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A fusion protein containing P450 and aldo-keto reductase domains is shown to catalyze reticuline isomerization, the critical branch point between the noscapine and morphine biosynthetic pathways. This discovery completes the enzymatic route to morphine and related compounds. The gateway to morphine biosynthesis in opium poppy ( Papaver somniferum ) is the stereochemical inversion of ( S )-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for ( R )-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S -to- R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum , which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas , which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of ( R )-reticuline and morphinan alkaloids and increased the overall abundance of ( S )-reticuline and its O -methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Sialylation, or the covalent addition of sialic acid to the terminal end of glycoproteins, is a biologically important modification that is involved in embryonic development, neurodevelopment, reprogramming, oncogenesis and immune responses. In this review, we have given a comprehensive overview of the current literature on the involvement of sialylation in cell fate decision during development, reprogramming and cancer progression. Sialylation is essential for early embryonic development and the deletion of UDP-GlcNAc 2-epimerase, a rate-limiting enzyme in sialic acid biosynthesis, is embryonically lethal. Furthermore, the sialyltransferase ST6GAL1 is required for somatic cell reprogramming, and its downregulation is associated with decreased reprogramming efficiency. In addition, sialylation levels and patterns are altered during cancer progression, indicating the potential of sialylated molecules as cancer biomarkers. Taken together, the current evidences demonstrate that sialylation is involved in crucial cell fate decision.

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ( Δ H ‡  = 54 kJ mol −1 ), significant loss in entropy ( − T Δ S ‡  = 20 kJ mol −1 ; 298 K) and negative activation heat capacity ( Δ C p ‡  = −0.64 kJ mol −1 K −1 ). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function. Enzymes involve structural flexibility in their function, but understanding enzyme catalysis as connected to protein motions is a major challenge. Here, the authors obtain energetic description of C-H activation in nicotinamide coenzyme-dependent UDP-glucuronic acid C4 epimerase based on temperature kinetic studies and isotope effect measurements.

Purpose Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. Methods Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. Results The highest blood galactose levels observed in each patient were 17.3–41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between β- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients’ peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. Conclusion Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.

Background d -Allulose is an ultra-low calorie sugar of multifarious health benefits, including anti-diabetic and anti-obesity potential. d -Allulose 3-epimerase family enzymes catalyze biosynthesis of d -allulose via epimerization of d -fructose. Results A novel d -allulose 3-epimerase (DaeB) was cloned from a plant probiotic strain, Bacillus sp. KCTC 13219, and expressed in Bacillus subtilis cells. The purified protein exhibited substantial epimerization activity in a broad pH spectrum, 6.0–11.0. DaeB was able to catalyze d -fructose to d -allulose bioconversion at the temperature range of 35 °C to 70 °C, exhibiting at least 50 % activity. It displaced excessive heat stability, with the half-life of 25 days at 50 °C, and high turnover number ( k cat 367 s − 1 ). The coupling of DaeB treatment and yeast fermentation of 700 g L − 1 d -fructose solution yielded approximately 200 g L − 1 d -allulose, and 214 g L − 1 ethanol. Conclusions The novel d -allulose 3-epimerase of Bacillus sp. origin discerned a high magnitude of heat stability along with exorbitant epimerization ability. This biocatalyst has enormous potential for the large-scale production of d -allulose.

Background D-psicose 3-epimerase (DPEase) is a potential catalytic enzyme for D-psicose production. D-psicose, also known as D-allulose, is a low-calorie sweetener that has gained considerable attention as a healthy alternative sweetener due to its notable physicochemical properties. This research focused on an in-depth investigation of the expression of the constructed DPEase gene from Agrobacterium tumefaciens in Escherichia coli for D-psicose synthesis. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, investigated the enzymatic characterization, and then optimized the D-psicose production. Finally, the produced D-psicose syrup underwent acute toxicity evaluation to provide scientific evidence supporting its safety. Results The optimization of DPEase expression involved the utilization of Mn 2+ as a cofactor, fine-tuning isopropyl β-D-1-thiogalactopyranoside induction, and controlling the induction temperature. The purification process was strategically designed by a nickel column and an elution buffer containing 200 mM imidazole, resulting in purified DPEase with a notable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions were at pH 7.5 and 55 °C with a final concentration of 10 mM Mn 2+ addition using purified DPEase to achieve the highest D-psicose concentration of 5.60% (w/v) using 25% (w/v) of fructose concentration with a conversion rate of 22.42%. Kinetic parameters of the purified DPEase were V max and K m values of 28.01 mM/min and 110 mM, respectively, which demonstrated the high substrate affinity and efficiency of DPEase conversion by the binding site of the fructose-DPEase-Mn 2+ structure. Strategies for maintaining stability of DPEase activity were glycerol addition and storage at -20 °C. Based on the results from the acute toxicity study, there was no toxicity to rats, supporting the safety of the mixed D-fructose–D-psicose syrup produced using recombinant DPEase. Conclusions These findings have direct and practical implications for the industrial-scale production of D-psicose, a valuable rare sugar with a broad range of applications in the food and pharmaceutical industries. This research should advance the understanding of DPEase biocatalysis and offers a roadmap for the successful scale-up production of rare sugars, opening new avenues for their utilization in various industrial processes.

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T m) and the urea midtransition concentration (C m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T m and C m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.

Because bacterial surface glycans are in direct contact with the environment they can provide essential protective functions during infections or against competing bacteria. But such structures are also “Achilles’ heels” since they can serve as primary receptors for bacteriophages. Bacteriophages are ubiquitous parasites of bacteria and major drivers of bacterial ecology and evolution. Despite an ever-growing interest in their biotechnological and therapeutic applications, detailed knowledge of the molecular mechanisms underlying phage-host interactions remains scarce. Here, we show that bacteriophage N4 exploits a novel surface glycan (NGR) as a receptor to infect its host Escherichia coli . We demonstrate that this process is regulated by the second messenger c-di-GMP and that N4 infection is specifically stimulated by the diguanylate cyclase DgcJ, while the phosphodiesterase PdeL effectively protects E. coli from N4-mediated killing. PdeL-mediated protection requires its catalytic activity to reduce c-di-GMP and includes a secondary role as a transcriptional repressor. We demonstrate that PdeL binds to and represses the promoter of the wec operon, which encodes components of the enterobacterial common antigen (ECA) exopolysaccharide pathway. However, only the acetylglucosamine epimerase WecB but none of the other ECA components is required for N4 infection. Based on this, we postulate that NGR is an N -acetylmannosamine-based carbohydrate polymer that is produced and exported to the cell surface of E. coli in a c-di-GMP-dependent manner, where it serves as a receptor for N4. This novel carbohydrate pathway is conserved in E. coli and other bacterial pathogens, serves as the primary receptor for various bacteriophages, and is induced at elevated temperature and by specific amino acid-based nutrients. These studies provide an entry point into understanding how bacteria use specific regulatory mechanisms to balance costs and benefits of highly conserved surface structures. IMPORTANCE Because bacterial surface glycans are in direct contact with the environment they can provide essential protective functions during infections or against competing bacteria. But such structures are also “Achilles’ heels” since they can serve as primary receptors for bacteriophages. Bacteria thus need to carefully control the exposure of conserved surface glycans to balance costs and benefits. Here, we identify a novel exopolysaccharide that is widely conserved in E. coli and is used by N4 and related bacteriophages as primary receptor. We demonstrate that the synthesis of NGR (N4 glycan receptor) is tightly controlled by the second messenger c-di-GMP in a highly specific manner and by a single diguanylate cyclase. These studies provide an example of how bacteria can alleviate the strong selective pressure imposed on them by bacteriophages entering through conserved surface structures by carefully regulating their synthesis and secretion.

Biosynthesis of heparin, a mast cell–derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D -glucuronic acid (GlcA) to L -iduronic acid (IdoA) residues. We have generated Hsepi -null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O -sulfation pattern.

Bacterial alginate initially consists of 1–4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α- L -guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum . The three A. chroococcum genes, here designated AcalgE1 , AcalgE2 and AcalgE3 , were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions − 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.