Characterising glycosaminoglycans in human breastmilk and their potential role in infant health.

Human breastmilk is composed of many well researched bioactive components crucial for infant nutrition and priming of the neonatal microbiome and immune system. Understanding these components gives us crucial insight to the health and wellbeing of infants. Research surrounding glycosaminoglycans (GAGs) previously focused on those produced endogenously; however, recent efforts have shifted to understanding GAGs in human breastmilk. The structural complexity of GAGs makes detection and analysis complicated therefore, research is time consuming and limited to highly specialised teams experienced in carbohydrate analysis. In breastmilk, GAGs are present in varying quantities in four forms; chondroitin sulphate, heparin/heparan sulphate, dermatan sulphate and hyaluronic acid, and are hypothesised to behave similar to other bioactive components with suspected roles in pathogen defense and proliferation of beneficial gut bacteria. Chondroitin sulphate and heparin, being the most abundant, are expected to have the most impact on infant health. Their decreasing concentration over lactation further indicates their role and potential importance during early life.

Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

RetroBioCat Database: A Platform for Collaborative Curation and Automated Meta-Analysis of Biocatalysis Data.

Despite the increasing use of biocatalysis for organic synthesis, there are currently no databases that adequately capture synthetic biotransformations. The lack of a biocatalysis database prevents accelerating biocatalyst characterization efforts from being leveraged to quickly identify candidate enzymes for reactions or cascades, slowing their development. The RetroBioCat Database (available at retrobiocat.com) addresses this gap by capturing information on synthetic biotransformations and providing an analysis platform that allows biocatalysis data to be searched and explored through a range of highly interactive data visualization tools. This database makes it simple to explore available enzymes, their substrate scopes, and how characterized enzymes are related to each other and the wider sequence space. Data entry is facilitated through an openly accessible curation platform, featuring automated tools to accelerate the process. The RetroBioCat Database democratizes biocatalysis knowledge and has the potential to accelerate biocatalytic reaction development, making it a valuable resource for the community.

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern.

The emergence of a polybasic cleavage motif for the protease furin in SARS-CoV-2 spike has been established as a major factor for human viral transmission. The region N-terminal to that motif is extensively mutated in variants of concern (VOCs). Besides furin, spikes from these variants appear to rely on other proteases for maturation, including TMPRSS2. Glycans near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, we identify that sialic acid-containing O-linked glycans on Thr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage and posit O-linked glycosylation as a likely driving force for the emergence of VOC mutations. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, an event that is suppressed by mutations in the VOCs Alpha, Delta, and Omicron. We found that the sole incorporation of N-acetylgalactosamine did not impact furin activity in synthetic O-glycopeptides, but the presence of sialic acid reduced the furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on TMPRSS2 cleavage. With a chemistry-centered approach, we substantiate O-glycosylation as a major determinant of spike maturation and propose disruption of O-glycosylation as a substantial driving force for VOC evolution.

Biocatalytic Cascades toward Iminosugar Scaffolds Reveal Promiscuous Activity of Shikimate Dehydrogenases.

Iminosugar scaffolds are highly sought-after pharmaceutical targets, but their chemical synthesis is lengthy and can suffer from poor scalability and purification. Here we report protecting-group-free chemoenzymatic and biocatalytic cascades to synthesize iminosugars from sugar-derived aminopolyols in two steps. Using galactose oxidase variant F2 followed by a chemical or enzymatic reduction provided an efficient one-pot route to these targets, with product formation >70%. Key to success of this strategy was the application of genome mining, which identified bacterial shikimate dehydrogenases as promiscuous iminosugar reductases. The cell-free protocols allowed for isolation of highly polar iminosugar products from biotransformations in a single step through development of a gradient-elution cation exchange purification. The two-step pathway provides a short synthetic route that can be used as a cell-free platform for broader iminosugar synthesis.

Biochemical characterization of a glycoside hydrolase family 43 ß-D-galactofuranosidase from the fungus Aspergillus niger.

ß-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members via conserved unique peptide patterns and phylogeny, revealed the occurrence of distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative ß-D-galactofuranosidase from A. niger which was recombinantly produced in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-nitrophenyl-ß-galactofuranoside (pNP-ß-Galf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 µM min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures.

The Time and Place for Nature in Drug Discovery.

The case for a renewed focus on Nature in drug discovery is reviewed; not in terms of natural product screening, but how and why biomimetic molecules, especially those produced by natural processes, should deliver in the age of artificial intelligence and screening of vast collections both in vitro and in silico. The declining natural product-likeness of licensed drugs and the consequent physicochemical implications of this trend in the context of current practices are noted. To arrest these trends, the logic of seeking new bioactive agents with enhanced natural mimicry is considered; notably that molecules constructed by proteins (enzymes) are more likely to interact with other proteins (e.g., targets and transporters), a notion validated by natural products. Nature's finite number of building blocks and their interactions necessarily reduce potential numbers of structures, yet these enable expansion of chemical space with their inherent diversity of physical characteristics, pertinent to property-based design. The feasible variations on natural motifs are considered and expanded to encompass pseudo-natural products, leading to the further logical step of harnessing bioprocessing routes to access them. Together, these offer opportunities for enhancing natural mimicry, thereby bringing innovation to drug synthesis exploiting the characteristics of natural recognition processes. The potential for computational guidance to help identifying binding commonalities in the route map is a logical opportunity to enable the design of tailored molecules, with a focus on "organic/biological" rather than purely "synthetic" structures. The design and synthesis of prototype structures should pay dividends in the disposition and efficacy of the molecules, while inherently enabling greener and more sustainable manufacturing techniques.

Three-Component Stereoselective Enzymatic Synthesis of Amino-Diols and Amino-Polyols.

Amino-polyols represent attractive chemical building blocks but can be challenging to synthesize because of the high density of asymmetric functionalities and the need for extensive protecting-group strategies. Here we present a three-component strategy for the stereoselective enzymatic synthesis of amino-diols and amino-polyols using a diverse set of prochiral aldehydes, hydroxy ketones, and amines as starting materials. We were able to combine biocatalytic aldol reactions, using variants of d-fructose-6-phosphate aldolase (FSA), with reductive aminations catalyzed by IRED-259, identified from a metagenomic library. A two-step process, without the need for intermediate isolation, was developed to avoid cross-reactivity of the carbonyl components. Stereoselective formation of the 2R,3R,4R enantiomers of amino-polyols was observed and confirmed by X-ray crystallography.

Cell-specific bioorthogonal tagging of glycoproteins.

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

Enzymatic elaboration of oxime-linked glycoconjugates in solution and on liposomes.

Oxime formation is a convenient one-step method for ligating reducing sugars to surfaces, producing a mixture of closed ring α- and ß-anomers along with open-chain (E)- and (Z)-isomers. Here we show that despite existing as a mixture of isomers, N-acetylglucosamine (GlcNAc) oximes can still be substrates for ß(1,4)-galactosyltransferase (ß4GalT1). ß4GalT1 catalysed the galactosylation of GlcNAc oximes by a galactose donor (UDP-Gal) both in solution and in situ on the surface of liposomes, with conversions up to 60% in solution and ca. 15-20% at the liposome surface. It is proposed that the ß-anomer is consumed preferentially but long reaction times allow this isomer to be replenished by equilibration from the remaining isomers. Adding further enzymes gave more complex oligosaccharides, with a combination of α-1,3-fucosyltransferase, ß4GalT1 and the corresponding sugar donors providing Lewis X coated liposomes. However, sialylation using T. cruzi trans-sialidase and sialyllactose provided only very small amounts of sialyl Lewis X (sLex) capped lipid. These observations show that combining oxime formation with enzymatic elaboration will be a useful method for the high-throughput surface modification of drug delivery vehicles, such as liposomes, with cell-targeting oligosaccharides.

One-Step Biocatalytic Synthesis of Sustainable Surfactants by Selective Amide Bond Formation.

N-alkanoyl-N-methylglucamides (MEGAs) are non-toxic surfactants widely used as commercial ingredients, but more sustainable syntheses towards these compounds are highly desirable. Here, we present a biocatalytic route towards MEGAs and analogues using a truncated carboxylic acid reductase construct tailored for amide bond formation (CARmm-A). CARmm-A is capable of selective amide bond formation without the competing esterification reaction observed in lipase catalysed reactions. A kinase was implemented to regenerate ATP from polyphosphate and by thorough reaction optimisation using design of experiments, the amine concentration needed for amidation was significantly reduced. The wide substrate scope of CARmm-A was exemplified by the synthesis of 24 commercially relevant amides, including selected examples on a preparative scale. This work establishes acyl-phosphate mediated chemistry as a highly selective strategy for biocatalytic amide bond formation in the presence of multiple competing alcohol functionalities.

Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios.

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees of drug-to-antibody ratio (DAR), in particular on native antibodies. Here, a glycoengineering methodology was developed to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This four-step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions.

Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor.

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.

A promiscuous glycosyltransferase generates poly-ß-1,4-glucan derivatives that facilitate mass spectrometry-based detection of cellulolytic enzymes.

Promiscuous activity of a glycosyltransferase was exploited to polymerise glucose from UDP-glucose via the generation of ß-1,4-glycosidic linkages. The biocatalyst was incorporated into biocatalytic cascades and chemo-enzymatic strategies to synthesise cello-oligosaccharides with tailored functionalities on a scale suitable for employment in mass spectrometry-based assays. The resulting glycan structures enabled reporting of the activity and selectivity of celluloltic enzymes.

Rapid Screening of Diverse Biotransformations for Enzyme Evolution.

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s per sample. The heat map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL) as a case study, enhancing its activity toward electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.

Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades*.

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof-of-principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross-reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O-methylnorbelladine.