Fructosyl Amino Oxidase as a Therapeutic Enzyme in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is an age-related disorder that is a global public health problem. The non-enzymatic Maillard reaction results in the formation of advanced glycation end products (AGEs). Accumulation of AGEs in drusen plays a key role in AMD. AGE-reducing drugs may contribute to the prevention and treatment of AGE-related disease. Fructosamine oxidase (FAOD) acts on fructosyl lysine and fructosyl valine. Based upon the published results of fructosamine 3-kinase (FN3K) and FAOD obtained in cataract and presbyopia, we studied ex vivo FAOD treatment as a non-invasive AMD therapy. On glycolaldehyde-treated porcine retinas, FAOD significantly reduced AGE autofluorescence (p = 0.001). FAOD treatment results in a breakdown of AGEs, as evidenced using UV fluorescence, near-infrared microspectroscopy on stained tissue sections of human retina, and gel permeation chromatography. Drusen are accumulations of AGEs that build up between Bruch's membrane and the retinal pigment epithelium. On microscopy slides of human retina affected by AMD, a significant reduction in drusen surface to 45 ± 21% was observed following FAOD treatment. Enzymatic digestion followed by mass spectrometry of fructose- and glucose-based AGEs (produced in vitro) revealed a broader spectrum of substrates for FAOD, as compared to FN3K, including the following: fructosyllysine, carboxymethyllysine, carboxyethyllysine, and imidazolone. In contrast to FN3K digestion, agmatine (4-aminobutyl-guanidine) was formed following FAOD treatment in vitro. The present study highlights the therapeutic potential of FAOD in AMD by repairing glycation-induced damage.

Nucleotide sugar dehydratases: Structure, mechanism, substrate specificity, and application potential.

Nucleotide sugar (NS) dehydratases play a central role in the biosynthesis of deoxy and amino sugars, which are involved in a variety of biological functions in all domains of life. Bacteria are true masters of deoxy sugar biosynthesis as they can produce a wide range of highly specialized monosaccharides. Indeed, deoxy and amino sugars play important roles in the virulence of gram-positive and gram-negative pathogenic species and are additionally involved in the biosynthesis of diverse macrolide antibiotics. The biosynthesis of deoxy sugars relies on the activity of NS dehydratases, which can be subdivided into three groups based on their structure and reaction mechanism. The best-characterized NS dehydratases are the 4,6-dehydratases that, together with the 5,6-dehydratases, belong to the NS-short-chain dehydrogenase/reductase superfamily. The other two groups are the less abundant 2,3-dehydratases that belong to the Nudix hydrolase superfamily and 3-dehydratases, which are related to aspartame aminotransferases. 4,6-Dehydratases catalyze the first step in all deoxy sugar biosynthesis pathways, converting nucleoside diphosphate hexoses to nucleoside diphosphate-4-keto-6-deoxy hexoses, which in turn are further deoxygenated by the 2,3- and 3-dehydratases to form dideoxy and trideoxy sugars. In this review, we give an overview of the NS dehydratases focusing on the comparison of their structure and reaction mechanisms, thereby highlighting common features, and investigating differences between closely related members of the same superfamilies.

The Conversion of UDP-Glc to UDP-Man: In Silico and Biochemical Exploration To Improve the Catalytic Efficiency of CDP-Tyvelose C2-Epimerases.

A promiscuous CDP-tyvelose 2-epimerase (TyvE) from Thermodesulfatator atlanticus (TaTyvE) belonging to the nucleotide sugar active short-chain dehydrogenase/reductase superfamily (NS-SDRs) was recently discovered. TaTyvE performs the slow conversion of NDP-glucose (NDP-Glc) to NDP-mannose (NDP-Man). Here, we present the sequence fingerprints that are indicative of the conversion of UDP-Glc to UDP-Man in TyvE-like enzymes based on the heptagonal box motifs. Our data-mining approach led to the identification of 11 additional TyvE-like enzymes for the conversion of UDP-Glc to UDP-Man. We characterized the top two wild-type candidates, which show a 15- and 20-fold improved catalytic efficiency, respectively, on UDP-Glc compared to TaTyvE. In addition, we present a quadruple variant of one of the identified enzymes with a 70-fold improved catalytic efficiency on UDP-Glc compared to TaTyvE. These findings could help the design of new nucleotide production pathways starting from a cheap sugar substrate like glucose or sucrose.

Topical Application of Deglycating Enzymes as an Alternative Non-Invasive Treatment for Presbyopia.

Presbyopia is an age-related vision disorder that is a global public health problem. Up to 85% of people aged ≥40 years develop presbyopia. In 2015, 1.8 billion people globally had presbyopia. Of those with significant near vision disabilities due to uncorrected presbyopia, 94% live in developing countries. Presbyopia is undercorrected in many countries, with reading glasses available for only 6-45% of patients living in developing countries. The high prevalence of uncorrected presbyopia in these parts of the world is due to the lack of adequate diagnosis and affordable treatment. The formation of advanced glycation end products (AGEs) is a non-enzymatic process known as the Maillard reaction. The accumulation of AGEs in the lens contributes to lens aging (leading to presbyopia and cataract formation). Non-enzymatic lens protein glycation induces the gradual accumulation of AGEs in aging lenses. AGE-reducing compounds may be effective at preventing and treating AGE-related processes. Fructosyl-amino acid oxidase (FAOD) is active on both fructosyl lysine and fructosyl valine. As the crosslinks encountered in presbyopia are mainly non-disulfide bridges, and based on the positive results of deglycating enzymes in cataracts (another disease caused by glycation of lens proteins), we studied the ex vivo effects of topical FAOD treatment on the power of human lenses as a new potential non-invasive treatment for presbyopia. This study demonstrated that topical FAOD treatment resulted in an increase in lens power, which is approximately equivalent to the correction obtained by most reading glasses. The best results were obtained for the newer lenses. Simultaneously, a decrease in lens opacity was observed, which improved lens quality. We also demonstrated that topical FAOD treatment results in a breakdown of AGEs, as evidenced by gel permeation chromatography and a marked reduction in autofluorescence. This study demonstrated the therapeutic potential of topical FAOD treatment in presbyopia.

A colorimetric assay for the screening and kinetic analysis of nucleotide sugar 4,6-dehydratases.

Nucleotide sugar 4,6-dehydratases belong to the Short-chain Dehydrogenase/Reductase (SDR) superfamily and catalyze the conversion of an NDP-hexose to an NDP-4-keto-6-deoxy hexose, a key step in the biosynthesis of a plethora of deoxy and amino sugars. Here, we present a colorimetric assay for the detection of their reaction products (NDP-4-keto-6-deoxy hexoses) using concentrated sulfuric acid and an ethanolic resorcinol solution. Under these conditions, the keto-function of the dehydratase product reacts specifically with resorcinol to form an orange-red or pink complex for NDP-glucose/GDP-mannose and UDP-N-acetylglucosamine, respectively, with an absorption maximum at 510 nm. The presented assay allows reliable product detection at low concentrations and can be applied in microtiter plates. It thus allows the determination of kinetic enzyme parameters like the optimal temperature, pH, Vmax, KM and kcat, as well as the miniaturization for screening purposes with crude cell extracts. As such, this detection assay opens new possibilities for the characterization and screening of these dehydratases in 96-well plates for different research goals.

Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol.

2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2-O-glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site-directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21-fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2-position of the substrate, while retaining its activity for several days at elevated temperatures.

Exploration of GH94 Sequence Space for Enzyme Discovery Reveals a Novel Glucosylgalactose Phosphorylase Specificity.

The substantial increase in DNA sequencing efforts has led to a rapid expansion of available sequences in glycoside hydrolase families. The ever-increasing sequence space presents considerable opportunities for the search for enzymes with novel functionalities. In this work, the sequence-function space of glycoside hydrolase family 94 (GH94) was explored in detail, using a combined approach of phylogenetic analysis and sequence similarity networks. The identification and experimental screening of unknown clusters led to the discovery of an enzyme from the soil bacterium Paenibacillus polymyxa that acts as a 4-O-ß-d-glucosyl-d-galactose phosphorylase (GGalP), a specificity that has not been reported to date. Detailed characterization of GGalP revealed that its kinetic parameters were consistent with those of other known phosphorylases. Furthermore, the enzyme could be used for production of the rare disaccharides 4-O-ß-d-glucosyl-d-galactose and 4-O-ß-d-glucosyl-l-arabinose. Our current work highlights the power of rational sequence space exploration in the search for novel enzyme specificities, as well as the potential of phosphorylases for rare disaccharide synthesis.

Expanding the Enzyme Repertoire for Sugar Nucleotide Epimerization: The CDP-Tyvelose 2-Epimerase from Thermodesulfatator atlanticus for Glucose/Mannose Interconversion.

Epimerization of sugar nucleotides is central to the structural diversification of monosaccharide building blocks for cellular biosynthesis. Epimerase applicability to carbohydrate synthesis can be limited, however, by the high degree of substrate specificity exhibited by most sugar nucleotide epimerases. Here, we discovered a promiscuous type of CDP-tyvelose 2-epimerase (TyvE)-like enzyme that promotes C2-epimerization in all nucleotide (CDP, UDP, GDP, ADP, TDP)-activated forms of d-glucose. This new epimerase, originating from Thermodesulfatator atlanticus, is a functional homodimer that contains one tightly bound NAD+/subunit and shows optimum activity at 70°C and pH 9.5. The enzyme exhibits a k cat with CDP-dglucose of ∼1.0 min-1 (pH 7.5, 60°C). To characterize the epimerase kinetically and probe its substrate specificity, we developed chemo-enzymatic syntheses for CDP-dmannose, CDP-6-deoxy-dglucose, CDP-3-deoxy-dglucose and CDP-6-deoxy-dxylo-hexopyranos-4-ulose. Attempts to obtain CDP-dparatose and CDP-dtyvelose were not successful. Using high-resolution carbohydrate analytics and in situ NMR to monitor the enzymatic conversions (60°C, pH 7.5), we show that the CDP-dmannose/CDP-dglucose ratio at equilibrium is 0.67 (± 0.1), determined from the kinetic Haldane relationship and directly from the reaction. We further show that deoxygenation at sugar C6 enhances the enzyme activity 5-fold compared to CDP-dglucose whereas deoxygenation at C3 renders the substrate inactive. Phylogenetic analysis places the T. atlanticus epimerase into a distinct subgroup within the sugar nucleotide epimerase family of SDR (short-chain dehydrogenases/reductases), for which the current study now provides the functional context. Collectively, our results expand an emerging toolbox of epimerase-catalyzed reactions for sugar nucleotide synthesis.IMPORTANCE Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest in carbohydrate (bio)chemistry, for the mechanistic challenges and the opportunities for synthesis involved in the reactions catalyzed. Discovery of new epimerases with expanded scope of sugar nucleotide substrates used is important to promote the mechanistic inquiry and can facilitate the development of new enzyme applications. Here, a CDP-tyvelose 2-epimerase-like enzyme from Thermodesulfatator atlanticus is shown to catalyze sugar C2 epimerization in CDP-glucose and other nucleotide-activated forms of dglucose. The reactions are new to nature in the context of enzymatic sugar nucleotide modification. The current study explores the substrate scope of the discovered C2-epimerase and, based on modeling, suggests structure-function relationships that may be important for specificity and catalysis.

ß-Glucan phosphorylases in carbohydrate synthesis.

ß-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of ß-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of ß-glucans as major bioactive compounds. Their preference for sugar phosphates (rather than nucleotide sugars) as donor substrates further underlines their significance for the carbohydrate industry. Presently, they are classified in the glycoside hydrolase families 94, 149, and 161 ( www.cazy.org ). Since the discovery of ß-1,3-oligoglucan phosphorylase in 1963, several other specificities have been reported that differ in linkage type and/or degree of polymerization. Here, we present an overview of the progress that has been made in our understanding of ß-glucan and associated ß-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules. KEY POINTS: • Discovery, characteristics, and applications of ß-glucan phosphorylases. • ß-Glucan phosphorylases in the production of functional carbohydrates.

Oral Microbiota Display Profound Differential Metabolic Kinetics and Community Shifts upon Incubation with Sucrose, Trehalose, Kojibiose, and Xylitol.

This study compares the metabolic properties of kojibiose, trehalose, sucrose, and xylitol upon incubation with representative oral bacteria as monocultures or synthetic communities or with human salivary bacteria in a defined medium. Compared to sucrose and trehalose, kojibiose resisted metabolism during a 48-h incubation with monocultures, except for Actinomyces viscosus Incubations with Lactobacillus-based communities, as well as salivary bacteria, displayed kojibiose metabolism, yet to a lesser extent than sucrose and trehalose. Concurring with our in vitro findings, screening for carbohydrate-active enzymes revealed that only Lactobacillus spp. and A. viscosus possess enzymes from glycohydrolase (GH) families GH65 and GH15, respectively, which are associated with kojibiose metabolism. Donor-dependent differences in salivary microbiome composition were noted, and differences in pH drop during incubation indicated different rates of sugar metabolism. However, functional analysis indicated that lactate, acetate, and formate evenly dominated the metabolic profile for all sugars except for xylitol. 16S rRNA gene sequencing analysis and α-diversity markers revealed that a significant shift of the microbiome community by sugars was more pronounced in sucrose and trehalose than in kojibiose and xylitol. In Streptococcus spp., a taxon linked to cariogenesis dominated in sucrose (mean ± standard deviation, 91.8 ± 6.4%) and trehalose (55.9 ± 38.6%), representing a high diversity loss. In contrast, Streptococcus (5.1 ± 3.7%) was less abundant in kojibiose, which instead was dominated by Veillonella (26.8 ± 19.6%), while for xylitol, Neisseria (29.4 ± 19.1%) was most abundant. Overall, kojibiose and xylitol incubations stimulated cariogenic species less yet closely maintained an abundance of key phyla and genera of the salivary microbiome, suggesting that kojibiose has low cariogenic properties.IMPORTANCE This study provides a detailed scientific insight on the metabolism of a rare disaccharide, kojibiose, whose mass production has recently been made possible. While the resistance of kojibiose was established with monocultures, delayed utilization of kojibiose was observed with communities containing lactobacilli and A. viscosus as well as with complex communities of bacteria from human saliva. Kojibiose is, therefore, less metabolizable than sucrose and trehalose. Moreover, although conventional sugars cause distinct shifts in salivary microbial communities, our study has revealed that kojibiose is able to closely maintain the salivary microbiome composition, suggesting its low cariogenic properties. This study furthermore underscores the importance and relevance of microbial culture and ex vivo mixed cultures to study cariogenicity and substrate utilization; this is in sharp contrast with tests that solely rely on monocultures such as Streptococcus mutans, which clearly fail to capture complex interactions between oral microbiota.

Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose.

Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Key points• Cellobiose phosphorylase is engineered for the production of cellotriose.• Cellotriose is synthesised with the highest purity and yield to date.• Both cellobiose and glucose can be used as acceptor for cellotriose production.

Synthesis, trehalase hydrolytic resistance and inhibition properties of 4- and 6-substituted trehalose derivatives.

Although trehalose has recently gained interest because of its pharmaceutical potential, its clinical use is hampered due to its low bioavailability. Hence, hydrolysis-resistant trehalose analogues retaining biological activity could be of interest. In this study, 34 4- and 6-O-substituted trehalose derivatives were synthesised using an ether- or carbamate-type linkage. Their hydrolysis susceptibility and inhibitory properties were determined against two trehalases, i.e. porcine kidney and Mycobacterium smegmatis. With the exception of three weakly hydrolysable 6-O-alkyl derivatives, the compounds generally showed to be completely resistant. Moreover, a number of derivatives was shown to be an inhibitor of one or both of these trehalases. For the strongest inhibitors of porcine kidney trehalase IC50 values of around 10 mM could be determined, whereas several compounds displayed sub-mM IC50 against M. smegmatis trehalase. Dockings studies were performed to explain the observed influence of the substitution pattern on the inhibitory activity towards porcine kidney trehalase.

Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase.

GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry.

Molecular Gating of an Engineered Enzyme Captured in Real Time.

Enzyme engineering tends to focus on the design of active sites for the chemical steps, while the physical steps of the catalytic cycle are often overlooked. Tight binding of a substrate in an active site is beneficial for the chemical steps, whereas good accessibility benefits substrate binding and product release. Many enzymes control the accessibility of their active sites by molecular gates. Here we analyzed the dynamics of a molecular gate artificially introduced into an access tunnel of the most efficient haloalkane dehalogenase using pre-steady-state kinetics, single-molecule fluorescence spectroscopy, and molecular dynamics. Photoinduced electron-transfer-fluorescence correlation spectroscopy (PET-FCS) has enabled real-time observation of molecular gating at the single-molecule level with rate constants ( kon = 1822 s-1, koff = 60 s-1) corresponding well with those from the pre-steady-state kinetics ( k-1 = 1100 s-1, k1 = 20 s-1). The PET-FCS technique is used here to study the conformational dynamics in a soluble enzyme, thus demonstrating an additional application for this method. Engineering dynamical molecular gates represents a widely applicable strategy for designing efficient biocatalysts.

Thermostable alpha-glucan phosphorylases: characteristics and industrial applications.

α-Glucan phosphorylases (α-GPs) catalyze the reversible phosphorolysis of α-1,4-linked polysaccharides such as glycogen, starch, and maltodextrins, therefore playing a central role in the usage of storage polysaccharides. The discovery of these enzymes and their role in the course of catalytic conversion of glycogen was rewarded with the Nobel Prize in Physiology or Medicine in 1947. Nowadays, however, thermostable representatives attract special attention due to their vast potential in the enzymatic production of diverse carbohydrates and derivatives such as (functional) oligo- and (non-natural) polysaccharides, artificial starch, glycosides, and nucleotide sugars. One of the most recently explored utilizations of α-GPs is their role in the multi-enzymatic process of energy production stored in carbohydrate biobatteries. Regardless of their use, thermostable α-GPs offer significant advantages and facilitated bioprocess design due to their high operational temperatures. Here, we present an overview and comparison of up-to-date characterized thermostable α-GPs with a special focus on their reported biotechnological applications.

Converting Galactose into the Rare Sugar Talose with Cellobiose 2-Epimerase as Biocatalyst.

Cellobiose 2-epimerase from Rhodothermus marinus (RmCE) reversibly converts a glucose residue to a mannose residue at the reducing end of ß-1,4-linked oligosaccharides. In this study, the monosaccharide specificity of RmCE has been mapped and the synthesis of d-talose from d-galactose was discovered, a reaction not yet known to occur in nature. Moreover, the conversion is industrially relevant, as talose and its derivatives have been reported to possess important antimicrobial and anti-inflammatory properties. As the enzyme also catalyzes the keto-aldo isomerization of galactose to tagatose as a minor side reaction, the purity of talose was found to decrease over time. After process optimization, 23 g/L of talose could be obtained with a product purity of 86% and a yield of 8.5% (starting from 4 g (24 mmol) of galactose). However, higher purities and concentrations can be reached by decreasing and increasing the reaction time, respectively. In addition, two engineering attempts have also been performed. First, a mutant library of RmCE was created to try and increase the activity on monosaccharide substrates. Next, two residues from RmCE were introduced in the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) (S99M/Q371F), increasing the kcat twofold.

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

Site-specific analysis of protein hydration based on unnatural amino acid fluorescence.

Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations.

Characterization and mutational analysis of the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis.

UDP-hexose 4-epimerases are important enzymes that play key roles in various biological pathways, including lipopolysaccharide biosynthesis, galactose metabolism through the Leloir pathway, and biofilm formation. Unfortunately, the determinants of their substrate specificity are not yet fully understood. They can be classified into three groups, with groups 1 and 3 preferring non-acetylated and acetylated UDP-hexoses, respectively, whereas members of group 2 are equally active on both types of substrates. In this study, the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis (mGalE) was functionally expressed in Escherichia coli and thoroughly characterized. The enzyme was found to be thermostable, displaying its highest activity at 70 °C and having a half-life of 23 min at 60 °C. Activity could be detected on both acetylated and non-acetylated UDP-hexoses, meaning that this epimerase belongs to group 2. This observation correlates well with the identity of the so-called "gatekeeper" residue (Ser279), which has previously been suggested to influence substrate specificity (Schulz et al., J Biol Chem 279:32796-32803, 2004). Furthermore, substituting this serine to a tyrosine brings about a significant preference for non-acetylated sugars, thereby demonstrating that a single residue can determine substrate specificity among type 1 and type 2 epimerases. In addition, two consecutive glycine residues (Gly118 and Gly119) were identified as a unique feature of GalE enzymes from Thermus species, and their importance for activity as well as affinity was confirmed by mutagenesis. Finally, homology modeling and mutational analysis has revealed that the enzyme's catalytic triad contains a threonine residue (Thr117) instead of the usual serine.

Rare Sugar Metabolism and Impact on Insulin Sensitivity along the Gut-Liver-Muscle Axis In Vitro.

Rare sugars have recently attracted attention as potential sugar replacers. Understanding the biochemical and biological behavior of these sugars is of importance in (novel) food formulations and prevention of type 2 diabetes. In this study, we investigated whether rare sugars may positively affect intestinal and liver metabolism, as well as muscle insulin sensitivity, compared to conventional sugars. Rare disaccharide digestibility, hepatic metabolism of monosaccharides (respirometry) and the effects of sugars on skeletal muscle insulin sensitivity (impaired glucose uptake) were investigated in, respectively, Caco-2, HepG2 and L6 cells or a triple coculture model with these cells. Glucose and fructose, but not l-arabinose, acutely increased extracellular acidification rate (ECAR) responses in HepG2 cells and impaired glucose uptake in L6 cells following a 24 h exposure at 28 mM. Cellular bioenergetics and digestion experiments with Caco-2 cells indicate that especially trehalose (α1-1α), D-Glc-α1,2-D-Gal, D-Glc-α1,2-D-Rib and D-Glc-α1,3-L-Ara experience delayed digestion and reduced cellular impact compared to maltose (α1-4), without differences on insulin-stimulated glucose uptake in a short-term setup with a Caco-2/HepG2/L6 triple coculture. These results suggest a potential for l-arabinose and specific rare disaccharides to improve metabolic health; however, additional in vivo research with longer sugar exposures should confirm their beneficial impact on insulin sensitivity in humans.