L-rhamnose isomerase: a crucial enzyme for rhamnose catabolism and conversion of rare sugars.

L-rhamnose isomerase (L-RhI) plays a key role in the microbial L-rhamnose metabolism by catalyzing the reversible isomerization of L-rhamnose to L-rhamnulose. Additionally, the enzyme exhibits activity on various other aldoses and ketoses, and its broad substrate specificity has attracted attention for its potential application in the production of rare sugars; however, improvement of the enzyme properties is desirable, such as thermal stability, enzymatic activity, and a pH optimum suitable for industrial usage. This review summarizes our current insights into L-RhIs with respect to their substrate recognition mechanism and their relationship with D-xylose isomerase (D-XI) based on structural and phylogenetic analyses. These two enzymes are inherently different, but recognize distinctly different substrates, and share common features that may be phylogenetically related. For example, they both have a flexible loop region that is involved in shaping active sites, and this region may also be responsible for various enzymatic properties of L-RhIs, such as substrate specificity and thermal stability. KEY POINTS: •L-RhIs share structural features with D-XI. •There are two types of L-RhIs: E. coli L-RhI-type and D-XI-type. •Flexible loop regions are involved in the specific enzyme properties.

L-arabinose isomerase from Lactobacillus fermentum C6: Enzymatic characteristics and its recombinant Bacillus subtilis whole cells achieving a significantly increased production of D-tagatose.

L-arabinose isomerase (L-AI) is a functional enzyme for the isomerizing of D-galactose to produce D-tagatose. In this study, L-AI-C6-encoding gene from the probiotic Lactobacillus fermentum C6 was cloned and expressed in Bacillus subtilis WB600 for investigating enzymatic characteristics and bioconverting D-tagatose by means of whole-cell catalysis. Results showed that the engineered B. subtilis WB600-pMA5-LAI achieved a maximum specific activity of L-AI-C6 (232.65 ± 15.54 U/mg protein) under cultivation in LB medium at 28 °C for 40 h. The recombinant L-AI-C6 was purified, and enzymatic characteristics test showed its optimum reaction temperature and pH at 60 °C and 8.0, respectively. In addition, L-AI-C6 exhibited good stability within the pH range of 5.5-9.0. By using B. subtilis WB600-pMA5-LAI cells as whole-cell catalyst, the highest D-tagatose yield reached 42.91 ± 0.28 % with D-galactose as substrate, which was 2.41 times that of L. fermentum C6 (17.79 ± 0.11 %). This suggested that the cloning and heterologous expression of L-AI-C6 was an effective strategy for improving D-tagatose conversion by whole-cell catalysis. In brief, the present study demonstrated that the reaction temperature, pH, and stability of L-AI-C6 from L. fermentum C6 meet the demands of industrial application, and the constructed B. subtilis WB600-pMA5-LAI shows promising potential for the whole-cell biotransformation of D-tagatose.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase as target for anti Toxoplasma gondii agents: crystal structure, biochemical characterization and biological evaluation of inhibitors.

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Šresolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.

Benchmarking predictive methods for small-angle X-ray scattering from atomic coordinates of proteins using maximum likelihood consensus data.

Stimulated by informal conversations at the XVII International Small Angle Scattering (SAS) conference (Traverse City, 2017), an international team of experts undertook a round-robin exercise to produce a large dataset from proteins under standard solution conditions. These data were used to generate consensus SAS profiles for xylose isomerase, urate oxidase, xylanase, lysozyme and ribonuclease A. Here, we apply a new protocol using maximum likelihood with a larger number of the contributed datasets to generate improved consensus profiles. We investigate the fits of these profiles to predicted profiles from atomic coordinates that incorporate different models to account for the contribution to the scattering of water molecules of hydration surrounding proteins in solution. Programs using an implicit, shell-type hydration layer generally optimize fits to experimental data with the aid of two parameters that adjust the volume of the bulk solvent excluded by the protein and the contrast of the hydration layer. For these models, we found the error-weighted residual differences between the model and the experiment generally reflected the subsidiary maxima and minima in the consensus profiles that are determined by the size of the protein plus the hydration layer. By comparison, all-atom solute and solvent molecular dynamics (MD) simulations are without the benefit of adjustable parameters and, nonetheless, they yielded at least equally good fits with residual differences that are less reflective of the structure in the consensus profile. Further, where MD simulations accounted for the precise solvent composition of the experiment, specifically the inclusion of ions, the modelled radius of gyration values were significantly closer to the experiment. The power of adjustable parameters to mask real differences between a model and the structure present in solution is demonstrated by the results for the conformationally dynamic ribonuclease A and calculations with pseudo-experimental data. This study shows that, while methods invoking an implicit hydration layer have the unequivocal advantage of speed, care is needed to understand the influence of the adjustable parameters. All-atom solute and solvent MD simulations are slower but are less susceptible to false positives, and can account for thermal fluctuations in atomic positions, and more accurately represent the water molecules of hydration that contribute to the scattering profile.

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.

A novel thermotolerant L-rhamnose isomerase variant for biocatalytic conversion of D-allulose to D-allose.

A novel L-rhamnose isomerase was identified and cloned from an extreme-temperature aquatic habitat metagenome. The deduced amino acid sequence homology suggested the possible source of this metagenomic sequence to be Chloroflexus islandicus. The gene expression was performed in a heterologous host, Escherichia coli, and the recombinant protein L-rhamnose isomerase (L-RIM) was extracted and purified. The catalytic function of L-RIM was characterized for D-allulose to D-allose bioconversion. D-Allose is a sweet, rare sugar molecule with anti-tumour, anti-hypertensive, cryoprotective, and antioxidative properties. The characterization experiments showed L-RIM to be a Co++- or Mn++-dependent metalloenzyme. L-RIM was remarkably active (~ 80%) in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges. Optimal L-RIM activity with D-allulose as the substrate occurred at pH 7.0 and 75 °C. The enzyme was found to be excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively. L-RIM catalysis conducted at slightly acidic pH of 6.0 and 70 °C achieved biosynthesis of about 30 g L-1 from 100 g L-1 D-allulose in 3 h. KEY POINTS: • The present study explored an extreme temperature metagenome to identify a novel gene that encodes a thermostable l-rhamnose isomerase (L-RIM) • L-RIM exhibits substantial (80% or more) activity in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges • L-RIM is excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively.

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.

X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase.

A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose. KEY POINTS: • Crystal structures of LrL-RhI in complexes with substrates were determined. • LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose. • The LrL-RhI is active in acidic conditions.

Engineered Saccharomyces cerevisiae harbors xylose isomerase and xylose transporter improves co-fermentation of xylose and glucose for ethanol production.

Saccharomyces cerevisiae cannot assimilate xylose, second to glucose derived from lignocellulosic biomass. Here, the engineered S. cerevisiae strains INVSc-XI and INVSc-XI/XT were constructed using xylA and Xltr1p to co-utilize xylose and glucose, achieving economic viability and sustainable production of fuels. The xylose utilization rate of INVSc-XI/XT was 2.3-fold higher than that of INVSc-XI, indicating that overexpressing Xltr1p could further enhance xylose utilization. In mixed sugar media, a small amount of glucose enhanced the consumption of xylose by INVSc-XI/XT. Transcriptome analysis showed that glucose increased the upregulation of acetate of coenzyme A synthetase (ACS), alcohol dehydrogenase (ADH), and transketolase (TKL) gene expression in INVSc-XI/XT, further promoting xylose utilization and ethanol yield. The highest ethanol titer of 2.91 g/L with a yield of 0.29 g/g at 96 h by INVSc-XI/XT was 56.9% and 63.0% of the theoretical ethanol yield from glucose and xylose, respectively. These results showed overexpression of xylA and Xltr1p is a promising strategy for improving xylose and glucose conversion to ethanol. Although the ability of strain INVSc-XI/XT to produce ethanol was not very satisfactory, glucose was discovered to influence xylose utilization in strain INVSc-XI/XT. Altering the glucose concentration is a promising strategy to improve the xylose and glucose co-utilization.

INVSc-XI and INVSc-XI/XT strains were newly constructed to utilize xylose and glucose.XylA, in combination with xylose transporter Xltr1p, enhances xylose consumption.A small amount of glucose enhanced xylose utilization in INVSc-XI/XT strain.The expression of ACS, ADH, and TKL genes is upregulated in the media containing mixed sugars.The highest ethanol yield of 0.29 g/g was produced in a 2-L scale-up fermenter.

Exploiting the biocatalytic potential of co-expressed l-fucose isomerase and d-tagatose 3-epimerase for the biosynthesis of 6-deoxy-l-sorbose.

6-Deoxy-l-sorbose (6-DLS) is an imperative rare sugar employed in food, agriculture, pharmaceutical and cosmetic industeries. However, it is a synthetic and very expensive rare sugars, previously synthesized by chemo-enzymatic methods through a long chain of chemical processes. Recently, enzymatic synthesis of rare sugars has attracted a lot of attention due to its advantages over synthetic methods. In this work, a promising approach for the synthesis of 6-DLS from an inexpensive sugar l-fucose was identified. The genes for l-fucose isomerase from Paenibacillus rhizosphaerae (Pr-LFI) and genes for d-tagatose-3-epimerase from Caballeronia fortuita (Cf-DTE) have been used for cloning and co-expression in Escherichia coli, developed a recombinant plasmid harboring pANY1-Pr-LFI/Cf-DTE vector. The recombinant co-expression system exhibited an optimum activity at 50 °C of temperature and pH 6.5 in the presence of Co2+ metal ion which inflated the catalytic activity by 6.8 folds as compared to control group with no metal ions. The recombinant co-expressed system was stable up to more than 50 % relative activity after 12 h and revealed a melting temperature (Tm) of 63.38 °C exhibiting half-life of 13.17 h at 50 °C. The co-expression system exhibited, 4.93, 11.41 and 16.21 g/L of 6-DLS production from initial l-fucose concentration of 30, 70 and 100 g/L, which equates to conversion yield of 16.44 %, 16.30 % and 16.21 % respectively. Generally, this study offers a promising strategy for the biological production of 6-DLS from an inexpensive substrate l-fucose in slightly acidic conditions with the aid of co-expression system harboring Pr-LFI and CF-DTE genes.

PbrbZIP15 promotes sugar accumulation in pear via activating the transcription of the glucose isomerase gene PbrXylA1.

The composition and abundance of soluble sugars in mature pear (Pyrus) fruit are important for its acceptance by consumers. However, our understanding of the genes responsible for soluble sugar accumulation remains limited. In this study, a S1-group member of bZIP gene family, PbrbZIP15, was characterized from pear genome through the combined analyses of metabolite and transcriptome data followed by experimental validation. PbrbZIP15, located in nucleus, was found to function in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli. After analyzing the expression profiles of sugar-metabolism-related genes and the distribution of cis-acting elements in their promoters, the glucose isomerase 1 gene (PbrXylA1), whose corresponding protein catalyzed the isomerization of glucose and fructose in vitro, was identified as a downstream target gene of PbrbZIP15. PbrbZIP15 could directly bind to the G-box element in PbrXylA1 promoter and activate its transcription, as evidenced by chromatin immunoprecipitation-quantitative PCR, yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase assay. PbrXylA1, featuring a leucine-rich signal peptide in its N-terminal, was localized to the endoplasmic reticulum. It was validated to play a significant role in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli, which was associated with the upregulated fructose/glucose ratio. Further studies revealed a positive correlation between the sucrose content and the expression levels of several sucrose-biosynthesis-related genes (PbrFRK3/8, PbrSPS1/3/4/8, and PbrSPP1) in PbrbZIP15-/PbrXylA1-transgenic fruit/calli. In conclusion, our results suggest that PbrbZIP15-induced soluble sugar accumulation during pear development is at least partly attributed to the activation of PbrXylA1 transcription.

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.

Exploring an l-arabinose isomerase from cryophile bacteria Arthrobacter psychrolactophilus B7 for d-tagatose production.

A novel l-arabinose isomerase (L-AI) from Arthrobacter psychrolactophilus (Ap L-AI) was successfully cloned and characterized. The enzyme catalyzes the isomerization of d-galactose into a rare sugar d-tagatose. The recombinant Ap L-AI had an approximate molecular weight of about 258 kDa, suggesting it was an aggregate of five 58 kDa monomers and became the first record as a homo-pentamer L-AI. The catalytic efficiency (kcat/Km) and Km for d-galactose were 0.32 mM-1 min-1 and 51.43 mM, respectively, while for l-arabinose, were 0.64 mM-1 min-1 and 23.41 mM, respectively. It had the highest activity at pH 7.0-7.5 and 60 °C in the presence of 0.250 mM Mn2+. Ap L-AI was discovered to be an outstanding thermostable enzyme that only lost its half-life value at 60 °C for >1000 min. These findings suggest that l-arabinose isomerase from Arthrobacter psychrolactophilus is a promising candidate for d-tagatose mass-production due to its industrially competitive temperature.

Identification of novel compounds and repurposing of FDA drugs for 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme of Plasmodium falciparum to combat malaria resistance.

The rise of Plasmodium falciparum resistance to Artemisinin-based combination therapies (ACTs) is a significant concern in the fight against malaria. This situation calls for the search for novel anti-malarial candidates. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) is a potential target involved in various cellular processes in P. falciparum (Pf). We screened ∼0.69 billion novel compounds from the ZINC20 library and repurposed ∼1400 FDA drugs using computational drug discovery methods against PfIspC. Following our computational pipeline, we found five novel ZINC20 compounds (Z-2, Z-3, Z-10, Z-13, and Z-14) and three FDA drugs (Aliskiren, Ceftolozane, and Ombitasvir) that showed striking docking energy (ranging from -8.405 to -10.834 kcal/mol), and strong interactions with key binding site residues (Ser269, Ser270, Ser306, Asn311, Lys312, and Met360) of PfIspC. The novel anti-malarial compounds also exhibited favorable pharmacokinetics and physicochemical properties. Furthermore, through molecular dynamics simulation, we observed the stable dynamics of PfIspC-inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. Notably, the binding free energy estimation confirmed high binding affinity (varied from -11.68 to -33.16 kcal/mol) of these compounds for PfIspC. Our findings could contribute to the ongoing efforts in combating malaria and invite experimental-lab researchers for validation.

Design, Synthesis and Bioactivity Evaluation of Heterocycle-Containing Mono- and Bisphosphonic Acid Compounds.

Fosmidomycin (FOS) is a naturally occurring compound active against the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) enzyme in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and using it as a template for lead structure design is an effective strategy to develop new active compounds. In this work, by replacing the hydroxamate unit of FOS with pyrazole, isoxazole and the related heterocycles that also have metal ion binding affinity, while retaining the monophosphonic acid in FOS or replacing it with a bisphosphonic acid group, heterocycle-containing mono- and bisphosphonic acid compounds as FOS analogs were designed. The key steps involved in the facile synthesis of these FOS analogs included the Michael addition of diethyl vinylphosphonate or tetraethyl vinylidenebisphosphonate to ß-dicarbonyl compounds and the subsequent cyclic condensation with hydrazine or hydroxylamine. Two additional isoxazolinone-bearing FOS analogs were synthesized via the Michaelis-Becker reaction with diethyl phosphite as a key step. The bioactivity evaluation on model plants demonstrated that several compounds have better herbicidal activities compared to FOS, with the most active compound showing a 3.7-fold inhibitory activity on Arabidopsis thaliana, while on the roots and stalks of Brassica napus L. and Echinochloa crus-galli in a pre-emergence inhibitory activity test, the activities of this compound were found to be 3.2- and 14.3-fold and 5.4- and 9.4-fold, respectively, and in a post-emergency activity test on Amaranthus retroflexus and Echinochloa crus-galli, 2.2- and 2.0-fold inhibition activities were displayed. Despite the significant herbicidal activity, this compound exhibited a DXR inhibitory activity lower than that of FOS but comparable to that of other non-hydroxamate DXR inhibitors, and the dimethylallyl pyrophosphate rescue assay gave no statistical significance, suggesting that a different target might be involved in the inhibiting process. This work demonstrates that using bioisosteric replacement can be considered as a valuable strategy to discover new FOS analogs that may have high herbicidal activities.

Xylitol binding to the M1 site of glucose isomerase induces a conformational change in the substrate binding channel.

Glucose isomerase (GI) is extensively used in the food industry for production of high-fructose corn syrup and for the production of biofuels and other renewable chemicals. Structure-based studies on GI inhibitors are important for improving its efficiency in industrial applications. Here, we report the subatomic crystal structure of Streptomyces rubiginosus GI (SruGI) complexed with its inhibitor, xylitol, at 0.99 Å resolution. Electron density map and temperature factor analysis showed partial binding of xylitol to the M1 metal binding site of SruGI, providing two different conformations of the metal binding site and the substrate binding channel. The xylitol molecule induced a conformational change in the M2 metal ion-interacting Asp255 residue, which subsequently led to a conformational change in the side chain of Asp181 residue. This led to the positional shift of Pro25 by 1.71 Å and side chain rotation of Phe26 by 21°, where located on the neighboring protomer in tetrameric SruGI. The conformation change of these two residues affect the size of the substrate-binding channel of GI. Therefore, xylitol binding to M1 site of SruGI induces not only a conformational changes of the metal-binding site, but also conformational change of substrate-binding channel of the tetrameric SruGI. These results expand our knowledge about the mechanism underlying the inhibitory effect of xylitol on GI.