Esketamine accelerates emergence from isoflurane general anaesthesia by activating the paraventricular thalamus glutamatergic neurones in mice.

BACKGROUND: Delayed emergence from general anaesthesia poses a significant perioperative safety hazard. Subanaesthetic doses of ketamine not only deepen anaesthesia but also accelerate recovery from isoflurane anaesthesia; however, the mechanisms underlying this phenomenon remain elusive. Esketamine exhibits a more potent receptor affinity and fewer adverse effects than ketamine and exhibits shorter recovery times after brief periods of anaesthesia. As the paraventricular thalamus (PVT) plays a pivotal role in regulating wakefulness, we studied its role in the emergence process during combined esketamine and isoflurane anaesthesia. METHODS: The righting reflex and cortical electroencephalography were used as measures of consciousness in mice during isoflurane anaesthesia with coadministration of esketamine. The expression of c-Fos was used to determine neuronal activity changes in PVT neurones after esketamine administration. The effect of esketamine combined with isoflurane anaesthesia on PVT glutamatergic (PVTGlu) neuronal activity was monitored by fibre photometry, and chemogenetic technology was used to manipulate PVTGlu neuronal activity. RESULTS: A low dose of esketamine (5 mg kg-1) accelerated emergence from isoflurane general anaesthesia (474 [30] s vs 544 [39] s, P=0.001). Esketamine (5 mg kg-1) increased PVT c-Fos expression (508 [198] vs 258 [87], P=0.009) and enhanced the population activity of PVTGlu neurones (0.03 [1.7]% vs 6.9 [3.4]%, P=0.002) during isoflurane anaesthesia (1.9 [5.7]% vs -5.1 [5.3]%, P=0.016) and emergence (6.1 [6.2]% vs -1.1 [5.0]%, P=0.022). Chemogenetic suppression of PVTGlu neurones abolished the arousal-promoting effects of esketamine (459 [33] s vs 596 [33] s, P<0.001). CONCLUSIONS: Our results suggest that esketamine promotes recovery from isoflurane anaesthesia by activating PVTGlu neurones. This mechanism could explain the rapid arousability exhibited upon treatment with a low dose of esketamine.

Is adherence to the 24-h movement guidelines associated with greater academic-related outcomes in children and adolescents? A systematic review and meta-analysis.

The purpose of this systematic review was to synthesise the evidence for the association of adherence to the 24-h movement guidelines with academic-related outcomes in children and adolescents. This systematic review was based on the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement. PubMed, PsycINFO, Scopus, WOS, SPORTDiscus, and EMBASE were searched from their inception to 12 December 2023. The Joanna Briggs Institute (JBI) Critical Appraisal Checklist was used to assess the risk of bias of included studies. In total, 4326 records were identified through database searches; 10 articles met the inclusion criteria and were included in this systematic review. There were eight cross-sectional studies and two longitudinal studies; the main academic-related outcomes were academic achievement and cognitive function. A small association between adherence to all three recommendations and academic achievement (k = 5, r = 0.17, 95% CI = 0.10-0.24, I2 = 49%) was found compared to those who did not adhere to any recommendations.   Conclusion: Findings from this systematic review and meta-analysis reveal a small association between adherence to all three recommendations and greater academic achievement in children and adolescents. Nevertheless, it is imperative to underscore the need for more studies to establish robust evidence underpinning this relationship.   Trial registration: PROSPERO (CRD42021295403). What is Known: • Regular physical activity, reduced screen time, and optimal sleep duration are independently associated with improved academic-related outcomes in children and adolescents. • The associations between adherence to the 24-h movement guidelines and academic-related outcomes in children and adolescents have not been quantitatively synthesised. What is New: • There is a small but positive association between adherence to all three recommendations of the 24-h movement guidelines and greater academic achievement in children and adolescents. • Further well-designed research is needed to focus on academic achievement, cognitive function and classroom behaviours in young individuals.

The Identification, Characterization, and Functional Analysis of the Sugar Transporter Gene Family of the Rice False Smut Pathogen, Villosiclava virens.

False smut, caused by Villosiclava virens, is becoming increasingly serious in modern rice production systems, leading to yield losses and quality declines. Successful infection requires efficient acquisition of sucrose, abundant in rice panicles, as well as other sugars. Sugar transporters (STPs) may play an important role in this process. STPs belong to a major facilitator superfamily, which consists of large multigenic families necessary to partition sugars between fungal pathogens and their hosts. This study identified and characterized the STP family of V. viren, and further analyzed their gene functions to uncover their roles in interactions with rice. Through genome-wide and systematic bioinformatics analyses, 35 STPs were identified from V.virens and named from VvSTP1 to VvSTP35. Transmembrane domains, gene structures, and conserved motifs of VvSTPs have been identified and characterized through the bioinformatic analysis. In addition, a phylogenetic analysis revealed relationship between VvSTPs and STPs from the other three reference fungi. According to a qRT-PCR and RNA-sequencing analysis, VvSTP expression responded differently to different sole carbon sources and H2O2 treatments, and changed during the pathogenic process, suggesting that these proteins are involved in interactions with rice and potentially functional in pathogenesis. In total, 12 representative VvSTPs were knocked out through genetic recombination in order to analyze their roles in pathogenicity of V. virens. The knock-out mutants of VvSTPs showed little difference in mycelia growth and conidiation, indicating a single gene in this family cannot influence vegetative growth of V. virens. It is clear, however, that these mutants result in a change in infection efficiency in a different way, indicating that VvSTPs play an important role in the pathogenicity of virens. This study is expected to contribute to a better understanding of how host-derived sugars contribute to V. virens pathogenicity.

State-of-the-art strategies and research advances for the biosynthesis of D-amino acids.

D-amino acids (D-AAs) are the enantiomeric counterparts of L-amino acids (L-AAs) and important functional factors with a wide variety of physiological activities and applications in the food manufacture industry. Some D-AAs, such as D-Ala, D-Leu, and D-Phe, have been favored by consumers as sweeteners and fragrances because of their unique flavor. The biosynthesis of D-AAs has attracted much attention in recent years due to their unique advantages. In this review, we comprehensively analyze the structure-function relationships, biosynthesis pathways, multi-enzyme cascade and whole-cell catalysis for the production of D-AAs. The state-of-the-art strategies, including immobilization, protein engineering, and high-throughput screening, are summarized. Future challenges and perspectives of strategies-driven by bioinformatics technologies and smart computing technologies, as well as enzyme immobilization, are also discussed. These new approaches will promote the commercial production and application of D-AAs in the food industry by optimizing the key enzymes for industrial biocatalysts.

Design, synthesis and evaluation of heterocyclic 2-phenylacetate derivatives as water-soluble rapid recovery hypnotics.

In this work, a series of novel heterocyclic 2-phenylacetate derivatives were designed and synthesized as water-soluble and rapid recovery hypnotic agents. After introducing heterocyclic ring to the amide group of propanidid, the obtained propanidid derivatives showed greatly improved hydrophilicity and good anesthetic activity. In three animal experiments (mice, rats, and rabbits), compounds 13-15 showed potent hypnotic potency (HD50 = 7.6, 6.5, 7.4 mg/kg in rabbits, respectively) and higher therapeutic indexes (TI = 17.3, 16.6, 15.2 in rabbits, respectively) than propanidid (TI = 14.7 in rabbits) or propofol (TI = 5.4 in rabbits). Moreover, the recovery time of compounds 13-15 (time to walk, 96.6, 79.6, 81.4 s in rabbits, respectively) were shorter than that of propanidid (124.5 s in rabbits) or propofol (425.3 s in rabbits). The experimental results suggested the potential of compounds 13-15 as water-soluble anesthetics with rapid recovery profile.

Transcriptome analysis preliminary reveals the immune response mechanism of golden cuttlefish (Sepia esculenta) larvae exposed to Cd.

As a well-known marine metal element, Cd can significantly affect bivalve mollusk life processes such as growth and development. However, the effects of Cd on the molecular mechanisms of the economically important cephalopod species Sepia esculenta remain unclear. In this study, S. esculenta larval immunity exposed to Cd is explored based on RNA-Seq. The analyses of GO, KEGG, and protein-protein interaction (PPI) network of 1,471 differentially expressed genes (DEGs) reveal that multiple immune processes are affected by exposure such as inflammatory reaction and cell adhesion. Comprehensive analyses of KEGG signaling pathways and the PPI network are first used to explore Cd-exposed S. esculenta larval immunity, revealing the presence of 16 immune-related key and hub genes involved in exposure response. Results of gene and pathway functional analyses increase our understanding of Cd-exposed S. esculenta larval immunity and improve our overall understanding of mollusk immune functions.

Immunotherapy of B cell lymphoma with CD22-redirected CAR NK-92 cells.

INTRODUCTION: Chimeric antigen receptor (CAR)-NK cells are considered safer than CAR-T cells due to their short lifetime and production of lower toxicity cytokines. By virtue of unlimited proliferative ability in vitro, NK-92 cells could be utilized as the source for CAR-engineered NK cells. CD22 is highly expressed in B cell lymphoma. The goal of our study was to determine whether CD22 could become an alternative target for CAR-NK-92 therapy against B cell lymphoma. MATERIAL AND METHODS: We first generated m971-BBZ NK-92 that expressed a CAR for binding CD22 in vitro. The expression of CAR was assessed by flow cytometric analysis as well as immunoblotting. The cytotoxicity of the m971-BBZ NK-92 cells towards target lymphoma cells was determined by the luciferase-based cytolysis assay. The production of cytokines in CAR NK-92 cells in response to target cells was evaluated by ELISA assay. Lastly, the cytolytic effect was evaluated by the cytolysis assay mentioned above following irradiation. The level of inhibitory receptor of CAR-expressing cells was assessed by flow cytometry. RESULTS: CD22-specific CAR was expressed on m971-BBZ NK-92 cells successfully. m971-BBZ NK-92 cells efficiently lysed CD22-expressing lymphoma cells and produced large amounts of cytokines after coculture with target cells. Meanwhile, irradiation did not apparently influence the cytotoxicity of m971-BBZ NK-92 cells. Inhibitory receptor detection exhibited a lower level of PD-1 in m971-BBZ NK-92 cells than FMC-63 BBZ T cells after repeated antigen stimulation. CONCLUSIONS: Our data show that adoptive transfer of m971-BBZ NK-92 could serve as a promising strategy for immunotherapy of B cell lymphoma.

Substantial Improvement of an Epimerase for the Synthesis of D-Allulose by Biosensor-Based High-Throughput Microdroplet Screening.

Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D-allulose 3-epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure-guided rational design and directed evolution, in which a mutant M3-2 was identified with 17-fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor-based microdroplet screening platform.

Structural insight and engineering of a plastic degrading hydrolase Ple629.

Polyethylene terephthalate (PET) is one of the most abundantly produced synthetic polyesters. The vast number of waste plastics including PET has challenged the waste management sector while also posing a serious threat to the environment due to improper littering. Recently, enzymatic PET degradation has been shown to be a viable option for a circular plastic economy, which can mitigate the plastic pollution. While protein engineering studies on specific PET degradation enzymes such as leaf-branch compost cutinase (LCC), Thermobifida sp. cutinases and Ideonella sakaiensis PETase (IsPETase) have been extensively published, other homologous PET degrading enzymes have received less attention. Ple629 is a polyester hydrolase identified from marine microbial consortium having activity on PET and the bioplastic polybutylene adipate terephthalate (PBAT). In order to explore its catalytic mechanism and improve its potential for PET hydrolysis, we solved its crystal structure in complex with a PET monomer analogue, and validated its structural and mechanistic similarity to known PET hydrolases. By structural comparisons, we identified some hot spot positions described in previous research on protein engineering of PET hydrolases. We substitute these amino acid residues in Ple629, and obtained variants with improved activity and thermo-stability. The most promising variant D226A/S279A exhibited a more than 5.5-fold improved activity on PET nanoparticles than the wild-type enzyme, suggesting its potential applicability in the biotechnological plastic recycling.

Morphological Transformation and In Situ Polymerization of Caspase-3 Responsive Diacetylene-Containing Lipidated Peptide Amphiphile for Self-Amplified Cooperative Antitumor Therapy.

In order to artificially regulate cell behaviors, intracellular polymerization as an emerging chemical technique has attracted much attention. Yet, it is still a challenge to achieve effective intracellular polymerization to conquer tumors in the complex cellular environment. Herein, this work develops a tumor-targeting and caspase-3 responsive nanoparticle composed of a diacetylene-containing lipidated peptide amphiphile and mitochondria-targeting photosensitizer (C3), which undergoes nanoparticle-to-nanofiber transformation and efficient in situ polymerization triggered by photodynamic treatment and activation of caspase-3. The locational nanofibers on the mitochondria membranes lead to mitochondrial reactive oxygen species (mtROS) burst and self-amplified circulation, offering persistent high oxidative stress to induce cell apoptosis. This study provides a strategy for greatly enhanced antitumor therapeutic efficacy through mtROS burst and self-amplified circulation induced by intracellular transformation and in situ polymerization.

The anti-fatigue activity of corn peptides and their effect on gut bacteria.

BACKGROUND: Corn peptides (CPs) are rich in branched-chain amino acids such as leucine and have a variety of biological activities such as antioxidant and improved lipid distribution. In this article, we prepared CPs by enzymatic digestion of corn proteins and evaluated their anti-fatigue activity. RESULTS: We evaluated the anti-fatigue effect of CPs through an exhaustive swimming experiment. The results showed that CPs were able to significantly reduce the rate of body weight gain and prolong the duration of exhaustive swimming. Besides, CPs reduced blood urea nitrogen (BUN) levels after exercise, while they significantly increased muscle glycogen and liver glycogen stores. They reduced muscle cell damage from exercise. In addition, CPs were effective in increasing AMPK, PGC-1α and PI3K protein expression levels and promoting Akt phosphorylation. Correlation analysis showed that CPs increased the abundance of probiotics such as Lactobacillus and Akkermansia in the gut microflora. CONCLUSION: CPs, which enhanced exercise performance in mice and could modulate gut microbial composition, had significant anti-fatigue activity. © 2021 Society of Chemical Industry.

Redesign of a novel D-allulose 3-epimerase from Staphylococcus aureus for thermostability and efficient biocatalytic production of D-allulose.

BACKGROUND: A novel D-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a D-allulose 3-epimerase family enzyme based on its high specificity for D-allulose. It usually converts both D-fructose and D-tagatose to respectively D-allulose and D-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars. RESULTS: SaDAE showed a high activity on D-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s-1 mM-1. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward D-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type. CONCLUSIONS: We redesigned SaDAE for thermostability and biocatalytic production of D-allulose. The research will aid the development of industrial biocatalysts for D-allulose.

Structure and Polymannuronate Specificity of a Eukaryotic Member of Polysaccharide Lyase Family 14.

Alginate is an abundant algal polysaccharide, composed of ß-d-mannuronate and its C5 epimer α-l-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the polysaccharide lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a ß-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 Å resolution and the putative substrate-binding model show that the enzyme adopts a ß-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the ß-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115-Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate lyases.

A novel l-leucine 5-hydroxylase from Nostoc piscinale unravels unexpected sulfoxidation activity toward l-methionine.

Hydroxy amino acids are produced by Fe(II)/αKG-dependent dioxygenases and used widely as medicinal intermediates for chemical synthesis. A novel l-leucine 5-hydroxylase gene from Nostoc piscinale (NpLDO) was cloned into pET28a (+), pColdI and pQE-80 L plasmids. Using a two-step purification process (Ni-affinity chromatography and gel filtration), highly purified recombinant NpLDO was obtained. Recombinant NpLDO displayed unexpectedly high sulfoxidation activity toward l-methionine. The reaction products were analyzed by high-performance liquid chromatography. Sequence alignment analysis implied that residues of His150, His236 and Asp152 constitute the catalytic triad of NpLDO, which is completely conserved in the Fe(II)/αKG-dependent dioxygenase superfamily. Biochemical data showed that NpLDO catalyzed regio- and stereoselective hydroxylation of l-leucine and sulfoxidation of l-methionine with Fe(II) and l-ascorbic acid as cofactor, and αKG as cosubstrate, respectively.

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates.

BACKGROUND: Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. RESULTS: Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively). CONCLUSIONS: The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.

Refolding of a novel cholesterol oxidase from Pimelobacter simplex reveals dehydrogenation activity.

Cholesterol oxidases, which catalyze the degradation of cholesterol to cholest-4-en-3-one, are widely used in the pharmaceutical and food processing industries. The cholesterol oxidase from Pimelobacter simplex (PsChO3) was transformed into E. coli BL21(DE3), but it was expressed mainly as inclusion bodies, and any soluble PsChO3 failed to bind to Ni-NTA resin. To overcome this obstacle, we devised a simple yet efficient purification and refolding process using 8 M urea for the solubilization of PsChO3 and achieved a high yield of the enzyme in its active form. Column-bound PsChO3 was refolded in situ through a gradient of successively decreased urea concentrations and purified using Ni-affinity chromatography, ionic exchange and gel filtration. This treatment converted the denatured PsChO3 into a soluble protein exhibiting an unexpected dehydrogenation activity amounting to 9.27 U/mg - an activity not reported for enzymes with noncovalently-linked FAD to date. The product, cholest-5-en-3-one, was confirmed using TLC, GC-MS and NMR. Structural analysis revealed a distinct binding mode in both FAD and substrate domain, which may explain the enzyme's unusual catalytic behavior.

Biochemical analysis and the preliminary crystallographic characterization of D-tagatose 3-epimerase from Rhodobacter sphaeroides.

BACKGROUND: D-Tagatose 3-epimerase epimerizes D-fructose to yield D-psicose, which is a rare sugar that exists in small quantities in nature and is difficult to synthesize chemically. We aim to explore potential industrial biocatalysts for commercial-scale manufacture of this rare sugar. A D-tagatose 3-epimerase from Rhodobacter sphaeroides (RsDTE) has recently been identified as a D-tagatose 3-epimerase that can epimerize D-fructose to yield D-psicose with a high conversion rate. RESULTS: The purified RsDTE by Ni-affinity chromatography, ionic exchange chromatography and gel filtration forms a tetramer in solution. The maximal activity was in Tris-HCl buffer pH 8.5, and the optimal temperature was at 35 °C. The product, D-psicose, was confirmed using HPLC and NMR. Crystals of RsDTE were obtained using crystal kits and further refined under crystallization conditions such as 10% PEG 8000,0.1 M HEPES pH 7.5, and 8% ethylene glycol at 20 °C using the sitting-drop vapor diffusion method. The RsDTE homology model showed that it possessed the characteristic TIM-barrel fold. Four residues, Glu156, Asp189, Gln215 and Glu250, forms a hydrogen bond network with the active Mn(II) for the hydride transfer reaction. These residues may constitute the catalytic tetrad of RsDTE. The residues around O1, O2 and O3 of the substrates were conserved. However, the binding-site residues are different at O4, O5 and O6. Arg118 formed the unique hydrogen bond with O4 of D-fructose which indicates RsDTE's preference of D-fructose more than any other family enzymes. CONCLUSIONS: RsDTE possesses a different metal-binding site. Arg118, forming unique hydrogen bond with O4 of D-fructose, regulates the substrate recognition. The research on D-tagatose 3-epimerase or D-psicose 3-epimerase enzymes attracts enormous commercial interest and would be widely used for rare sugar production in the future.

Multienzymatic cascade synthesis of fucosyloligosaccharide via a two-step fermentation strategy in Escherichia coli.

OBJECTIVES: To achieve multienzymatic cascade synthesis of fucosyl oligosaccharide from D-mannose by two-step fermentation pathway in Escherichia coli. RESULTS: E. coli BL21(DE3) harboring pET-22b(+) vectors with six genes, i.e., glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate guanylytransferase (ManC), GDP-mannose 4,6-dehydratase (Gmd), GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (WcaG), and α-1,2-fucosyltransferase (Fuct) were co-inoculated, and the multienzyme synthetic pathway was constructed to produce fucosyloligosaccharide using D-mannose as substrate. The product, analyzed by LC/MS, fucosyloligosaccharide was formed under the catalysis of Fuct using GDP-fucose as donor substrate and lactose as acceptor substrate. Fucosyloligosaccharides reached 22 mM by a two-step fermentation compared to 3.7 mM with a one-pot fermentation. CONCLUSIONS: Fucosyloligosaccharide was produced by a two-step fermentation to avoid the inhibitory effect of GDP-fucose on Gmd. Two-step fermentation is a rational synthetic pathway for accumulating fucosyloligosaccharide.

L-allo-threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity.

L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Šresolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Šoutwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.

Structural optimization of SadA, an Fe(II)- and α-ketoglutarate-dependent dioxygenase targeting biocatalytic synthesis of N-succinyl-L-threo-3,4-dimethoxyphenylserine.

L-threo-3,4-Dihydroxyphenylserine (l-DOPS, Droxidopa) is a psychoactive drug and synthetic amino acid precursor that acts as a prodrug to the neurotransmitters. SadA, a dioxygenase from Burkholderia ambifaria AMMD, is an Fe(II)- and α-ketoglutarate (KG)-dependent enzyme that catalyzes N-substituted branched-chain or aromatic l-amino acids. SadA is able to produce N-succinyl-l-threo-3,4-dimethoxyphenylserine (NSDOPS), which is a precursor of l-DOPS, by catalyzing the hydroxylation of N-succinyl-3,4-dimethoxyphenylalanine (NSDOPA). However, the catalytic activity of SadA toward NSDOPS is much lower than that toward N-succinyl branched-chain l-amino acids. Here, we report an improved biocatalytic synthesis of NSDOPS with SadA. Structure-based protein engineering was applied to improve the α-KG turnover activity for the synthesis of NSDOPS. The G79A, G79A/F261W or G79A/F261R mutant showed a more than 6-fold increase in activity compared to that of the wild-type enzyme. The results provide a new insight into the substrate specificity toward NSDOPA and will be useful for the rational design of SadA mutants as a target of industrial biocatalysts.