Enhancing PET Degrading Enzymes: A Combinatory Approach.

Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.

Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii.

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.

Discovery of a highly potent, selective, orally bioavailable inhibitor of KAT6A/B histone acetyltransferases with efficacy against KAT6A-high ER+ breast cancer.

KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

Moving beyond MARCO.

The use of imaging systems in protein crystallisation means that the experimental setups no longer require manual inspection to determine the outcome of the trials. However, it leads to the problem of how best to find images which contain useful information about the crystallisation experiments. The adoption of a deeplearning approach in 2018 enabled a four-class machine classification system of the images to exceed human accuracy for the first time. Underpinning this was the creation of a labelled training set which came from a consortium of several different laboratories. The MARCO classification model does not have the same accuracy on local data as it does on images from the original test set; this can be somewhat mitigated by retraining the ML model and including local images. We have characterized the image data used in the original MARCO model, and performed extensive experiments to identify training settings most likely to enhance the local performance of a MARCO-dataset based ML classification model.

Molecular characterization of cyanobacterial short-chain prenyltransferases and discovery of a novel GGPP phosphatase.

Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short-chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type-II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP; C20 ) primarily but also significant amounts of farnesyl pyrophosphate (FPP, C15 ) and geranyl pyrophosphate (GPP, C10 ); whereas SeCrtE appears to produce only GGPP. The crystal structures were solved to 2.02 and 1.37 Å, respectively, and the superposition of the structures against the GGPPS of Synechococcus elongatus sp. PCC 7002 yield a root mean square deviation of 0.8 Å (SeCrtE) and 1.1 Å (SyCrtE). We also discovered that SeCrtE is co-encoded in an operon with a functional GGPP phosphatase, suggesting metabolic pairing of these two activities and a putative function in tocopherol biosynthesis. This work sheds light on the activity of SPTs and terpenoid synthesis in cyanobacteria. Understanding native prenyl phosphate metabolism is an important step in developing approaches to engineering the production of different chain-length terpenoids in cyanobacteria.

Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors.

The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.

Over the rainbow: structural characterization of the chromoproteins gfasPurple, amilCP, spisPink and eforRed.

Anthozoan chromoproteins are highly pigmented, diversely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp2-hybridized N-acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp3-hybridized trans-peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.

Submission of structural biology data for review purposes.

The editors discuss the submission of structural biology data.

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices.

Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

Data- and diversity-driven development of a Shotgun crystallization screen using the Protein Data Bank.

Protein crystallization has for decades been a critical and restrictive step in macromolecular structure determination via X-ray diffraction. Crystallization typically involves a multi-stage exploration of the available chemical space, beginning with an initial sampling (screening) followed by iterative refinement (optimization). Effective screening is important for reducing the number of optimization rounds required, reducing the cost and time required to determine a structure. Here, an initial screen (Shotgun II) derived from analysis of the up-to-date Protein Data Bank (PDB) is proposed and compared with the previously derived (2014) Shotgun I screen. In an update to that analysis, it is clarified that the Shotgun approach entails finding the crystallization conditions that cover the most diverse space of proteins by sequence found in the PDB, which can be mapped to the well known maximum coverage problem in computer science. With this realization, it was possible to apply a more effective algorithm for selecting conditions. In-house data demonstrate that compared with alternatives, the Shotgun I screen has been remarkably successful over the seven years that it has been in use, indicating that Shotgun II is also likely to be a highly effective screen.

Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Crystal structure of fungal tannase from Aspergillus niger.

Tannases are serine esterases that were first discovered in fungi more than one and half centuries ago. They catalyze the hydrolysis of the gallolyl ester bonds in gallotannins to release gallic acid, which is an important intermediate in the chemical and pharmaceutical industries. Since their discovery, fungal tannases have found wide industrial applications, although there is scarce knowledge about these enzymes at the molecular level, including their catalytic and substrate-binding sites. While this lack of knowledge hinders engineering efforts to modify the enzymes, many tannases have been isolated from various fungal strains in a search for the desired enzymatic properties. Here, the first crystal structure of a fungal tannase, that from Aspergillus niger, is reported. The enzyme possesses a typical α/ß-hydrolase-fold domain with a large inserted cap domain, which together form a bowl-shaped hemispherical shape with a surface concavity surrounded by N-linked glycans. Gallic acid is bound at the junction of the two domains within the concavity by forming two hydrogen-bonding networks with neighbouring residues. One is formed around the carboxyl group of the gallic acid and involves residues from the hydrolase-fold domain, including those from the catalytic triad, which consists of Ser206, His485 and Asp439. The other is formed around the three hydroxyl groups of the compound, with the involvement of residues mainly from the cap domain, including Gln238, Gln239, His242 and Ser441. Gallic acid is bound in a sandwich-like mode by forming a hydrophobic contact with Ile442. All of these residues are found to be highly conserved among fungal and yeast tannases.

The X-ray crystal structure of the N-terminal domain of Ssr4, a Schizosaccharomyces pombe chromatin-remodelling protein.

Ssr4 is a yeast protein from Schizosaccharomyces pombe and is an essential part of the chromatin-remodelling [SWI/SNF and RSC (remodelling the structure of chromatin)] complexes found in S. pombe. These complexes (or their homologues) regulate gene expression in eukaryotic organisms, affecting a large number of genes both positively and negatively. The downstream effects are seen in development, and in humans have implications for disease such as cancer. The chromatin structure is altered by modifying the DNA-histone contacts, thus opening up or closing down sections of DNA to specific transcription factors that regulate the transcription of genes. The Ssr4 sequence has little homology to other sequences in the Protein Data Bank, so the structure was solved using an iodine derivative with SAD phasing. The structure of the N-terminal domain is an antiparallel ß-sheet of seven strands with α-helices on one side and random coil on the other. The structure is significantly different to deposited structures and was used as a target in the most recent Critical Assessment of Techniques for Protein Structure Prediction (CASP; https://predictioncenter.org/) competition.

Crystal structures of human ENPP1 in apo and bound forms.

Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design campaigns against this attractive cancer therapeutic target.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.