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.

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.

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.

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.

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.

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.

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.

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.

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.

Enhanced Thermostability of an l-Rhamnose Isomerase for d-Allose Synthesis by Computation-Based Rational Redesign of Flexible Regions.

d-Allose is a low-calorie rare sugar with great application potential in the food and pharmaceutical industries. The production of d-allose has been accomplished using l-rhamnose isomerase (L-RI), but concomitantly increasing the enzyme's stability and activity remains challenging. Here, we rationally engineered an L-RI from Clostridium stercorarium to enhance its stability by comprehensive computation-aided redesign of its flexible regions, which were successively identified using molecular dynamics simulations. The resulting combinatorial mutant M2-4 exhibited a 5.7-fold increased half-life at 75 °C while also exhibiting improved catalytic efficiency. Especially, by combining structure modeling and multiple sequence alignment, we identified an α0 region that was universal in the L-RI family and likely acted as a "helix-breaker". Truncating this region is crucial for improving the thermostability of related enzymes. Our work provides a significantly stable biocatalyst with potential for the industrial production of d-allose.

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.

The Arabinose 5-Phosphate Isomerase KdsD Is Required for Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, which is endemic primarily in Southeast Asia and northern Australia but is increasingly being seen in other tropical and subtropical regions of the world. Melioidosis is associated with high morbidity and mortality rates, which is mediated by the wide range of virulence factors encoded by B. pseudomallei. These virulence determinants include surface polysaccharides such as lipopolysaccharide (LPS) and capsular polysaccharides (CPS). Here, we investigated a predicted arabinose-5-phosphate isomerase (API) similar to KdsD in B. pseudomallei strain K96243. KdsD is required for the production of the highly conserved 3-deoxy-d-manno-octulosonic acid (Kdo), a key sugar in the core region of LPS. Recombinant KdsD was expressed and purified, and API activity was determined. Although a putative API paralogue (KpsF) is also predicted to be encoded, the deletion of kdsD resulted in growth defects, loss of motility, reduced survival in RAW 264.7 murine macrophages, and attenuation in a BALB/c mouse model of melioidosis. Suppressor mutations were observed during a phenotypic screen for motility, revealing single nucleotide polymorphisms or indels located in the poorly understood CPS type IV cluster. Crucially, suppressor mutations did not result in reversion of attenuation in vivo. This study demonstrates the importance of KdsD for B. pseudomallei virulence and highlights further the complex nature of the polysaccharides it produces. IMPORTANCE The intrinsic resistance of B. pseudomallei to many antibiotics complicates treatment. This opportunistic pathogen possesses a wide range of virulence factors, resulting in severe and potentially fatal disease. Virulence factors as targets for drug development offer an alternative approach to combat pathogenic bacteria. Prior to initiating early drug discovery approaches, it is important to demonstrate that disruption of the target gene will prevent the development of disease. This study highlights the fact that KdsD is crucial for virulence of B. pseudomallei in an animal model of infection and provides supportive phenotypic characterization that builds a foundation for future therapeutic development.

Production of d-aldotetrose from l-erythrulose using various isomerases.

d-Aldotetroses are rare sugars that are obtained via chemical synthesis in low yield. In this study, we demonstrated that d-aldotetroses could be produced using 3 isomerases. First, l-erythrulose was epimerized using d-tagatose 3-epimerase from Pseudomonas cichorii ST-24. The specific optical rotation of the reaction solution gradually decreased to zero, indicating that approximately 50% of the l-erythrulose was converted to d-erythrulose. d, l-Erythrulose mixture was isomerized with d-arabinose isomerase from Klebsiella pneumoniae 40bXX to produce d-threose, resulting in a conversion rate of 9.35%. d-Erythrose production using l-rhamnose isomerase from Pseudomonas stutzeri LL172 resulted in a conversion rate of 12.9%. Because of the low purity of the purchased d-erythrose, the product was reduced by the Raney nickel catalyst compared with authentic erythritol. We confirmed the products using HPLC and 13C-NMR spectra. This is the first report of d-aldotetrose production using an enzymatic reaction.

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.

[Rational design of L-arabinose isomerase from Lactobacillus fermentum and its application in D-tagatose production].

L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The Km and kcat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.