Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.     

Engineering Glyco-Enzymes for Substrate Identification and Targeting

Glycoconjugates are assembled by the coordinate actions of glycosyltransferases, which add sugars, and glycosidases, which remove sugars. These glyco-enzymes comprise families of enzymes that catalyze the same reaction, making it difficult to identify the direct substrates of each isozyme. To solve this challenge, mutagenesis of glyco-enzymes has been used to enable incorporation of unnatural sugar analogs that can be employed to tag and isolate the protein substrates of an individual glycosyltransferase. A second challenge arises in efforts to determine which substrates mediate biological effects. Engineering a glycosyltransferase to target its activity toward select acceptor substrates allows deconvolution of the roles of specific glycosylation events. Similarly, glycosidases can be engineered to target specific substrates, with basic science and translational applications. This review describes recent efforts at engineering glyco-enzymes to identify and target their distinct substrates.     

Enzymatic synthesis of manno- and heteromanno-oligosaccharides using α-mannosidases: A comparative study of linkage-specific and non-linkage-specific enzymes

Two α-mannosidase enzymes (EC 3.2.1.24) and an α-1,2-mannosidase enzyme (EC 3.2.1.113) were compared for their ability to synthesise hetero-oligosaccharides by the equilibrium approach (condensation reaction). A panel of disaccharide acceptors was used and product yields and product spectra determined. Choice of enzyme had a significant influence on product yield, with yield being dependent on enzyme and substrate combination. Most of the enzyme/substrate combinations tested gave 1→6 linked manno-oligosaccharides as the principal product. Mannose was linked to lactose, cellobiose and lactulose by both 1→3 and 1→6 linkages in ratios that depended upon the enzyme used. Homo-oligosaccharide (mannotriose) formation was found in all but one of the enzyme/substrate combinations tested and, in the case of the α1,2-mannosidase, the structure of the mannotrioses depended upon the acceptor used. Formation of hetero-oligosaccharides over homo-oligosaccharides could be favoured by use of low mannose: acceptor ratios. © 1998 Society of Chemical Industry     

Comparing the Catalytic and Structural Characteristics of a ‘Short’ Unspecific Peroxygenase (UPO) Expressed in Pichia pastoris and Escherichia coli

Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an ‘artificial’ peroxygenase (artUPO), close in sequence to the ‘short’ UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)-alcohols, also given by a variant of the ‘long’ UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related'short’ UPOs.     

Novel fluorescent phosphonic acid esters for discrimination of lipases and esterases

Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)selective modification of synthetic substrates. Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations. The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis. The selectivities of the inhibitors were determined by using different (phospho)lipase, esterase and cholesterol esterase preparations. The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor. We identified inhibitors with very low specificity, and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases. A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.     

Specificity fingerprinting of retaining beta-1,4-glycanases in the Cellulomonas fimi secretome using two fluorescent mechanism-based probes

Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.     

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

Cytochrome P450 enzymes (P450s, CYPs) catalyze the oxidative transformation of a wide range of organic substrates. Their functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The enzymes are promising for synthetic biology applications but limited by several drawbacks including low turnover rates, poor stability, the dependance of expensive cofactors and redox partners, and the narrow substrate scope. To conquer these obstacles, emerging strategies including substrate engineering, usage of decoy and decoy-based small molecules auxiliaries, designing of artificial enzyme cascades and the incorporation of materials have been explored based on the unique properties of P450s. These strategies can be applied to a wide range of P450s and can be combined with protein engineering to improve the enzymatic activities. This minireview will focus on some recent developments of these strategies which have been used to leverage P450 catalysis. Remaining challenges and future opportunities will also be discussed.     

Comparison of various methods to determine kinetic constants for β-galactosidase in soluble and immobilised states

The graphical methods of Lineweaver—Burk, Eadie, Hanes and the ‘direct linear plot’ have been used to determine the kinetic constants of β-galactosidase in soluble and immobilised states. Because some of these methods require a reciprocal concentration scale, the activity values obtained from dilute substrate solutions are weighted more than when using more concentrated substrates. However, in the case of immobilised enzymes the values from dilute substrate are more affected by diffusion from the bulk solution into the pores where the enzyme is fixed, and vice versa. Therefore it, seems preferable to use methods with direct substrate and velocity scales. The direct linear plot method is recommended because it does not require any transformation of the measurements, the area of intersection points indicates the accuracy of the measurement and runaway points are easily detected.     

The effect of immobilization on the hydrolytic/interesterification activity of lipase from Rhizopus niveus

Lipase from Rhizopus niveus was immobilized by physical adsorption on Celite 545 and glass beads. The results showed that the highest immobilization efficiency and specific hydrolytic activity of 96% and 9.2 meq/mg protein/min, respectively, were obtained with Celite as the carrier. However, the specific hydrolytic activity of lipase adsorbed on glass beads by acetone precipitation was similar to that obtained by the Celite carrier, although the protein loading capacity was relatively low. The results showed that lipase immobilized on glass beads exhibited similar activity profiles with respect to reaction time, different enzyme concentrations, and water content, using trimyristin and tripalmitin as substrates, to those obtained with the free enzyme. In contrast, the immobilized lipase on Celite exhibited a considerably lower hydrolytic activity. However, the results also showed that the lipase activities of the free enzyme and the immobilized Celite enzyme were similar when the more hydrophilic triolein was used as the substrate. The interesterification of a mixture of tripalmitin and trimyristin or triolein was carried out using both the free and immobilized enzymes. The results indicated that the hydrolytic activity of lipase was similar in both cases for the first 24 h, after which it decreased dramatically. These findings suggest that at this late stage an equilibrium between the hydrolytic and interesterification reactions was reached.     

N‐Alkyl‐, 1‐C‐Alkyl‐, and 5‐C‐Alkyl‐1,5‐dideoxy‐1,5‐imino‐(l)‐ribitols as Galactosidase Inhibitors

A series of 1,5‐dideoxy‐1,5‐imino‐(l )‐ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4‐epi‐isofagomine (4‐epi‐IFG) mimics and were expected to behave as selective inhibitors of β‐galactosidases. All compounds were indeed found to be highly selective for β‐galactosidases versus α‐glycosidases, as they generally did not inhibit coffee bean α‐galactosidase or other α‐glycosidases. Some compounds were also found to be inhibitors of almond β‐glucosidase. The N‐alkyl DIR derivatives were only modest inhibitors of bovine β‐galactosidase, with IC₅₀ values in the 30–700 μm range. Likewise, imino‐l ‐ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced β‐galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the ‘pseudo‐anomeric’ configuration in this series does not appear to play a role. Human lysosomal β‐galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC₅₀ values in the 100 μm range), while 4‐epi‐IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1‐gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7‐fold.     

Analyzing the Substrate Specificity of a Class of Long-Horned-Beetle-Derived Xylanases by Using Synthetic Arabinoxylan Oligo- and Polysaccharides

Xylophagous long-horned beetles thrive in challenging environments. To access nutrients, they secrete plant-cell-wall-degrading enzymes in their gut fluid; among them are cellulases of the subfamily 2 of glycoside hydrolase family 5 (GH5_2). Recently, we discovered that several beetle-derived GH5_2s use xylan as a substrate instead of cellulose, which is unusual for this family of enzymes. Here, we analyze the substrate specificity of a GH5_2 xylanase from the beetle Apriona japonica (AJAGH5_2-1) using commercially available substrates and synthetic arabinoxylan oligo- and polysaccharides. We demonstrate that AJAGH5_2-1 processes arabinoxylan polysaccharides in a manner distinct from classical xylanase families such as GH10 and GH11. AJAGH5_2-1 is active on long oligosaccharides and cleaves at the non-reducing end of a substituted xylose residue (position +1) only if: 1) three xylose residues are present upstream and downstream of the cleavage site, and 2) xylose residues at positions −1, −2, +2 and +3 are not substituted.     

Stability and Stabilization of Enzymes from Mesophilic and Thermophilic Organisms in Respect to Their Use in FIA-Systems for the Determination of L-Lactate and Acetate

The stabilization effect of ‘bilayer encagement’ on enzymes from mesophilic organisms and their ‘thermophilic’ counterparts was compared. Lactate dehydrogenases from pig heart and from a thermophilic bacterium (Clostridium thermohydrosulfuricum), respectively, showed stabilization factors of 4·5 (at 47°C) and 12·8 (at 70°C), respectively. For ‘thermophilic’ acetate kinase no stabilization effect of encagement was observed. Lactate dehydrogenase and acetate kinase from Clostridium thermohydrosulfuricum were immobilized to controlled porous glass and tested for their long-term stability. The ‘thermophilic’ enzymes showed by far a longer half-life as compared with the corresponding enzymes from pig heart and Escherichia coli, respectively, the half-life time of the flow injection system response with thermophilic lactate dehydrogenase at 35°C attaining 250 h (mesophilic enzyme 89 h), and with thermophilic acetate kinase 79 h (mesophilic enzyme 24 h). © 1997 SCI.     

Natural dyes in madder (Rubia spp.) and their extraction and analysis in historical textiles

Textiles coloration using extracts from the roots of various madder species (Rubia spp.) has been performed for centuries. To date, 68 anthraquinone colorants have been detected in Rubia spp. used to dye textiles. Many of these dyes are sensitive to hydrolysis and degradation from enzymes, extraction chemicals and processing temperatures, and are often overlooked as colorants in historical textiles. Conclusions in literature of the past 30 years concerning colorants present in planta and, particularly, in madder‐dyed artefacts are being challenged as new analysis methods are developed. The recent advent of ‘soft’ extraction techniques has demonstrated that anthraquinone glycosides and other sensitive molecules, such as carboxylated compounds, need to be preserved; this valuable chemical information embedded in the dye structure may be lost if extraction and analysis is too harsh. Some compounds thought to be present in madder and madder‐dyed artefacts are in fact degradation products resultant from the extraction process, and degradation pathways have been developed to better understand the reactivity and stability of these compounds. Detailed analysis of dyes in textile artefacts can reveal important cultural and heritage information concerning historical textiles relative to the specific dye species, the area of the world where this may have grown, how and where it was dyed, and, perhaps, where it was traded. Understanding the precise molecular structure of these dyes and their chemical reactivity is important to provide knowledge of their interactions with physical substrates, such as textile fibres, which could be used to develop superior techniques for analysis of artefacts.     

Biocatalytic Synthesis of Nitriles through Dehydration of Aldoximes: The Substrate Scope of Aldoxime Dehydratases

Nitriles, which are mostly needed and produced by the chemical industry, play a major role in various industry segments, ranging from high‐volume, low‐price sectors, such as polymers, to low‐volume, high‐price sectors, such as chiral pharma drugs. A common industrial technology for nitrile production is ammoxidation as a gas‐phase reaction at high temperature. Further popular approaches are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed with aldoxime dehydratases, which are capable of converting an aldoxime in one step through dehydration into nitriles. Because the aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria for application as a “synthetic platform technology”. Thus, the scope of this review is to give an overview of the current state of the substrate scope of this enzymatic method for synthesizing nitriles with aldoxime dehydratases. As a recently emerging enzyme class, a range of substrates has already been studied so far, comprising nonchiral and chiral aldoximes. This enzyme class of aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic aldoximes, as well as arylaliphatic aldoximes. Furthermore, aldoximes with a stereogenic center are also recognized and high enantioselectivities are found for 2‐arylpropylaldoximes, in particular. It is further noteworthy that the enantiopreference depends on the E and Z isomers. Thus, opposite enantiomers are accessible from the same racemic aldehyde and the same enzyme.     

Towards Tuneable Retaining Glycosidase‐Inhibiting Peptides by Mimicry of a Plant Flavonol Warhead

Retaining glycosidases are an important class of enzymes involved in glycan degradation. To study better the role of specific enzymes in deglycosylation processes, and thereby the importance of particular glycosylation patterns, a set of potent inhibitors, each specific to a particular glycosidase, would be an invaluable toolkit. Towards this goal, we detail here a more in‐depth study of a prototypical macrocyclic peptide inhibitor of the model retaining glycosidase human pancreatic α‐amylase (HPA). Notably, incorporation of l ‐DOPA into this peptide affords an inhibitor of HPA with potency that is tenfold higher (K_i=480 pm ) than that of the previously found consensus sequence. This represents a first successful step in converting a recently discovered natural‐product‐derived motif, already specific for the catalytic side‐chain arrangement conserved in the active sites of retaining glycosidases, into a tuneable retaining glycosidase inhibition warhead.     

Molecular basis of lipase stereoselectivity

Lipases exhibit specific catalytic properties that make them attractive to biotechnological applications. Most important are the broad substrate specificity and the regio‐ and stereoselectivity of lipases. Despite mechanistic and structural similarities lipases differ significantly with respect to stereoselectivity toward natural and synthetic substrates. Models developed to describe and predict stereoselectivity toward certain types of synthetic substrates, e. g., secondary alcohols cannot be applied to natural acylglycerols, that are hydrolyzed by several animal and microbial lipases in a regioselective or stereoselective manner. Therefore, computer‐aided molecular modeling studies were used in order to predict the stereopreference of lipases toward triradylglycerols. Lipase variants with modified stereoselectivity properties toward triacylglycerols were engineered by re‐designing the recombinant enzyme. To understand the interactions governing lipase stereoselectivity towards natural substrates, knowledge of the structure of enzyme‐substrate complexes at the atomic level is essential. Such information can be obtained by X‐ray or NMR analysis of covalent enzyme‐inhibitor complexes. The crystal structures of enzymes complexed with triacylglycerol analog inhibitors allowed the identification of distinct binding sites for the three hydrophobic chains of the inhibitor.     

Enzymatic Thioxyloside Synthesis: Characterization of Thioglycoligase Variants Identified from A Site‐Saturation Mutagenesis Library of Bacillus Circulans Xylanase

Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site‐saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.     

Fermentation inhibitors in wood hydrolysates derived from the softwood Pinus radiata

Sugar solutions obtained by dilute acid hydrolysis of the softwood Pinus radiata contain various wood-derived components which are inhibitory to the ethanolic fermentation by the yeast Saccharomyces cerevisiae. A detailed examination of these compounds indicated that a range of low molecular weight phenolics, related in structure to ‘Hibberts ketones’, may be identified as the most inhibitory materials. These lignin-based compounds, although present at low levels compared with the carbohydrate-degradation compounds, are approximately 10 times more inhibitory.     

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Sortase‐catalyzed transacylation reactions are widely used for the construction of non‐natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase‐mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site‐specific modification of the N‐terminal serine residue in the naturally occurring antimicrobial peptide DCD‐1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.