A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

Novel ß-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154.

Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-ß-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-ß-Gal showed exo-ß-galactosidase activity with regiopreference for hydrolyzing ß-d-(1,6) glycosidic linkages. Notably, it exhibited a preference for d-glucopyranosyl (d-Glcp) over d-galactopyranosyl (d-Galp) and d-fructofuranosyl (d-Fruf) at the reducing end of the investigated disaccharides. In addition, we determined the high resolution crystal structure of BD-ß-Gal, thus providing the first structural characterization of a GH154 enzyme. Surprisingly, this revealed an (α/α)6 topology, which has not been observed before for ß-galactosidases. BD-ß-Gal displayed low structural homology with characterized CAZymes, but conservation analysis suggested that the active site was located in a central cavity, with conserved E73, R252, and D253 as putative catalytic residues. Interestingly, BD-ß-Gal has a tetrameric structure and a flexible loop from a neighboring protomer may contribute to its reaction specificity. Finally, we showed that the founding member of GH154, BT3677 from Bacteroides thetaiotaomicron, described as ß-glucuronidase, displayed exo-ß-galactosidase activity like BD-ß-Gal but lacked a tetrameric structure.

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans.

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a ß-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.

A tripartite carbohydrate-binding module to functionalize cellulose nanocrystals.

The development of protein and microorganism engineering have led to rising expectations of biotechnology in the design of emerging biomaterials, putatively of high interest to reduce our dependence on fossil carbon resources. In this way, cellulose, a renewable carbon based polysaccharide and derived products, displays unique properties used in many industrial applications. Although the functionalization of cellulose is common, it is however limited in terms of number and type of functions. In this work, a Carbohydrate-Binding Module (CBM) was used as a central core to provide a versatile strategy to bring a large diversity of functions to cellulose surfaces. CBM3a from Clostridium thermocellum, which has a high affinity for crystalline cellulose, was flanked through linkers with a streptavidin domain and an azide group introduced through a non-canonical amino acid. Each of these two extra domains was effectively produced and functionalized with a variety of biological and chemical molecules. Structural properties of the resulting tripartite chimeric protein were investigated using molecular modelling approaches, and its potential for the multi-functionalization of cellulose was confirmed experimentally. As a proof of concept, we show that cellulose can be labelled with a fluorescent version of the tripartite protein grafted to magnetic beads and captured using a magnet.

Enantioselective Reductive Oligomerization of Carbon Dioxide into l-Erythrulose via a Chemoenzymatic Catalysis.

A cell-free enantioselective transformation of the carbon atom of CO2 has never been reported. In the urgent context of transforming CO2 into products of high value, the enantiocontrolled synthesis of chiral compounds from CO2 would be highly desirable. Using an original hybrid chemoenzymatic catalytic process, we report herein the reductive oligomerization of CO2 into C3 (dihydroxyacetone, DHA) and C4 (l-erythrulose) carbohydrates, with perfect enantioselectivity of the latter chiral product. This was achieved with the key intermediacy of formaldehyde. CO2 is first reduced selectively by 4e- by an iron-catalyzed hydroboration reaction, leading to the isolation and complete characterization of a new bis(boryl)acetal compound derived from dimesitylborane. In an aqueous buffer solution at 30 °C, this compound readily releases formaldehyde, which is then involved in selective enzymatic transformations, giving rise either (i) to DHA using a formolase (FLS) catalysis or (ii) to l-erythrulose with a cascade reaction combining FLS and d-fructose-6-phosphate aldolase (FSA) A129S variant. Finally, the nature of the synthesized products is noteworthy, since carbohydrates are of high interest for the chemical and pharmaceutical industries. The present results prove that the cell-free de novo synthesis of carbohydrates from CO2 as a sustainable carbon source is a possible alternative pathway in addition to the intensely studied biomass extraction and de novo syntheses from fossil resources.

Synthesis of α-l-Araf and ß-d-Galf series furanobiosides using mutants of a GH51 α-l-arabinofuranosidase.

The GH-51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) possesses versatile catalytic properties, displaying not only the ability to hydrolyze glycosidic linkages but also to synthesize furanobiosides in α-l-Araf and ß-d-Galf series. Herein, mutants are investigated to evaluate their ability to perform self-condensation, assessing both yield improvements and changes in regioselectivity. Overall yields of oligo-α-l-arabino- and oligo-ß-d-galactofuranosides were increased up to 4.8-fold compared to the wild-type enzyme. In depth characterization revealed that the mutants exhibit increased transfer rates and thus a hydrolysis/self-condensation ratio in favor of synthesis. The consequence of the substitution N216W is the creation of an additional binding subsite that provides the basis for an alternative acceptor substrate binding mode. As a result, mutants bearing N216W synthesize not only (1,2)-linked furanobiosides, but also (1,3)- and even (1,5)-linked furanobiosides. Since the self-condensation is under kinetic control, the yield of homo-disaccharides was maximized using higher substrate concentrations. In this way, the mutant R69H-N216W produced oligo-ß-d-galactofuranosides in > 70% yield. Overall, this study further demonstrates the potential usefulness of TxAbf mutants for glycosynthesis and shows how these might be used to synthesize biologically-relevant glycoconjugates.

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans.

BACKGROUND: Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized 'treasure troves' in the era of exponentially growing numbers of sequenced genomes. RESULTS: We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/ß-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. CONCLUSIONS: The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

Regioselective chemoenzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases.

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. For feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl 5-O-feruloyl-α-ʟ-arabinofuranosides were significantly shortened (from 7 or 8 to 4-6 steps), and the transesterification yields were enhanced (from 46 to 73% and from 47 to 86%, respectively). This was achieved using enzymatic (immobilized Lipozyme® TL IM from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxy group of α-ʟ-arabinofuranosides. Moreover, a novel feruloylated 4-nitrocatechol-1-yl-substituted butanetriol analog, containing a cleavable hydroxylated linker, was also synthesized in 32% overall yield in 3 steps (convergent synthesis). The latter route combined the regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of this strategy to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d/l-, α/ß-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.

Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-l-arabinofuranosidase.

The use of retaining glycoside hydrolases as synthetic tools for glycochemistry is highly topical and the focus of considerable research. However, due to the incomplete identification of the molecular determinants of the transglycosylation/hydrolysis partition (t/h), rational engineering of retaining glycoside hydrolases to create transglycosylases remains challenging. Therefore, to understand better the factors that underpin transglycosylation in a GH51 retaining α-l-arabinofuranosidase from Thermobacillus xylanilyticus, the investigation of this enzyme's active site was pursued. Specifically, the properties of two mutants, F26L and L352M, located in the vicinity of the active site are described, using kinetic and 3D structural analyses and molecular dynamics simulations. The results reveal that the presence of L352M in the context of a triple mutant (also containing R69H and N216W) generates changes both in the donor and acceptor subsites, the latter being the result of a domino-like effect. Overall, the mutant R69H-N216W-L352M displays excellent transglycosylation activity (70 % yield, 78 % transfer rate and reduced secondary hydrolysis of the product). In the course of this study, the central role played by the conserved R69 residue was also reaffirmed. The mutation R69H affects both the catalytic nucleophile and the acid/base, including their flexibility, and has a determinant effect on the t/h partition. Finally, the results reveal that increased loop flexibility in the acceptor subsites creates new interactions with the acceptor, in particular with a hydrophobic binding platform composed of N216W, W248 and W302.

Synthetic Derivatives of (+)-epi-α-Bisabolol Are Formed by Mammalian Cytochromes P450 Expressed in a Yeast Reconstituted Pathway.

Identification of the enzyme(s) involved in complex biosynthetic pathways can be challenging. An alternative approach might be to deliberately diverge from the original natural enzyme source and use promiscuous enzymes from other organisms. In this paper, we have tested the ability of a series of human and animal cytochromes P450 involved in xenobiotic detoxification to generate derivatives of (+)-epi-α-bisabolol and attempt to produce the direct precursor of hernandulcin, a sweetener from Lippia dulcis for which the last enzymatic steps are unknown. Screening steps were implemented in vivo in S. cerevisiae optimized for the biosynthesis of oxidized derivatives of (+)-epi-α-bisabolol by coexpressing two key enzymes: the (+)-epi-α-bisabolol synthase and the NADPH cytochrome P450 reductase. Five out of 25 cytochromes P450 were capable of producing new hydroxylated regioisomers of (+)-epi-α-bisabolol. Of the new oxidized bisabolol products, the structure of one compound, 14-hydroxy-(+)-epi-α-bisabolol, was fully elucidated by NMR while the probable structure of the second product was determined. In parallel, the production of (+)-epi-α-bisabolol derivatives was enhanced through the addition of a supplementary genomic copy of (+)-epi-α-bisabolol synthase that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism cytochromes P450 can be used to produce novel compounds from a terpene scaffold.

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides.

Combined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities. In this review, we intend to give a brief overview of the advantageous use of engineered glycoenzymes, sometimes in combination with chemical steps, for the synthesis of natural bioactive oligosaccharides or their precursors. The focus will be on examples resulting from the three main classes of glycoenzymes specialized in carbohydrate synthesis: glycosyltransferases, glycoside hydrolases and glycoside phosphorylases.

Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants.

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a ß-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.

Biochemical identification of the catalytic residues of a glycoside hydrolase family 120 ß-xylosidase, involved in xylooligosaccharide metabolisation by gut bacteria.

The ß-xylosidase B from Bifidobacterium adolescentis ATCC15703 belongs to the newly characterized family 120 of glycoside hydrolases. In order to investigate its catalytic mechanism, an extensive kinetic study of the wild-type enzyme and mutants targeting the three highly conserved residues Asp(393), Glu(416) and Glu(364) was performed. NMR analysis of the xyloside hydrolysis products, the change of the reaction rate-limiting step for the Glu(416) mutants, the pH dependency of E416A activity and its chemical rescue allowed to demonstrate that this GH120 enzyme uses a retaining mechanism of glycoside hydrolysis, Glu(416) playing the role of acid/base catalyst and Asp(393) that of nucleophile.

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases.

Carbohydrates are ubiquitous in Nature and play vital roles in many biological systems. Therefore the synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, carbohydrates are challenging, due to the large number of sugar subunits and the multiple ways in which these can be linked together. Therefore, to tackle the challenge of glycosynthesis, chemists are increasingly turning their attention towards enzymes, which are exquisitely adapted to the intricacy of these biomolecules. In Nature, glycosidic linkages are mainly synthesized by Leloir glycosyltransferases, but can result from the action of non-Leloir transglycosylases or phosphorylases. Advantageously for chemists, non-Leloir transglycosylases are glycoside hydrolases, enzymes that are readily available and exhibit a wide range of substrate specificities. Nevertheless, non-Leloir transglycosylases are unusual glycoside hydrolases in as much that they efficiently catalyse the formation of glycosidic bonds, whereas most glycoside hydrolases favour the mechanistically related hydrolysis reaction. Unfortunately, because non-Leloir transglycosylases are almost indistinguishable from their hydrolytic counterparts, it is unclear how these enzymes overcome the ubiquity of water, thus avoiding the hydrolytic reaction. Without this knowledge, it is impossible to rationally design non-Leloir transglycosylases using the vast diversity of glycoside hydrolases as protein templates. In this critical review, a careful analysis of literature data describing non-Leloir transglycosylases and their relationship to glycoside hydrolase counterparts is used to clarify the state of the art knowledge and to establish a new rational basis for the engineering of glycoside hydrolases.

Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 α-L-arabinofuranosidase.

Random mutagenesis was performed on the α-l-arabinofuranosidase of Thermobacillus xylanilyticus in order to enhance its ability to perform transarabinofuranosylation using natural xylo-oligosaccharides as acceptors. To achieve this goal, a two-step, high-throughput digital imaging protocol involving a colorimetric substrate was used to screen a library of 30,000 mutants. In the first step this screen selected for hydrolytically-impaired mutants, and in the second step the screen identified mutants whose global activity was improved in the presence of a xylo-oligosaccharide mixture. Thereby, 199 mutants displaying lowered hydrolytic activity and modified properties were detected. In the presence of these xylo-oligosaccharides, most of the 199 (i.e., 70%) enzymes were less inhibited and some (18) mutants displayed an unambiguous alleviation of inhibition (<25% loss of activity). More precise monitoring of reactions catalyzed by the most promising mutants revealed a significant improvement of the synthesis yields of transglycosylation products (up to 18% compared to 9% for the parental enzyme) when xylobiose was present in the reaction. Genetic analysis of improved mutants revealed that many of the amino acid substitutions that correlate with the modified phenotype are located in the vicinity of the active site, particularly in subsite -1. Consequently, we hypothesize that these mutations modify the active site topology or the molecular interaction network of the l-arabinofuranoside donor substrate, thus impairing the hydrolysis and concomitantly favoring transglycosylation onto natural acceptors.

A ¹H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates.

BACKGROUND: The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs). METHODS: Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs. RESULTS: The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups. CONCLUSIONS AND GENERAL SIGNIFICANCE: The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.

Mutation of a pH-modulating residue in a GH51 α-l-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products.

BACKGROUND: The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition. METHODS: Detailed kinetic analysis (kcat, KM, pKa determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution. RESULTS: The mutants N344P and N344Y displayed crippled hydrolytic abilities, and thus procured improved transglycosylation yields. This behavior was correlated with an increased pKa of the catalytic nucleophile (E298), the pKa of the acid/base catalyst remaining unaffected. Finally, mutations at position 344 provoked a pH-dependent product inhibition phenomenon, which is likely to be the result of a significant modification of the proton sharing network in the mutants. CONCLUSIONS AND GENERAL SIGNIFICANCE: Using a combination of biochemical and biophysical methods, we have studied TxAbf-N344 mutants, thus revealing some fundamental details concerning pH modulation. Although these results concern a GH51 α-l-arabinofuranosidase, it is likely that the general lessons that can be drawn from them will be applicable to other glycoside hydrolases. Moreover, the effects of mutations at position 344 on the transglycosylation/hydrolysis partition provide clues as to how TxAbf can be further engineered to obtain an efficient transfuranosidase.