A heavy layer of glycans forms a brush matrix bound to the outside of all the cells in our bodies; it is referred to as the "sugar forest" or glycocalyx. Beyond the increased appreciation of the glycocalyx over the past two decades, recent advances in engineering the glycocalyx on live cells have spurred the creation of cellular drugs and novel medical treatments. The development of new tools and techniques has empowered scientists to manipulate the structures and functions of cell-surface glycans on target cells and endow target cells with desired properties. Herein, we provide an overview of live-cell glycocalyx engineering strategies for controlling the cell-surface molecular repertory to suit therapeutic applications, even though the realm of this field remains young and largely unexplored.

The design of artificial enzymes has emerged as a promising tool for the generation of potent biocatalysts able to promote new-to-nature reactions with improved catalytic performances, providing a powerful platform for wide-ranging applications and a better understanding of protein functions and structures. The selection of an appropriate protein scaffold plays a key role in the design process. This review aims to give a general overview of the most common protein scaffolds that can be exploited for the generation of artificial enzymes. Several examples are discussed and categorized according to the strategy used for the design of the artificial biocatalyst, namely the functionalization of natural enzymes, the creation of a new catalytic site in a protein scaffold bearing a wide hydrophobic pocket and de novo protein design. The review is concluded by a comparison of these different methods and by our perspective on the topic.

In nature, enzymes that catalyze sequential reactions are often assembled as clusters or complexes. The formation of multienzyme complexes, or metabolons, brings the enzyme active sites into proximity to promote intermediate transfer, decrease intermediate leakage, and streamline the metabolic flux towards the desired products. We and others have developed synthetic versions of metabolons through various strategies to enhance the catalytic rates for synthesizing valuable chemicals inside microbes. Synthetic multienzyme complexes range from static enzyme nanostructures to dynamic enzyme coacervates. Enzyme complexation optimizes the metabolic fluxes inside microbes, increases the product titer, and supplies the field with high-yield microbe strains that are amenable to large-scale fermentation. Enzyme complexes constructed inside microbial cells can be separated as independent entities and catalyze biosynthetic reactions ex vivo; such a feature gains these complexes another name, "synthetic organelles" - new subcellular entities with independent structures and functions. Still, the field is seeking new strategies to better balance dynamicity and confinement and to achieve finer control of local compartmentalization in the cells, as the natural multienzyme complexes do. Industrial applications of synthetic multienzyme complexes for the large-scale production of valuable chemicals are yet to be realized. This review focuses on synthetic multienzyme complexes that are constructed and function inside microbial cells.

Sonodynamic therapy (SDT) for cancer treatment is gaining attention owing to its non-invasive property and ultrasound's (US) deep tissue penetration ability. In SDT, US activates the sonosensitizer at the target deep-seated tumors to generate reactive oxygen species (ROS), which ultimately damage tumors. However, drawbacks such as insufficient ROS production, aggregation of sonosensitizer, off-target side effects, etc., of the current organic/nanomaterial-based sonosensitizers limit the effectiveness of cancer SDT. Very recently, metal complexes with tunable physiochemical properties (such as sonostability, HOMO to LUMO energy gap, ROS generation ability, aqueous solubility, emission, etc.) have been devised as effective sonosensitizers, which could overcome the limitations of organic/nanomaterial-based sonosensitizers. This concept introduces all the reported metal-based sonosensitizers and delineates the prospects of metal complexes in cancer sonodynamic therapy. This new concept of metal-based sonosensitizer can deliver next-generation cancer drugs.

The discovery of immunogenic cell death (ICD) by small molecules (e. g., chemotherapeutic drugs) intrigued medicinal chemists and led them to exploit anticancer agents with such a trait because ICD agents provoke anticancer immune responses in addition to their cytotoxicity. However, the unclear molecular mechanism of ICD hampers further achievements in drug development. Fortunately, increasing efforts have been made in this area in recent years by using either chemical or biological approaches. Here, we review the current achievements towards understanding the mechanisms of small molecule-induced ICD effects. Based on the established role of the unfolded protein response (UPR) in ICD, we classify the mechanisms of different inducers by their dependency on UPR. Key proteins and pathways with important implications are discussed in depth. We also give our perspectives on the research strategies for future investigation in this field.

The short- and medium-chain dehydrogenase/reductase superfamilies are responsible for most chiral alcohol production in laboratories and industries. In nature, they participate in diverse roles such as detoxification, housekeeping, secondary metabolite production, and catalysis of several chemicals with commercial and environmental significance. As a result, they are used in industries to create biopolymers, active pharmaceutical intermediates (APIs), and are also used as components of modular enzymes like polyketide synthases for fabricating bioactive molecules. Consequently, random, semi-rational and rational engineering have helped transform these enzymes into product-oriented efficient catalysts. The rise of newer synthetic chemicals and their enantiopure counterparts has proved challenging, and engineering them has been the subject of numerous studies. However, they are frequently limited to the synthesis of a single chiral alcohol. The study attempts to defragment and describe hotspots of engineering short- and medium-chain dehydrogenases/reductases for the production of chiral synthons.

Structure determination lies at the heart of many biochemical research programs. However, the "giants": X-ray diffraction, electron microscopy, molecular dynamics simulations, and nuclear magnetic resonance, among others, leave quite a few dark spots on the structural pictures drawn of proteins, nucleic acids, membranes, and other biomacromolecules. For example, structural models under physiological conditions or of short-lived intermediates often remain out of reach of the established experimental methods. This account frames the possibility of including hyperpolarized, that is, dramatically signal-enhanced NMR in existing workflows to fill these spots with detailed depictions. We highlight how integrating methods based on dissolution dynamic nuclear polarization can provide valuable complementary information about formerly inaccessible conformational spaces for many systems. A particular focus will be on hyperpolarized buffers to facilitate the NMR structure determination of challenging systems.

Guanine quadruplexes (G4s) are nucleic acid structures exhibiting a complex structural behavior and exerting crucial biological functions in both cells and viruses. The specific interactions of peptides with G4s, as well as an understanding of the factors driving the specific recognition are important for the rational design of both therapeutic and diagnostic agents. In this review, we examine the most important studies dealing with the interactions between G4s and peptides, highlighting the strengths and limitations of current analytic approaches. We also show how the combined use of high-level molecular simulation techniques and experimental spectroscopy is the best avenue to design specifically tuned and selective peptides, thus leading to the control of important biological functions.

Engineered luciferase-luciferin pairs have expanded the number of cellular targets that can be visualized in tandem. While light production relies on selective processing of synthetic luciferins by mutant luciferases, little is known about the origin of selectivity. The development of new and improved pairs requires a better understanding of the structure-function relationship of bioluminescent probes. In this work, we report a biochemical approach to assessing and optimizing two popular bioluminescent pairs: Cashew/d-luc and Pecan/4'-BrLuc. Single mutants derived from Cashew and Pecan revealed key residues for selectivity and thermal stability. Stability was further improved through a rational addition of beneficial residues. In addition to providing increased stability, the known stabilizing mutations surprisingly also improved selectivity. The resultant improved pair of luciferases are >100-fold selective for their respective substrates and highly thermally stable. Collectively, this work highlights the importance of mechanistic insight for improving bioluminescent pairs and provides significantly improved Cashew and Pecan enzymes which should be immediately suitable for multicomponent imaging applications.

Fusarium mangiferae causes the mango malformation disease (MMD) on young mango trees and seedlings resulting in economically significant crop losses. In addition, F. mangiferae produces a vast array of secondary metabolites (SMs), including mycotoxins that may contaminate the harvest. Their production is tightly regulated at the transcriptional level. Here, we show that lack of the H3 K9-specific histone methyltransferase, FmKmt1, influences the expression of the F. mangiferae polyketide synthase (PKS) 8 (FmPKS8), a so far cryptic PKS. By a combination of reverse genetics, untargeted metabolomics, bioinformatics and chemical analyses including structural elucidation, we determined the FmPKS8 biosynthetic gene cluster (BGC) and linked its activity to the production of fusamarins (FMN), which can be structurally classified as dihydroisocoumarins. Functional characterization of the four FMN cluster genes shed light on the biosynthetic pathway. Cytotoxicity assays revealed moderate toxicities with IC50 values between 1 and 50 μM depending on the compound.

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.

The glycopeptide antibiotics (GPAs) are a clinically approved class of antimicrobial agents that classically function through the inhibition of bacterial cell-wall biosynthesis by sequestration of the precursor lipid II. The oxidative crosslinking of the core peptide by cytochrome P450 (Oxy) enzymes during GPA biosynthesis is both essential to their function and the source of their synthetic challenge. Thus, understanding the activity and selectivity of these Oxy enzymes is of key importance for the future engineering of this important compound class. Recent reports of GPAs that display an alternative mode of action and a wider range of core peptide structures compared to classic lipid II-binding GPAs raises the question of the tolerance of Oxy enzymes for larger changes in their peptide substrates. In this work, we explore the ability of Oxy enzymes from the biosynthesis pathways of lipid II-binding GPAs to accept altered peptide substrates based on a vancomycin template. Our results show that Oxy enzymes are more tolerant of changes at the N terminus of their substrates, whilst C-terminal extension of the peptide substrates is deleterious to the activity of all Oxy enzymes. Thus, future studies should prioritise the study of Oxy enzymes from atypical GPA biosynthesis pathways bearing C-terminal peptide extension to increase the substrate scope of these important cyclisation enzymes.

Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activity. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without any supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon-elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.

Group B Streptococcus (GBS) is an encapsulated Gram-positive bacterial pathogen that causes severe perinatal infections. Human milk oligosaccharides (HMOs) are short-chain sugars that have recently been shown to possess antimicrobial and anti-biofilm activity against a variety of bacterial pathogens, including GBS. We have expanded these studies to demonstrate that HMOs can inhibit and dismantle biofilm in both invasive and colonizing strains of GBS. A cohort of 30 diverse strains of GBS were analyzed for susceptibility to HMO-dependent biofilm inhibition or destruction. HMOs were significantly effective at inhibiting biofilm in capsular-type- and sequence-type-specific fashion, with significant efficacy in CpsIb, CpsII, CpsIII, CpsV, and CpsVI strains as well as ST-1, ST-12, ST-19, and ST-23 strains. Interestingly, CpsIa as well as ST-7 and ST-17 were not susceptible to the anti-biofilm activity of HMOs, underscoring the strain-specific effects of these important antimicrobial molecules against the perinatal pathogen Streptococcus agalactiae.

Post-transcriptional modifications of tRNA nucleotide are important determinants in folding, structure and function. We have successfully identified and characterized a new modified base named 2-methylthio-methylenethio-N6 -(cis-4-hydroxyisopentenyl)adenosine, which is present at position 37 in some tRNAs. We also showed that this new modified adenosine is derived from the known 2-methylthio-methylenethio-N6 -(isopentenyl)adenosine nucleoside by a catalytic cycle of the tRNA-diiron monooxygenase, MiaE, present in Salmonella typhimurium.

Reverse transcriptases are DNA polymerases that can use RNA as a template for DNA synthesis. They thus catalyze the reverse of transcription. Although discovered in 1970, reverse transcriptases are still of great interest and are constantly being further developed for numerous modern research approaches. They are frequently used in biotechnological and molecular diagnostic applications. In this review, we describe the discovery of these fascinating enzymes and summarize research results and applications ranging from molecular cloning, direct virus detection, and modern sequencing methods to xenobiology.

The use of polymerase enzymes in biotechnology has allowed us to gain unprecedented control over the manipulation of DNA, opening up new and exciting applications in areas such as biosensing, polynucleotide synthesis, and DNA storage, aptamer development and DNA-nanotechnology. One of the most intriguing enzymes which has gained prominence in the last decade is terminal deoxynucleotidyl transferase (TdT), which is one of the only polymerase enzymes capable of catalysing the template independent stepwise addition of nucleotides onto an oligonucleotide chain. This unique enzyme has seen a significant increase in a variety of different applications. In this review, we give a comprehensive discussion of the unique properties and applications of TdT as a biotechnology tool, and the application in the enzymatic synthesis of poly/oligonucleotides. Finally, we look at the increasing role of TdT enzyme in biosensing, DNA storage, synthesis of DNA nanostructures and aptamer development, and give a future outlook for this technology.

Esterases are among the most studied enzymes, and their applications expand into several branches of industrial biotechnology. Yet, despite the fact that information on their substrate specificity is crucial for selecting or designing the best fitted biocatalyst for the desired application, it cannot be predicted from their amino acid sequence. In this work, we studied the substrate scope of the newly discovered hydrolytic extremozyme, EstDZ3, against a library of esters with variable carbon chain lengths in an effort to understand the crucial amino acids for the substrate selectivity of this enzyme. EstDZ3 appears to be active against a wide range of esters with high selectivity towards medium- to long-carbon chain vinyl esters. In-silico studies of its 3D structure revealed that the selectivity might arise from the mainly hydrophobic nature of the active site's environment.

Heparin is a commonly used anticoagulant in clinical practice; however, excessive heparin can cause serious adverse reactions. Convenient and accurate detection of heparin levels is thus very important. In this research, a pyrene-based self-assembling fluorescent peptide PyFFRRR was designed for simple, selective, and efficient heparin detection. The guanidine groups in the arginine residues of PyFFRRR bind tightly with heparin, which is highly sulfated, through electrostatic interactions. Charge neutralization facilitated the self-assembly of PyFFRRR, resulting in its spectral response changing from deep blue monomer fluorescence to green excimer fluorescence. PyFFRRR exhibited excellent sensitivity and selectivity for ratiometric detection of heparin. The binding mechanism was investigated by using spectral and simulation tools, and structural observation. Finally, PyFFRRR was employed in human serum samples for ratiometric detection of heparin.

Benzoxazoles are important structural motifs in pharmaceutical drugs. Here, we present the heterologous production of 3-hydroxyanthranilate-derived benzoxazoles in the host bacterium Myxococcus xanthus following the expression of two genes from the nataxazole biosynthetic gene cluster of Streptomyces sp. Tü 6176. The M. xanthus expression strain achieved a benzoxazole titer of 114.6±7.4 mg L-1 upon precursor supplementation, which is superior to other bacterial production systems. Crosstalk between the heterologously expressed benzoxazole pathway and the endogenous myxochelin pathway led to the combinatorial biosynthesis of benzoxazoles featuring a 2,3-dihydroxybenzoic acid (2,3-DHBA) building block. Subsequent in vitro studies confirmed that this crosstalk is not only due to the availability of 2,3-DHBA in M. xanthus, rather, it is promoted by the adenylating enzyme MxcE from the myxochelin pathway, which contributes to the activation of aryl carboxylic acids and delivers them to benzoxazole biosynthesis.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.

The chemistry of DNA endows it with certain functional properties that facilitate the generation of self-assembled nanostructures, offering precise control over their geometry and morphology, that can be exploited for advanced biological applications. Despite the structural promise of these materials, their applications are limited owing to lack of functional capability to interact favourably with biological systems, which has been achieved by functional proteins or peptides. Herein, we outline a strategy for functionalizing DNA structures with short-peptides, leading to the formation of DNA-peptide hybrid materials. This proposition offers the opportunity to leverage the unique advantages of each of these bio-molecules, that have far reaching emergent properties in terms of better cellular interactions and uptake, better stability in biological media, an acceptable and programmable immune response and high bioactive molecule loading capacities. We discuss the synthetic strategies for the formation of these materials, namely, solid-phase functionalization and solution-coupling functionalization. We then proceed to highlight selected biological applications of these materials in the domains of cell instruction & molecular recognition, gene delivery, drug delivery and bone & tissue regeneration. We conclude with discussions shedding light on the challenges that these materials pose and offer our insights on future directions of peptide-DNA research for targeted biomedical applications.

The identification of pseudo- and N1 -methylpseudo-uridine (Ψ and mΨ, respectively) as immunosilent uridine analogues has propelled the development of mRNA-based vaccines and therapeutics. Here, we have characterised another uridine analogue, 5-ethynyluridine (EU), which has an ethynyl moiety. We show that this uridine analogue does not cause immune activation in human macrophages, as it does not induce interleukin-6 secretion or expression of the inflammatory and antiviral genes MX1, PKR, and TAP2. Moreover, EU allows for prolonged expression, as shown with mRNA coding for yellow fluorescent protein (YFP). Side-by-side comparisons of EU with unmodified, Ψ, and mΨ revealed that EU-modified mRNA is expressed at lower levels, but confers similar stability and low immunogenicity to the other uridine analogues. Furthermore, structure analysis of modified mRNAs suggests that the observed phenotype is largely independent of RNA folding. Thus, EU is a potential candidate for RNA-based vaccines and therapeutics.

Many dimeric natural products containing bisanthraquinone and related xanthones with diverse structures and versatile bioactivities have been isolated over the years. However, the complicated biosynthetic pathways of such natural products, which have remained elusive until recently, negatively impact their mass bioproduction and biosynthetic structural modification for drug discovery. In this concept, we summarize the recent progress in gene cluster mining and biosynthetic pathway elucidation of natural products containing bisanthraquinone and related xanthones. These pioneering works may pave the way for further biosynthetic pathway elucidation and structure modification of dimeric natural products through gene and protein engineering.

The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site-specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.

Proteomics, or the large-scale study of proteomes, has benefitted from many recent advances in chemical biology, mass spectrometry, and machine learning. The Proteomics in Cell Biology and Disease Mechanisms conference showcased the synergy between these elements and the vast range of biological questions that proteomics can now help us to answer.

Peroxynitrite (ONOO- ) is an essential endogenous reactive oxygen species (ROS) generated in mitochondria under various pathological and physiological conditions. An increase in its level in mitochondria is related to numerous diseases. Herein, we report a series of hemicyanine-derived water-soluble colorimetric probes (1-4) and the reactivity of which was studied with various reactive oxygen, nitrogen, and sulfur species. Probes 1-4 are formed by conjugating 1,2,3,3-tetramethyl-3H-indolium iodide and 4-hydroxybenzaldehyde or its derivatives through an alkene linkage formed by the Knoevenagel reaction. Oxidative cleavage of the electron-rich double bond of the conjugated hemicyanine dye revealed a discerning affinity of probe 3 towards peroxynitrite among all reactive oxygen species. The rapid change in color of 3 provides a sensitive and selective method for detecting peroxynitrite with a low detection limit of 180 nM. Notably, the water solubility of the probe displays excellent performance for the selective detection of peroxynitrite among ROS and reactive nitrogen (RNS)/sulfur species (RSS). UV-vis, 1 H NMR, and 13 C NMR spectroscopic data and results from theoretical calculations provide further information on the interaction of peroxynitrite with probe 3.

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between βS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.

Ultraviolet C (UV-C) radiation induces apoptosis in mammalian cells via the mitochondrion-mediated pathway. The Bcl-2 family of proteins are the regulators of the mitochondrial pathway of apoptosis and appears responsive to UV-C radiation. It is unknown how the structure and, effectively, the function of these proteins are directly impacted by UV-C exposure. Here, we present the effect of UV-C irradiation on the structure and function of pro-apoptotic Bid-FL and anti-apoptotic Bcl-xlΔC proteins. Using a variety of biophysical tools, we show that, following UV-C irradiation, the structures of Bcl-xlΔC and Bid-FL are irreversibly altered. Bcl-xLΔC is found to be more sensitive to UV stress than Bid-FL Interestingly, UV-C exposure shows dramatic chemical shift perturbations in consequence of dramatic structural perturbations (α-helix to β-sheet) in the BH3- binding region, a crucial segment of Bcl-xlΔC. Furter it has been shown that UV-exposed Bcl-xlΔC has reduced efficacy of its interactions with pro-apoptotic tBid.

Combining natural product fragments to design new scaffolds with unprecedented bioactivity is a powerful strategy for the discovery of tool compounds and potential therapeutics. However, the choice of fragments to couple and the biological screens to employ remain open questions in the field. By choosing a primary fragment containing the A/B ring system of estradiol and fusing it to nine different secondary fragments, we were able to identify compounds that modulated four different phenotypes: inhibition of autophagy and osteoblast differentiation, as well as potassium channel and tubulin modulation. The latter two were uncovered by using unbiased morphological profiling with a cell-painting assay. The number of hits and variety in bioactivity discovered validates the use of recombining natural product fragments coupled to phenotypic screening for the rapid identification of biologically diverse compounds.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-β-l-Araf-(1→2)-β-l-Araf-(1→2)-β-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 β-l-arabinobiosidase (HypBA2), producing β-l-Araf-(1→2)-l-Ara (β-l-arabinobiose) and mono-β-l-Araf linked to the HRGP backbone. In bifidobacteria, the β-l-arabinobiose is then hydrolyzed by GH127 β-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 β-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

The gene cluster in Streptomyces calvus associated with the biosynthesis of the fluoro- and sulfamyl-metabolite nucleocidin was interrogated by systematic gene knockouts. Out of the 26 gene deletions, most did not affect fluorometabolite production, nine abolished sulfamylation but not fluorination, and three precluded fluorination, but had no effect on sulfamylation. In addition to nucI, nucG, nucJ, nucK, nucL, nucN, nucO, nucQ and nucP, we identified two genes (nucW, nucA), belonging to a phosphoadenosine phosphosulfate (PAPS) gene cluster, as required for sulfamyl assembly. Three genes (orf(-3), orf2 and orf3) were found to be essential for fluorination, although the activities of their protein products are unknown. These genes as well as nucK, nucN, nucO and nucPNP, whose knockouts produced results differing from those described in a recent report, were also deleted in Streptomyces virens - with confirmatory outcomes. This genetic profile should inform biochemistry aimed at uncovering the enzymology behind nucleocidin biosynthesis.