GJ , Lalk M , Lammers M , Damborsky J , Weber G , Liu W , Bornscheuer UT , Wei R
Ref : ACS Catal , 12 :15259 , 2022
Abstract : TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structurefunction relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.
ESTHER : von Haugwitz_2022_ACS.Catal_12_15259
PubMedSearch : von Haugwitz_2022_ACS.Catal_12_15259
PubMedID: 36570084
Gene_locus related to this paper: thefu-1831

Title : Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase - Pfaff_2022_ACS.Catalysis_12_9790
Author(s) : Pfaff L , Gao J , Li Z , Jackering A , Weber G , Mican J , Chen Y , Dong W , Han X , Feiler CG , Ao YF , Badenhorst CPS , Bednar D , Palm GJ , Lammers M , Damborsky J , Strodel B , Liu W , Bornscheuer UT , Wei R
Ref : ACS Catal , 12 :9790 , 2022
Abstract : Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substratebinding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/ Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic r"cycling processes.
ESTHER : Pfaff_2022_ACS.Catalysis_12_9790
PubMedSearch : Pfaff_2022_ACS.Catalysis_12_9790
PubMedID: 35966606
Gene_locus related to this paper: 9firm-PHL7

Title : Promiscuous Dehalogenase Activity of the Epoxide Hydrolase CorEH from Corynebacterium sp. C12 - Schuiten_2021_ACS.Catal_11_6113
Author(s) : Schuiten ED , Badenhorst CPS , Palm GJ , Berndt L , Lammers M , Mican J , Bednar D , Damborsky J , Bornscheuer UT
Ref : ACS Catal , 11 :6113 , 2021
Abstract : Haloalkane dehalogenases and epoxide hydrolases are phylogenetically related and structurally homologous enzymes that use nucleophilic aspartate residues for an SN2 attack on their substrates. Despite their mechanistic similarities, no enzymes are known that exhibit both epoxide hydrolase and dehalogenase activity. We screened a subset of epoxide hydrolases, closely related to dehalogenases, for dehalogenase activity and found that the epoxide hydrolase CorEH from Corynebacterium sp. C12 exhibits promiscuous dehalogenase activity. Compared to the hydrolysis of epoxides like cyclohexene oxide (1.41 micromol min-1 mg-1), the dehalogenation of haloalkanes like 1-bromobutane (0.25 nmol min-1 mg-1) is about 5000-fold lower. In addition to the activity with 1-bromobutane, dehalogenase activity was detected with other substrates like 1-bromohexane, 1,2-dibromoethane, 1-iodobutane, and 1-iodohexane. This study shows that dual epoxide hydrolase and dehalogenase activity can be present in one naturally occurring protein scaffold.
ESTHER : Schuiten_2021_ACS.Catal_11_6113
PubMedSearch : Schuiten_2021_ACS.Catal_11_6113
PubMedID:
Gene_locus related to this paper: corsp-cEH

Title : Structural analysis of PET-degrading enzymes PETase and MHETase from Ideonella sakaiensis - Graf_2021_Methods.Enzymol_648_337
Author(s) : Graf LG , Michels EAP , Yew Y , Liu W , Palm GJ , Weber G
Ref : Methods Enzymol , 648 :337 , 2021
Abstract : The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes: cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand.
ESTHER : Graf_2021_Methods.Enzymol_648_337
PubMedSearch : Graf_2021_Methods.Enzymol_648_337
PubMedID: 33579411
Gene_locus related to this paper: idesa-mheth , idesa-peth

Title : Discovery and Design of Family VIII Carboxylesterases as Highly Efficient Acyltransferases - Muller_2021_Angew.Chem.Int.Ed.Engl_60_2013
Author(s) : Muller H , Godehard SP , Palm GJ , Berndt L , Badenhorst CPS , Becker AK , Lammers M , Bornscheuer UT
Ref : Angew Chem Int Ed Engl , 60 :2013 , 2021
Abstract : Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity-based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20% conversion). We solved the crystal structure of EstCE1 and detailed structure-function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase".
ESTHER : Muller_2021_Angew.Chem.Int.Ed.Engl_60_2013
PubMedSearch : Muller_2021_Angew.Chem.Int.Ed.Engl_60_2013
PubMedID: 33140887

Title : Sequence-Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases - Muller_2020_Angew.Chem.Int.Ed.Engl_59_11607
Author(s) : Muller H , Becker AK , Palm GJ , Berndt L , Badenhorst CPS , Godehard SP , Reisky L , Lammers M , Bornscheuer U
Ref : Angew Chem Int Ed Engl , 59 :11607 , 2020
Abstract : Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, molecular and structural reasons for this phenomenon are still unclear. Here we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple, yet versatile, colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificities and potential applications.
ESTHER : Muller_2020_Angew.Chem.Int.Ed.Engl_59_11607
PubMedSearch : Muller_2020_Angew.Chem.Int.Ed.Engl_59_11607
PubMedID: 32243661
Gene_locus related to this paper: 9bact-Est8.6Y9K

Title : Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate - Palm_2019_Nat.Commun_10_1717
Author(s) : Palm GJ , Reisky L , Bottcher D , Muller H , Michels EAP , Walczak MC , Berndt L , Weiss MS , Bornscheuer UT , Weber G
Ref : Nat Commun , 10 :1717 , 2019
Abstract : The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus alpha/beta-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic alpha/beta-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.
ESTHER : Palm_2019_Nat.Commun_10_1717
PubMedSearch : Palm_2019_Nat.Commun_10_1717
PubMedID: 30979881
Gene_locus related to this paper: idesa-mheth

Title : The crystal structure of an esterase from the hyperthermophilic microorganism Pyrobaculum calidifontis VA1 explains its enantioselectivity - Palm_2011_Appl.Microbiol.Biotechnol_91_1061
Author(s) : Palm GJ , Fernandez-Alvaro E , Bogdanovic X , Bartsch S , Sczodrok J , Singh RK , Bottcher D , Atomi H , Bornscheuer UT , Hinrichs W
Ref : Applied Microbiology & Biotechnology , 91 :1061 , 2011
Abstract : The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E > 100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E = 2-4). To explain these unique properties, its crystal structure has been determined at 2.0 A resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical alpha/beta-hydrolase fold as core domain with a cap structure at the C-terminal end of the beta-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in alpha/beta-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83-87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.
ESTHER : Palm_2011_Appl.Microbiol.Biotechnol_91_1061
PubMedSearch : Palm_2011_Appl.Microbiol.Biotechnol_91_1061
PubMedID: 21614503
Gene_locus related to this paper: pyrca-PCEST

Title : Cloning, functional expression, biochemical characterization, and structural analysis of a haloalkane dehalogenase from Plesiocystis pacifica SIR-1 - Hesseler_2011_Appl.Microbiol.Biotechnol_91_1049
Author(s) : Hesseler M , Bogdanovic X , Hidalgo A , Berenguer J , Palm GJ , Hinrichs W , Bornscheuer UT
Ref : Applied Microbiology & Biotechnology , 91 :1049 , 2011
Abstract : A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 A resolution to an R-factor of 21.93%. The enzyme is composed of an alpha/beta-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37 degrees C with a pH optimum between pH 8 and 9. The K(m) and k(cat) values for 1-bromobutane were 24.0 mM and 8.08 s(-1). Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences.
ESTHER : Hesseler_2011_Appl.Microbiol.Biotechnol_91_1049
PubMedSearch : Hesseler_2011_Appl.Microbiol.Biotechnol_91_1049
PubMedID: 21603934
Gene_locus related to this paper: 9delt-a6g7b1

Title : Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-I - Bogdanovic_2010_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_66_828
Author(s) : Bogdanovic X , Hesseler M , Palm GJ , Bornscheuer UT , Hinrichs W
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 66 :828 , 2010
Abstract : DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P2(1)2(1)2. The DppA crystal diffracted X-rays to 1.9 A resolution using an in-house X-ray generator.
ESTHER : Bogdanovic_2010_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_66_828
PubMedSearch : Bogdanovic_2010_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_66_828
PubMedID: 20606284
Gene_locus related to this paper: 9delt-a6g7b1