Characterization of Camphene- and Fenchol-Based Hydrophobic Eutectic Solvents and Their Application in Aldehyde Extraction.

Binary terpenoid-based eutectic systems consisting of the natural substances camphene (CA), fenchol (FE), thymol (TH), menthol (ME), dodecanoic acid (DA), and 1-dodecanol (DO) are synthesized and screened for their Solid-Liquid Equilibrium (SLE) and eutectic compositions. Out of nine eutectic systems, 13 solvent compositions at eutectic points and next to them, in addition to the reference solvent, TH:ME, are synthesized and applied for the solvent extraction of the aromatic aldehydes vanillin (VAN), syringaldehyde (SYR), and p-hydroxybenzaldehyde (HYD) from an acidic aqueous model solution. The extraction efficiency is determined from aldehyde concentrations measured by High-Performance Liquid Chromatography (HPLC), taking into consideration mutual solubility measured by Karl Fischer titration (KF) and a Total Organic Carbon-analysis (TOC). Physicochemical properties, such as the density, viscosity, and stability of the solvents, are evaluated and discussed. Additionally, 1H-NMR measurements are performed to verify hydrogen bonding present in some of the solvents. The results show that all synthesized eutectic systems have a strong hydrophobic character with a maximum water saturation of ≤2.21 vol.% and solvent losses of ≤0.12 vol.% per extraction step. The hydrophobic eutectic solvents based on CA exhibit lower viscosities, lower mutual solubility, and lower extraction efficiency for the aromatic aldehydes when compared with FE-based solvents. The highest extraction efficiencies for VAN (>95%) and for SYR (>93%) at an extraction efficiency of 92.61% for HYD are achieved by the reference solvent TH:ME (50:50 mol.%). With an extraction efficiency of 93.08%, HYD is most preferably extracted by the FE-DO-solvent (80:20 mol.%), where the extraction efficiencies for VAN and SYR reach their maximum at 93.37% and 90.75%, respectively. The drawbacks of the high viscosities of 34.741 mPas of the TH:ME solvent and 31.801 mPas of the FE-DO solvent can be overcome by the CA-TH solvent, which has a viscosity of 3.436 mPas, while exhibiting extraction efficiencies of 71.92% for HYD, >95% for VAN, and >93% for SYR, respectively.

Enzyme Machinery for Bacterial Glucoside Metabolism through a Conserved Non-hydrolytic Pathway.

Flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free-living and symbiotic bacteria have developed, alongside hydrolysis, a non-hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase-catalyzed reactions. Here, based on study of enzymes from the phytopathogen Agrobacterium tumefaciens, we reveal a conserved Mn2+ metallocenter active site in both lyases and identify the structural requirements for specific catalysis to elimination of 3-keto-glucosides and water addition to the resulting 2-hydroxy-3-keto-glycal product, yielding 3-keto-glucose. Extending our search of GUL-encoded putative lyases to the human gut commensal Bacteroides thetaiotaomicron, we discover a Ca2+ metallocenter active site in a putative glycoside hydrolase-like protein and demonstrate its catalytic function in the eliminative cleavage of 3-keto-glucosides of opposite (alpha) anomeric configuration as preferred by the A. tumefaciens enzyme (beta). Findings identify a basic set of GUL-encoded lyases for glucoside metabolism and assign physiological significance to GUL genetic diversity in bacteria.

Discovery, characterization, and comparative analysis of new UGT72 and UGT84 family glycosyltransferases.

Glycosylated derivatives of natural product polyphenols display a spectrum of biological activities, rendering them critical for both nutritional and pharmacological applications. Their enzymatic synthesis by glycosyltransferases is frequently constrained by the limited repertoire of characterized enzyme-catalyzed transformations. Here, we explore the glycosylation capabilities and substrate preferences of newly identified plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) within the UGT72 and UGT84 families, with particular focus on natural polyphenol glycosylation from UDP-glucose. Four UGTs are classified according to their phylogenetic relationships and reaction products, identifying them as biocatalysts for either glucoside (UGT72 enzymes) or glucose ester (UGT84 members) formation from selected phenylpropanoid compounds. Detailed kinetic evaluations expose the unique attributes of these enzymes, including their specific activities and regio-selectivities towards diverse polyphenolic substrates, with product characterizations validating the capacity of UGT84 family members to perform di-O-glycosylation on flavones. Sequence analysis coupled with structural predictions through AlphaFold reveal an unexpected absence of a conserved threonine residue across all four enzymes, a trait previously linked to pentosyltransferases. This comparative analysis broadens the understood substrate specificity range for UGT72 and UGT84 enzymes, enhancing our understanding of their utility in the production of natural phenolic glycosides. The findings from this in-depth characterization provide valuable insights into the functional versatility of UGT-mediated reactions.

The 'Myth of Zero-COVID' Nation: A Digital Ethnography of Expats' Survival Amid Shanghai Lockdown during the Omicron Variant Outbreak.

This study presents a digital ethnography of expats' survival amid the Shanghai lockdown during the Omicron variant outbreak. This study drew insights from studies on resilience and secondary coping within the context of global migration to comprehend the diverse emotional challenges faced by expats in a series of lockdowns and persistent nucleic acid amplification tests. Thus, this study asks what the major emotional challenges expats faced and what sources of social support they could draw from citizens in their host country during the Shanghai lockdown. Accordingly, this study collected WeChat group conversations to draw empirical findings, promoted scholarly conversations about fundamental survival necessity, and traced the process for establishing intercultural collective resilience with citizens from their host country. Overall, this study emphasized the significance of host country members who can promote certain coping mechanisms for their visitors in the specific regional and geographical context of China.

Regiospecific 7-hydroxylation of ten-carbon monoterpenes by detoxifying CYP5035S7 monooxygenase of the white-rot fungus Polyporus arcularius.

In a previous study, we identified CYP5035S7 of the white-rot fungus Polyporus arcularius with a broad activity towards monoterpenes such as p-cymene. Therefore, in this study we aimed at further exploring the substrate scope of detoxifying CYP5035S7 towards terpenes and semi-preparatively isolating some of the products via whole-cell biotransformation, in order to obtain information about the enzyme's reactivity. We noticed a clear preference for the monoterpene skeleton and elucidated a distinct regioselectivity pattern based on key structural and electronic features of its substrates. This study illustrates how minimal characterisation effort may already suffice to provide vital information on enzymatic reactivity by the comparison of structural derivatives.

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.

Mechanistic characterization of UDP-glucuronic acid 4-epimerase.

UDP-glucuronic acid (UDP-GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4-epimerases and decarboxylases releasing UDP-galacturonic acid (UDP-GalA) and UDP-pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short-chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme-bound NAD+ initiates the catalytic pathway. Here, we present mechanistic characterization of the C4-epimerization of UDP-GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP-GlcA 4-epimerase from Bacillus cereus functions as a homodimer and contains one NAD+ /subunit (kcat  = 0.25 ± 0.01 s-1 ). The epimerization of UDP-GlcA proceeds via hydride transfer from and to the substrate's C4 while retaining the enzyme-bound cofactor in its oxidized form (≥ 97%) at steady state and without trace of decarboxylation. The kcat for UDP-GlcA conversion shows a kinetic isotope effect of 2.0 (±0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP-4-keto-hexose-uronic acid intermediate whose formation is rate-limiting overall, and is governed by a conformational step before hydride abstraction from UDP-GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo-electronic constraints in which decarboxylation of the labile ß-keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo-electronic control may be a distinguishing feature of catalysis by SDR-type epimerases and decarboxylases active on UDP-GlcA.

The impact of LuxF on light intensity in bacterial bioluminescence.

The enzymes involved in bacterial bioluminescence are encoded in the lux operon with a conserved gene order of luxCDABEG. Some photobacterial strains carry an additional gene, termed luxF, which produces the LuxF protein, whose function and influence on bacterial bioluminescence is still uncertain. The LuxF protein binds the flavin derivative 6-(3'-(R)-myristyl)-flavin mononucleotide (myrFMN), which is generated as a side product in the luciferase-catalyzed reaction. This study utilized an Escherichia coli (E. coli) based lux operon expression system where the lux operons of Photobacterium leiognathi subsp. mandapamensis 27561 or of Photobacterium leiognathi subsp. leiognathi 25521, namely luxCDAB(F)EG, were cloned into a single expression vector. Exclusion of luxF gene from the lux operon enabled novel insights into the role of LuxF protein in light emission. E. coli cultures harboring and expressing the genes of the lux operon including luxF gene emit more light than without luxF gene. Furthermore, isolation of the tightly bound flavin derivative revealed the presence of at least three different flavin derivatives. Analysis by UV/Vis absorption and NMR spectroscopy as well as mass spectrometry showed that the flavin derivatives bear fatty acids of various chain lengths. This distribution of FMN derivatives is vastly different to what was found in bioluminescent bacteria and indicates that the luciferase is supplied with a range of aldehyde substrates in E. coli.

Pd/BIPHEPHOS is an Efficient Catalyst for the Pd-Catalyzed S-Allylation of Thiols with High n-Selectivity.

The Pd-catalyzed S-allylation of thiols with stable allylcarbonate and allylacetate reagents offers several advantages over established reactions for the formation of thioethers. We could demonstrate that Pd/BIPHEPHOS is a catalyst system which allows the transition metal-catalyzed S-allylation of thiols with excellent n-regioselectivity. Mechanistic studies showed that this reaction is reversible under the applied reaction conditions. The excellent functional group tolerance of this transformation was demonstrated with a broad variety of thiol nucleophiles (18 examples) and allyl substrates (9 examples), and could even be applied for the late-stage diversification of cephalosporins, which might find application in the synthesis of new antibiotics.

Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.

D-Apiose is a C-branched pentose sugar important for plant cell wall development. Its biosynthesis as UDP-D-apiose involves decarboxylation of the UDP-D-glucuronic acid precursor coupled to pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalyzed within a single active site by UDP-D-apiose/UDP-D-xylose synthase (UAXS). Here, we decipher the UAXS catalytic mechanism based on crystal structures of the enzyme from Arabidopsis thaliana, molecular dynamics simulations expanded by QM/MM calculations, and mutational-mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide coenzyme with retro-aldol/aldol chemistry for the sugar ring contraction. They further demonstrate that decarboxylation occurs only after the sugar ring opening and identify the thiol group of Cys100 in steering the sugar skeleton rearrangement by proton transfer to and from the C3'. The mechanistic features of UAXS highlight the evolutionary expansion of the basic catalytic apparatus of short-chain dehydrogenases/reductases for functional versatility in sugar biosynthesis.

Evolving the Promiscuity of Elizabethkingia meningoseptica Oleate Hydratase for the Regio- and Stereoselective Hydration of Oleic Acid Derivatives.

The addition of water to non-activated carbon-carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio- and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D-structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18-fold improved conversion of OA derivatives, while retaining the excellent regio- and stereoselectivity in the olefin hydration reaction.

Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca.

Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F. solani, based on in silico sequence analyses. After heterologous expression of the enzyme in the methylotrophic yeast Pichia pastoris, we have confirmed its kievitone hydration activity and have assessed its biochemical properties and substrate specificity. Purified recombinant NhKHS is obviously a homodimeric glycoprotein. Due to its good activity for the readily available chalcone derivative xanthohumol (XN), this compound was selected as a model substrate for biochemical studies. The optimal pH and temperature for hydratase activity were 6.0 and 35°C, respectively, and apparent Vmax and Km values for hydration of XN were 7.16 µmol min-1 mg-1 and 0.98 ± 0.13 mM, respectively. Due to its catalytic properties and apparent substrate promiscuity, NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols.

Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi.

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-ß-d-glucopyranose) in the presence of Mg2+ -ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting ß-glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20-50 mm), the apparent binding of levoglucosan (Km =180 mm) and ATP (Km =1.0 mm), as well as the inhibition by ADP (Ki =0.45 mm) and d-glucose-6-phosphate (IC50 =56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho-α-d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4 C1 →1 C4 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.

Mechanisms and Specificity of Phenazine Biosynthesis Protein PhzF.

Phenazines are bacterial virulence and survival factors with important roles in infectious disease. PhzF catalyzes a key reaction in their biosynthesis by isomerizing (2 S,3 S)-2,3-dihydro-3-hydroxy anthranilate (DHHA) in two steps, a [1,5]-hydrogen shift followed by tautomerization to an aminoketone. While the [1,5]-hydrogen shift requires the conserved glutamate E45, suggesting acid/base catalysis, it also shows hallmarks of a sigmatropic rearrangement, namely the suprafacial migration of a non-acidic proton. To discriminate these mechanistic alternatives, we employed enzyme kinetic measurements and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations revealed that the activation barrier of a proton shuttle mechanism involving E45 is significantly lower than that of a sigmatropic [1,5]-hydrogen shift. QM/MM also predicted a large kinetic isotope effect, which was indeed observed with deuterated substrate. For the tautomerization, QM/MM calculations suggested involvement of E45 and an active site water molecule, explaining the observed stereochemistry. Because these findings imply that PhzF can act only on a limited substrate spectrum, we also investigated the turnover of DHHA derivatives, of which only O-methyl and O-ethyl DHHA were converted. Together, these data reveal how PhzF orchestrates a water-free with a water-dependent step. Its unique mechanism, specificity and essential role in phenazine biosynthesis may offer opportunities for inhibitor development.

Isotope Probing of the UDP-Apiose/UDP-Xylose Synthase Reaction: Evidence of a Mechanism via a Coupled Oxidation and Aldol Cleavage.

The C-branched sugar d-apiose (Api) is essential for plant cell-wall development. An enzyme-catalyzed decarboxylation/pyranoside ring-contraction reaction leads from UDP-α-d-glucuronic acid (UDP-GlcA) to the Api precursor UDP-α-d-apiose (UDP-Api). We examined the mechanism of UDP-Api/UDP-α-d-xylose synthase (UAXS) with site-selectively 2 H-labeled and deoxygenated substrates. The analogue UDP-2-deoxy-GlcA, which prevents C-2/C-3 aldol cleavage as the plausible initiating step of pyranoside-to-furanoside conversion, did not give the corresponding Api product. Kinetic isotope effects (KIEs) support an UAXS mechanism in which substrate oxidation by enzyme-NAD+ and retro-aldol sugar ring-opening occur coupled in a single rate-limiting step leading to decarboxylation. Rearrangement and ring-contracting aldol addition in an open-chain intermediate then give the UDP-Api aldehyde, which is intercepted via reduction by enzyme-NADH.

Additive-Free Pd-Catalyzed α-Allylation of Imine-Containing Heterocycles.

An additive-free Pd-catalyzed α-allylation of different imino-group-ontaining heterocycles is reported. The activation of α-CH pronucleophiles (pKa (DMSO) > 25) occurs without the addition of strong bases or Lewis acids using only the Pd/Xantphos catalyst system. The reaction scope has been studied for various 5- and 6-membered nitrogen-containing heterocycles (yields up to 96%). Mechanistic investigations suggest an initial allylation of the imine-N followed by a Pd-catalyzed formal aza-Claisen rearrangement.

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa.

The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate-reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5-6.0, a Tm of 45 °C and inhibition by the co-product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole-cell biotransformation using the Escherichia coli K-12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.

From secondary alcohols to tertiary fluoro substituents: A simple route to hydroxymethyl branched sugars with a fluorine substituent at the branching point.

From a secondary hydroxyl group, by the simple sequence of oxidation, Wittig reaction of the obtained ulose with methoxymethylene triphenyl phosphorane, exposure of the resulting exocyclic enol ether to Selectfluor and subsequent reduction of the α-fluoro aldehyde thus obtained, tertiary fluoro substituents can be introduced into carbohydrate and carbohydrate-related scaffolds at a branching point now bearing a new hydroxymethyl group.

Rapid determination of γ-value and xanthate group distribution on viscose by liquid-state (1)H NMR spectroscopy.

A method for the determination of the γ-value and more importantly the distribution of xanthate groups on cellulose xanthate produced during the viscose process is presented. The method is based upon stabilization of xanthate groups attached to the cellulose chain by reaction with 4-methylbenzyl bromide and analysis of the resulting product by liquid-state (1)H NMR. Careful analysis of the proton-spectrum using deconvolution gave a very fast method for the measurement of the γ-value which compared well with the data obtained by IR spectroscopy. In addition it could be shown that the distribution of the xanthate groups on the anhydroglucose monomeric unit (xanthation at position 2, 3 or 6) changes significantly during ripening. The method gave useful results even for viscose with low γ-values of about 25.