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An NMR look at an engineered PET depolymerase.

ABSTRACT: Plastic environmental pollution is a major issue that our generation must face to protect our planet. Plastic recycling has the potential not only to reduce the pollution but also to limit the need for fossil-fuel-based production of new plastics. Enzymes capable of breaking down plastic could thereby support such a circular economy. Polyethylene terephthalate (PET) degrading enzymes have recently attracted considerable interest and have been subjected to intensive enzyme engineering to improve their characteristics. A quadruple mutant of Leaf-branch Compost Cutinase (LCC) was identified as a most efficient and promising enzyme. Here, we use NMR to follow the initial LCC enzyme through its different mutations that lead to its improved performance. We experimentally define the two calcium-binding sites and show their importance on the all-or-nothing thermal unfolding process, which occurs at a temperature of 72°C close to the PET glass transition temperature. Using various NMR probes such as backbone amide, methyl group, and histidine side-chain resonances, we probe the interaction of the enzymes with mono-(2-hydroxyethyl)terephthalic acid. The latter experiments are interpreted in terms of accessibility of the active site to the polymer chain.

SUBMITTER: Charlier C

PROVIDER: S-EPMC9388554 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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An NMR look at an engineered PET depolymerase.

Charlier Cyril C Gavalda Sabine S Borsenberger Vinciane V Duquesne Sophie S Marty Alain A Tournier Vincent V Lippens Guy G

Biophysical journal 20220706 15

Plastic environmental pollution is a major issue that our generation must face to protect our planet. Plastic recycling has the potential not only to reduce the pollution but also to limit the need for fossil-fuel-based production of new plastics. Enzymes capable of breaking down plastic could thereby support such a circular economy. Polyethylene terephthalate (PET) degrading enzymes have recently attracted considerable interest and have been subjected to intensive enzyme engineering to improve ...[more]

PMID: 35794828

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Project description:Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases.

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An NMR look at an engineered PET depolymerase.

Publications

An NMR look at an engineered PET depolymerase.

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