Promotion of lactose isomerization to fructose and lactulose in one batch by immobilized enzymes on bacterial cellulose membranes.

The production of the sugars fructose and lactulose from lactose using the enzymes ß-galactosidase and glucose isomerase immobilized on bacterial cellulose (BC) membranes has been investigated. Lactose is hydrolyzed by ß-galactosidase at 30 °C to glucose and galactose at a high conversion rate, while at the same temperature, glucose isomerase is not effective in converting the produced glucose to fructose. The rate of the isomerization reaction of glucose to fructose at 70 °C has been studied. Two types of enzyme immobilization were investigated: immobilization in one stage and immobilization in two stages. The results showed that BC membrane increased three-fold the yield and the reaction rate of fructose and lactulose production from lactose. The noteworthy enhancement of BC membranes' impact on the isomerization reaction by immobilized enzymes grants permission for a novel research avenue within the context of white biotechnology development. Additionally, this effect amplifies the role of BC in sustainability and the circular economy.

The metal cofactor: stationary or mobile?

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: • Metal ions are essential cofactors that can move during catalysis. • In class II aldolases, the metal cofactors can reside in a resting state and an active state. • In MDR, the movement of the metal cofactor is essential for substrate docking.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Involvement of Glucosamine 6 Phosphate Isomerase 2 (GNPDA2) Overproduction in ß-Amyloid- and Tau P301L-Driven Pathomechanisms.

Alzheimer's disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine- or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas of AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signaling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slowdown in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects or neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed dementia and amyotrophic lateral sclerosis) (n = 215). These data shed new light on GNPDA2-dependent mechanisms associated with the neurodegenerative process beyond the hexosamine route.

Reverse N-Substituted Hydroxamic Acid Derivatives of Fosmidomycin Target a Previously Unknown Subpocket of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase (DXR).

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

Hydroxamate-Containing Bisphosphonates as Fosmidomycin Analogues: Design, Synthesis, and Proherbicide Activity.

Fosmidomycin (FOS) is a natural product inhibiting the DXR enzyme in the MEP pathway and has stimulated interest for finding more suitable FOS analogues. Herein, two series of FOS analogue hydroxamate-containing bisphosphonates as proherbicides were designed, with bisphosphonate replacing the phosphonic unit in FOS while retaining the hydroxamate (BPF series) or replacing it with retro-hydroxamate (BPRF series). The BPF series were synthesized through a three-step reaction sequence including Michael addition of vinylidenebisphosphonate, N-acylation, and deprotection, and the BPRF series were synthesized with a retro-Claisen condensation incorporated into the reaction sequence. Evaluation on model plants demonstrated several compounds having considerable herbicidal activities, and in particular, compound 8m exhibited multifold activity enhancement as compared to the control FOS. The proherbicide properties were comparatively validated. Furthermore, DXR enzyme assay, dimethylallyl pyrophosphate rescue, and molecular docking verified 8m to be a promising proherbicide candidate targeting the DXR enzyme. In addition, 8m also displayed good antimalarial activities.

Characterization of a novel recombinant D-mannose isomerase from Bifidobacterium bifidum and its catalytic mechanism.

Due to the increasing demand for health-conscious and environmentally friendly products, D-mannose has gained significant attention as a natural, low-calorie sweetener. The use of D-mannose isomerases (D-MIases) for D-mannose production has emerged as a prominent area of research, offering superior advantages compared with conventional methods such as plant extraction and chemical synthesis. In this study, a gene encoding D-MIase was cloned from Bifidobacterium and expressed in E. coli BL21 (DE3). The heterologously expressed enzyme, Bifi-mannose, formed a trimer with a molecular weight of 146.3 kDa and a melting temperature (Tm) of 63.39 ± 1.3 °C. Bifi-mannose exhibited optimal catalytic activity at pH 7.5 and 55 °C, and retained more than 80% of its activity after a 3-hour incubation at 55 °C, demonstrating excellent thermal stability. The Km, Vmax, and kcat/Km values of Bifi-mannose for D-fructose isomerization were determined as 538.7 ± 62.5 mM, 11.7 ± 0.9 µmol·mg1·s1, and 1.02 ± 0.3 mM1·s1, respectively. Notably, under optimized conditions, catalytic yields of 29.4, 87.1, and 148.5 mg·mL1 were achieved when using 100, 300, and 500 mg·mL1 of D-fructose as substrates, resulting in a high conversion rate (29%). Furthermore, kinetic parameters and molecular docking studies revealed that His387 residue primarily participates in the opening of the pyranose ring, while His253 acts as a basic catalyst in the isomerization process.

A study of targeted mutation of l-rhamnose isomerase to improve the conversion efficiency of D-allose.

D-Allose is a rare cis-caprose with a wide range of physiological functions, which has a wide range of applications in medicine, food, and other industries. L-Rhamnose isomerase (L-Rhi) is the earliest enzyme found to catalyze the production of D-allose from D-psicose. This catalyst has a high conversion rate, but its specificity for substrates is limited; thus, it cannot fulfill the requirements of industrial production of D-allose. In this study, L-Rhi derived from Bacillus subtilis was employed as the research subject, and D-psicose as the conversion substrate. Two mutant libraries were constructed through alanine scanning, saturation mutation, and rational design based on the analysis of the secondary structure, tertiary structure, and interactions with ligands of the enzyme. The yield of D-allose produced by these mutants was assessed; it was found that the conversion rate of mutant D325M to D-allose was increased by 55.73 %, and the D325S improved by 15.34 %, while mutant W184H increased by 10.37 % at 55 °C, respectively. According to modeling analysis, manganese (Mn2+) had no significant effect on the production of D-psicose from D-psicose by L-Rhi. The results of molecular dynamics simulation demonstrated that the mutants W184H, D325M, and D325S had more stable protein structures while binding with the substrate D-psicose, as evidenced by its root mean square deviation (RMSD), root mean square fluctuation (RMSF), and binding free energy values. It was more conducive to binding D-psicose and facilitating its conversion to D-allose, providing the basis for the production of D-allose.

Kinetic characterization of methylthio-d-ribose-1-phosphate isomerase.

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-d-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-d-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose-ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [32P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct.

Not Every Hit-Identification Technique Works on 1-Deoxy-d-Xylulose 5-Phosphate Synthase (DXPS): Making the Most of a Virtual Screening Campaign.

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.

Isomerization of Galactose to Tagatose: Recent Advances in Non-enzymatic Isomerization.

The valorization of galactose derived from acid whey to low-calorie tagatose has gained increasing attention. Enzymatic isomerization is of great interest but faces several challenges, such as poor thermal stability of enzymes and a long processing time. In this work, non-enzymatic (supercritical fluids, triethylamine, arginine, boronate affinity, hydrotalcite, Sn-ß zeolite, and calcium hydroxide) pathways for galactose to tagatose isomerization were critically discussed. Unfortunately, most of these chemicals showed poor tagatose yields (<30%), except for calcium hydroxide (>70%). The latter is able to form a tagatose-calcium hydroxide-water complex, which stimulates the equilibrium toward tagatose and prevents sugar degradation. Nevertheless, the excessive use of calcium hydroxide may pose challenges in terms of economic and environmental feasibility. Moreover, the proposed mechanisms for the base (enediol intermediate) and Lewis acid (hydride shift between C-2 and C-1) catalysis of galactose were elucidated. Overall, it is crucial to explore novel and effective catalysts as well as integrated systems for isomerizing of galactose to tagatose.

Robust nano-enzyme conjugates for the sustainable synthesis of a rare sugar D-tagatose.

Aim of present study was to develop biological catalysts of L-arabinose isomerase (L-AI) by immobilizing on four different supports such as multiwalled carbon nanotube (MWCNT), graphene oxide (GOx), Santa Barbara Amorphous (SBA-15) and mobile composite matter (MCM-41). Also, comparative analysis of the developed catalysts was performed to evolve the best in terms of transformation efficiency for D-tagatose production. The developed nano-enzyme conjugates (NECs) were characterized using the high resolution transmission electron microscopy (HR-TEM) and elemental analysis was performed by energy dispersive X-ray spectroscopy (EDS). The functional groups were investigated by Fourier transform infra red spectroscopy. Also, the thermo gravimetric analysis (TGA) was employed to plot a thermal degradation weight loss profile of NECs. The conjugated L-AI with MWCNT and GOx were found to be more promising immobilized catalysts due to their ability to provide more surface area. Conversion of D-Galactose to D-Tagatose at moderate temperature and pH was observed to attain the equilibrium level of transformation (~50%). On the contrary, NECs prepared using SBA-15 and MCM-41 as support matrix were unable to reach the equilibrium level of conversion. Additionally, the developed NECs were suitable for reuse in multiple batch cycles. Thus, promising nanotechnology coupled with biocatalysis made the transformation of D-Galactose into D-tagatose more economically sustainable.

Novel Recyclable UCST-Type Immobilized Glucose Isomerase Biocatalyst with Excellent Performance for Isomerization of Glucose to Fructose.

The development of a suitable immobilization strategy to improve the performance of immobilized glucose isomerase for the isomerization of glucose to fructose is crucial to promoting the industrial production of high-fructose syrup. In this work, a novel recyclable upper critical solution temperature (UCST)-type mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst (PVAA@GI) was prepared, and the immobilized glucose isomerase could capture the glucose substrate through the affinity of 4-vinylbenzeneboronic acid (4-VBA) and the glucose substrate, which led to the enhanced substrate affinity and catalytic efficiency of the PVAA@GI. The biocatalyst exhibited excellent stability in pH, thermal, storage, and recycling compared to the free enzyme. The mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst displayed reversibly soluble-insoluble characteristics with temperature change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the temperature after the reaction. The highest fructose production rate reached 62.79%, which would have potential application in the industrial production of high-fructose syrup.

Altering the substrate specificity of recombinant l-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 to favor d-allose production.

l-Rhamnose isomerase (l-RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l-RhI (TsRhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R TsRhIs can increase the preference against d-allose in comparison with the wild-type enzyme. The catalytic efficiencies of the purified I102N, I102Q, and I102R TsRhIs against d-allose are 148 %, 277 %, and 191 %, respectively, of that of wild-type enzyme, while those against l-rhamnose are 100 %, 167 % and 87 %, respectively. Mutant I102N, I102Q, and I102R TsRhIs were noted to have the altered substrate specificity, and I102Q TsRhI has the highest catalytic efficiency against d-allose presumably through the formation of an additional hydrogen bond with d-allose. The purified wild-type and mutant TsRhIs were further used to produce d-allose from 100 g/L d-fructose in the presence of d-allulose 3-epimerase, and the yields can reach as high as 22 % d-allulose and 12 % d-allose upon equilibrium. I102Q TsRhI takes only around half of the time to reach the same 12 % d-allose yield, suggesting that this mutant enzyme has a potential to be applied in d-allose production.

Enhancement of L-ribulose Production from L-ribose Through Modification of Ochrobactrum sp. CSL1 Ribose-5-phosphate Isomerase A.

L-ribulose, a kind of high-value rare sugar, could be utilized to manufacture L-form sugars and antiviral drugs, generally produced from L-arabinose as a substrate. However, the production of L-ribulose from L-arabinose is limited by the equilibrium ratio of the catalytic reaction, hence, it is necessary to explore a new biological enzymatic method to produce L-ribulose. Ribose-5-phosphate isomerase (Rpi) is an enzyme that can catalyze the reversible isomerization between L-ribose and L-ribulose, which is of great significance for the preparation of L-ribulose. In order to obtain highly active ribose-5-phosphate isomerase to manufacture L-ribulose, ribose-5-phosphate isomerase A (OsRpiA) from Ochrobactrum sp. CSL1 was engineered based on structural and sequence analyses. Through a rational design strategy, a triple-mutant strain A10T/T32S/G101N with 160% activity was acquired. The enzymatic properties of the mutant were systematically investigated, and the optimum conditions were characterized to achieve the maximum yield of L-ribulose. Kinetic analysis clarified that the A10T/T32S/G101N mutant had a stronger affinity for the substrate and increased catalytic efficiency. Furthermore, molecular dynamics simulations indicated that the binding of the substrate to A10T/T32S/G101N was more stable than that of wild type. The shorter distance between the catalytic residues of A10T/T32S/G101N and L-ribose illuminated the increased activity. Overall, the present study provided a solid basis for demonstrating the complex functions of crucial residues in RpiAs as well as in rare sugar preparation.

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae.

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.

Nucleation of glucose isomerase protein crystals in a nonclassical disguise: The role of crystalline precursors.

Protein crystallization is an astounding feat of nature. Even though proteins are large, anisotropic molecules with complex, heterogeneous surfaces, they can spontaneously group into two- and three-dimensional arrays with high precision. And yet, the biggest hurdle in this assembly process, the formation of a nucleus, is still poorly understood. In recent years, the two-step nucleation model has emerged as the consensus on the subject, but it still awaits extensive experimental verification. Here, we set out to reconstruct the nucleation pathway of the candidate protein glucose isomerase (GI), for which there have been indications that it may follow a two-step nucleation pathway under certain conditions. We find that the precursor phase present during the early stages of the reaction process is nanoscopic crystallites that have lattice symmetry equivalent to the mature crystals found at the end of a crystallization experiment. Our observations underscore the need for experimental data at a lattice-resolving resolution on other proteins so that a general picture of protein crystal nucleation can be formed.

Characterization of a Novel Mannose Isomerase from Stenotrophomonas rhizophila and Identification of Its Possible Catalytic Residues.

D-Mannose has great value in the treatment of chronic diseases. D-Mannose isomerase can catalyze the bioconversion of D-fructose to D-mannose. Therefore, a novel D-mannose isomerase gene (Strh-MIase) from Stenotrophomonas rhizophila strain IS26 was expressed, purified, and characterized for the industrial production of D-mannose. The specific activities of the Strh-MIase for D-mannose and D-fructose were 437.5 ± 0.8 U/mg and 267.2 ± 0.7 U/mg. Its optimal temperature and pH were 50 °C and 7.0. The enzymatic bioconversion produced 25 g/L D-mannose from concentration D-fructose (100 g/L) in 6 h by recombinant Strh-MIase, resulting in a final yield of 25%. Sodium phosphate inhibition has little influence on D-mannose production when a high concentration of D-fructose is used as substrate. We demonstrate that the metal ions improve the efficiency of D-mannose isomerase because of the enhancement of its thermostability. Moreover, the possible catalytic residues of Strh-MIase were identified by site-directed mutagenesis.

Paradigm shift in xylose isomerase usage: a novel scenario with distinct applications.

Isomerases are enzymes that induce physical changes in a molecule without affecting the original molecular formula. Among this class of enzymes, xylose isomerases (XIs) are the most studied to date, partly due to their extensive application in industrial processes to produce high-fructose corn sirups. In recent years, the need for sustainable initiatives has triggered efforts to improve the biobased economy through the use of renewable raw materials. In this context, D-xylose usage is crucial as it is the second-most abundant sugar in nature. The application of XIs in biotransforming xylose, enabling downstream metabolism in several microorganisms, is a smart strategy for ensuring a low-carbon footprint and producing several value-added biochemicals with broad industrial applications such as in the food, cosmetics, pharmaceutical, and polymer industries. Considering recent advancements that have expanded the range of applications of XIs, this review provides a comprehensive and concise overview of XIs, from their primary sources to the biochemical and structural features that influence their mechanisms of action. This comprehensive review may help address the challenges involved in XI applications in different industries and facilitate the exploitation of xylose bioprocesses.

ZIP10 is a negative determinant for anti-tumor effect of mannose in thyroid cancer by activating phosphate mannose isomerase.

BACKGROUND: Mannose, a natural hexose existing in daily food, has been demonstrated to preferentially inhibit the progression of tumors with low expression of phosphate mannose isomerase (PMI). However, its function in thyroid cancer still remains elusive. METHODS: MTT, colony formation and flow cytometry assays were performed to determine the response of thyroid cancer cells to mannose. Meanwhile, mouse models of subcutaneous xenograft and primary papillary thyroid cancer were established to determine in vivo anti-tumor activity of mannose. The underlying mechanism of mannose selectively killing thyroid cancer cells was clarified by a series of molecular and biochemical experiments. RESULTS: Our data demonstrated that mannose selectively suppressed the growth of thyroid cancer cells, and found that enzyme activity of PMI rather than its protein expression was negatively associated with the response of thyroid cancer cells to mannose. Besides, our data showed that zinc ion (Zn2+) chelator TPEN clearly increased the response of mannose-insensitive cells to mannose by inhibiting enzyme activity of PMI, while Zn2+ supplement could effectively reverse this effect. Further studies found that the expression of zinc transport protein ZIP10, which transport Zn2+ from extracellular area into cells, was negatively related to the response of thyroid cancer cells to mannose. Knocking down ZIP10 in mannose-insensitive cells significantly inhibited in vitro and in vivo growth of these cells by decreasing intracellular Zn2+ concentration and enzyme activity of PMI. Moreover, ectopic expression of ZIP10 in mannose-sensitive cells decrease their cellular response to mannose. Mechanistically, mannose exerted its anti-tumor effect by inhibiting cellular glycolysis; however, this effect was highly dependent on expression status of ZIP10. CONCLUSION: The present study demonstrate that mannose selectively kills thyroid cancer cells dependent on enzyme activity of PMI rather than its expression, and provide a mechanistic rationale for exploring clinical use of mannose in thyroid cancer therapy.