Assessment of Four Engineered PET Degrading Enzymes Considering Large-Scale Industrial Applications.

In recent years, enzymatic recycling of the widely used polyester polyethylene terephthalate (PET) has become a complementary solution to current thermomechanical recycling for colored, opaque, and mixed PET. A large set of promising hydrolases that depolymerize PET have been found and enhanced by worldwide initiatives using various methods of protein engineering. Despite the achievements made in these works, it remains difficult to compare enzymes' performance and their applicability to large-scale reactions due to a lack of homogeneity between the experimental protocols used.

Structural Insights into (Tere)phthalate-Ester Hydrolysis by a Carboxylesterase and Its Role in Promoting PET Depolymerization.

TfCa, a promiscuous carboxylesterase from , 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 structure-function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths.

A growth selection system for the directed evolution of amine-forming or converting enzymes.

Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes.

Structural and functional investigation of the human snRNP assembly factor AAR2 in complex with the RNase H-like domain of PRPF8.

Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of the snRNP core particles has been well characterized, comparably little is known of the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling. U5 snRNP assembly in yeast requires binding of the the Aar2 protein to Prp8p as a placeholder to preclude premature assembly of the SNRNP200 helicase, but the role of the human AAR2 homolog has not yet been investigated in detail.

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase.

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.

Mechanism-Based Design of Efficient PET Hydrolases.

Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages and food, contributing considerably to the global solid waste stream and environmental plastic pollution. While enzymatic PET recycling and upcycling have recently emerged as viable disposal methods for a circular plastic economy, only a handful of benchmark enzymes have been thoroughly described and subjected to protein engineering for improved properties over the last 16 years. By analyzing the specific material properties of PET and the reaction mechanisms in the context of interfacial biocatalysis, this Perspective identifies several limitations in current enzymatic PET degradation approaches.

A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing.

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identify additional C9ORF78 interactors in spliceosomes.

A human kinase yeast array for the identification of kinases modulating phosphorylation-dependent protein-protein interactions.

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities.

The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling.

Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites.

DYW domain structures imply an unusual regulation principle in plant organellar RNA editing catalysis.

RNA editosomes selectively deaminate cytidines to uridines in plant organellar transcripts-mostly to restore protein functionality and consequently facilitate mitochondrial and chloroplast function. The RNA editosomal pentatricopeptide repeat proteins serve target RNA recognition, whereas the intensively studied DYW domain elicits catalysis. Here we present structures and functional data of a DYW domain in an inactive ground state and activated.

Yeast cell surface display of bacterial PET hydrolase as a sustainable biocatalyst for the degradation of polyethylene terephthalate.

Enzymatic hydrolysis of polyethylene terephthalate (PET) is considered to be an environmentally friendly method for the recycling of plastic waste. Recently, a bacterial enzyme named IsPETase was found in Ideonella sakaiensis with the ability to degrade amorphous PET at ambient temperature suggesting its possible use in recycling of PET. However, applying the purified IsPETase in large-scale PET recycling has limitations, i.

Structural analysis of PET-degrading enzymes PETase and MHETase from Ideonella sakaiensis.

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.

Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate.

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.

Ectopic Transplastomic Expression of a Synthetic MatK Gene Leads to Cotyledon-Specific Leaf Variegation.

Chloroplasts (and other plastids) harbor their own genetic material, with a bacterial-like gene-expression systems. Chloroplast RNA metabolism is complex and is predominantly mediated by nuclear-encoded RNA-binding proteins. In addition to these nuclear factors, the chloroplast-encoded intron maturase MatK has been suggested to perform as a splicing factor for a subset of chloroplast introns.

Molecular principles underlying dual RNA specificity in the Drosophila SNF protein.

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A' protein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A' or U2 stem-loop IV and U2A', SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies.

Plasmacytoid dendritic cells and RNA-containing immune complexes drive expansion of peripheral B cell subsets with an SLE-like phenotype.

Background: Hyperactive B cells and a continuous interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs) play a key role in systemic lupus erythematosus (SLE). We asked whether the interaction between B cells and pDCs stimulated with RNA-containing immune complexes affects peripheral B cell subsets.

Methods: B cells and pDCs were isolated from blood of healthy individuals and stimulated with immune complexes consisting of SLE-IgG and U1snRNP (RNA-IC).

Structural basis for λN-dependent processive transcription antitermination.

λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a λN-NusA-NusB-NusE-nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and λN, validated by crosslinking/mass spectrometry.

Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9.

In flowering plant plastids and mitochondria, multiple organellar RNA editing factor (MORF/RIP) proteins are required at most sites for efficient C to U RNA editing catalyzed by the RNA editosome. MORF proteins harbor a conserved stretch of residues (MORF-box), form homo- and heteromers and interact with selected PPR (pentatricopeptide repeat) proteins, which recognize each editing site. The molecular function of the MORF-box remains elusive since it shares no sequence similarity with known domains.

Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38.

Crystallization and biochemical characterization of the human spliceosomal Aar2-Prp8(RNaseH) complex.

In eukaryotes, the removal of nuclear noncoding sequences (pre-mRNA splicing) is catalyzed by the spliceosome, which consists of five ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs, each with a respective snRNA) and a plethora of protein factors that aid spliceosomal maturation, assembly, activation and disassembly. Recently, the U5 snRNP maturation factor Aar2p from baker's yeast has been characterized structurally and biochemically. Aar2p binds to the RNaseH (RH) and Jab1/MPN domains of the highly conserved U5-specific Prp8p, which forms a framework for the spliceosomal catalytic centre.

IFN-α production by plasmacytoid dendritic cell associations with polymorphisms in gene loci related to autoimmune and inflammatory diseases.

The type I interferon (IFN) system is persistently activated in systemic lupus erythematosus (SLE) and many other systemic autoimmune diseases. Studies have shown an association between SLE and several gene variants within the type I IFN system. We investigated whether single-nucleotide polymorphisms (SNPs) associated with SLE and other autoimmune diseases affect the IFN-α production in healthy individuals.

Structural basis for dual roles of Aar2p in U5 snRNP assembly.

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains.

Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome.

Assembly of a spliceosome, catalyzing precursor-messenger RNA splicing, involves multiple RNA-protein remodeling steps, driven by eight conserved DEXD/H-box RNA helicases. The 250-kDa Brr2 enzyme, which is essential for U4/U6 di-small nuclear ribonucleoprotein disruption during spliceosome catalytic activation and for spliceosome disassembly, is the only member of this group that is permanently associated with the spliceosome, thus requiring its faithful regulation. At the same time, Brr2 represents a unique subclass of superfamily 2 nucleic acid helicases, containing tandem helicase cassettes.

B lymphocytes enhance interferon-α production by plasmacytoid dendritic cells.

Objective: The type I interferon (IFN) system and B cells are activated in many autoimmune diseases, such as systemic lupus erythematosus (SLE). The IFNα produced by plasmacytoid dendritic cells (PDCs) stimulates several B cell functions, including autoantibody production. However, not much is known about how B cells influence PDC function.