Search

Background: The development of biofuels, especially liquid hydrocarbon fuels, has been widely concerned due to the depletion of fossil resources. In order to obtain fuel precursors, the reaction of C–C bond formation is usually carried out with biomass derived ketones/aldehydes as reactants. Acetoin and 2,3-butanediol are two platform chemicals, which are co-existed in fermentation broth and traditionally separated by distillation, and then acetoin could be use as C4 building block to prepare hydrocarbon fuels. In order to mitigate the process complexity, direct aldol condensation reaction of acetoin in fermentation broth was studied in this work. Results: A one-pot process of product separation and acetoin derivative synthesis was proposed based on salting-out extraction (SOE). Aldol condensation reaction of acetoin and 5-methyl furfural in different SOE systems was compared, and the results showed that the synthesis of C10 fuel precursors and separation of C10 products and 2,3-butanediol from fermentation broth were achieved in one-pot with ethanolammonium butyrate (EOAB) and K2HPO4 as SOE reagents and catalysts. The SOE and reaction conditions such as the concentrations of EOAB and K2HPO4, reaction temperature and time were optimized. When the system was composed of 6 wt% EOAB-44 wt% K2HPO4 and the mixture was stirred for 6 h at 200 rpm, 40 ℃, the yield of C10 products was 80.7%, and 95.5% 2,3-butanediol was distributed to the top EOAB-rich phase. The exploration of reaction mechanism showed that an imine intermediate was rapidly formed and the subsequent C10 product formation was the key step for aldol condensation reaction. Conclusions: With EOAB and K2HPO4 as SOE reagents and catalysts, one-pot synthesis of fuel precursor from acetoin fermentation broth was achieved without prior purification. A yield of 80.7% for C10 products was obtained which was accumulated at the interface of two aqueous-phase, and 95.5% 2,3-BD was distributed to the top EOAB-rich phase. This work provides a new integration process of product separation and derivative synthesis from fermentation broth based on ionic liquid SOE. [ABSTRACT FROM AUTHOR]

Since most electrocatalysts for oxygen evolution reaction (OER), except for precious metal oxides RuO2 and IrO2, are unstable in harsh acidic solutions, it is highly desirable to develop high‐performance OER electrocatalysts for acidic media, though it is still a big challenge. Herein, we report a simple strategy to produce carboxyl‐enriched multiwalled carbon nanotubes (COOH‐MWNTs) that exhibit stable and high electrocatalytic activities for OER in acidic solutions, showing an overpotential at a current density of 10 mA cm–2 and a Tafel slope as low as of 265 mV and 82 mV dec–1, respectively. As far as we are aware, these results represent the best OER performance for metal‐free electrocatalysts, even comparable to those of RuO2 and IrO2. We have further revealed the catalytic mechanism, which involves one electron lose from the COOH‐MWNTs catalyst at the beginning of the OER process to trigger H2O molecule oxidation by forming peralcohol, followed by the recapture of one electron from water molecule to oxidize water and to recover the initial state for the COOH‐MWNTs catalyst. The unravel of this new OER mechanism is important as it provides new insights into the crucial role of organic functional groups in electrocatalytic processes. Also, the mechanistic understanding can be used to guide the design and development of novel metal‐free catalysts for acidic OER electrocatalysis and beyond. [ABSTRACT FROM AUTHOR]

The role of the conserved Gly in the structure and activities of the cap domain loop of the haloacid dehalogenase (HAD) super family members, the Gly was mutated to Pro, Val, or Ala is evaluated. The studies implies that the flexibility afforded by the conserved Gly is important to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be seen within the amino acid sequence of HAD family members.

Background: The relationship between dysbiosis of the gastrointestinal microbiota and gastric cancer (GC) has been extensively studied. However, microbiota alterations in GC patients vary widely across studies, and reproducible diagnostic biomarkers for early GC are still lacking in multiple populations. Thus, this study aimed to characterize the gastrointestinal microbial communities involved in gastric carcinogenesis through a meta-analysis of multiple published and open datasets., Methods: We analyzed 16S rRNA sequencing data from 1,642 gastric biopsy samples and 394 stool samples across 11 independent studies. VSEARCH, QIIME and R packages such as vegan, phyloseq, cooccur, and random forest were used for data processing and analysis. PICRUSt software was employed to predict functions., Results: The α-diversity results indicated significant differences in the intratumoral microbiota of cancer patients compared to non-cancer patients, while no significant differences were observed in the fecal microbiota. Network analysis showed that the positive correlation with GC-enriched bacteria increased, and the positive correlation with GC-depleted bacteria decreased compared to healthy individuals. Functional analyses indicated that pathways related to carbohydrate metabolism were significantly enriched in GC, while biosynthesis of unsaturated fatty acids was diminished. Additionally, we investigated non- Helicobacter pylori (HP) commensals, which are crucial in both HP -negative and HP -positive GC. Random forest models, constructed using specific taxa associated with GC identified from the LEfSe analysis, revealed that the combination of Lactobacillus and Streptococcus included alone could effectively discriminate between GC patients and healthy individuals in fecal samples (area under the curve (AUC) = 0.7949). This finding was also validated in an independent cohort (AUC = 0.7712)., Conclusions: This study examined the intratumoral and fecal microbiota of GC patients from a dual microecological perspective and identified Lactobacillus, Streptococcus, Roseburia, Faecalibacterium and Phascolarctobacterium as intratumoral and intestinal-specific co-differential bacteria. Furthermore, it confirmed the validity of the combination of Lactobacillus and Streptococcus as GC-specific microbial markers across multiple populations, which may aid in the early non-invasive diagnosis of GC., Competing Interests: Author XL is employed by Liaoning Kanghui Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Wang, Han, Han, Liu, Dai, Dong, Sun and Xu.)

Aims: The aim of this meta-analysis was to assess the diagnostic performance of EUS-FNA/B in patients with panNETs., Methods: We conducted a computerized search of the MEDLINE and Embase databases to identify relevant articles. The primary outcomes involved grading concordance rate, diagnostic rate, and correlation coefficient (Cohen's κ) for FNA/B samples compared with surgical specimens. Secondary outcomes included sample adequacy, mean number of passes, and adverse events., Results: Forty-five studies involving 2978 patients were finally included. The pooled concordance rate between EUS-FNA/B and surgical grading was 0.77 (95% CI: 0.73-0.80; I2=48.2%). A significantly higher level of concordance was observed in G1 subgroup (0.88, 95% CI: 0.84-0.91), whereas the G2 subgroup revealed the lowest level of agreement (0.59, 95% CI: 0.52-0.65; P < 0.001). Pooled diagnostic rate for FNA/B sampling was 0.83 (95% CI: 0.79-0.86; I2=63.3%). In addition, FNB outperformed FNA in terms of sample adequacy (0.93 for FNB vs. 0.81 for FNA; P=0.007) and number of needle passes required (2.53 for FNB vs. 3.32 for FNA; P=0.013). Moreover, the overall level of agreement for grading was moderate (κ=0.59, 95% CI: 0.49-0.68; I2=84.5%). There were a limited number of adverse events that had minor influence on patient outcomes (0.03, 95% CI: 0.02-0.05; I2=19.2%)., Conclusions: EUS-FNA/B is a reliable approach for the diagnosis and preoperative grading of panNET, with FNB demonstrating superior performance compared with FNA., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Bio-refinery using cheap biomass focuses mainly on strain improvement and fermentation strategies whereas less effort is made on down-stream processing. Using cheap biomass more impurities are introduced into the fermentation broths than mono-sugar substrate, thus down-stream processing for bio-based chemicals becomes the key problem in industrial production. The technique called salting-out extraction (SOE) was introduced in this review, which is used to separate target products from fermentation broth on the basis of partition difference of chemicals in two phases formed by mixing salts and organic solvents (or amphipathic chemicals) with broth at suitable ratios. The effect of solvents and salts on the formation of two aqueous phases, especially short chain alcohols and inorganic salts, and the application of SOE in recovery of bio-based chemicals, such as lactic acid, 1,3-propanediol, 2,3-butanediol and acetoin were summarized. The bio-chemicals were efficiently recovered from fermentation broth, and most of the impurities (cells and proteins) were removed in the same step. This technique is promising in the separation of bio-based chemicals, especially the recovery of hydrophilic molecules with low molecular weights.

Tauhe beta-phosphoglucomutase (beta-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of beta-glucose 1-phosphate (betaG1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active site to mediate phosphoryl transfer from beta-glucose 1,6-(bis)phosphate (betaG1,6bisP) to betaG1P. Herein, we explore the mechanism by which hydrolysis of the beta-PGM phospho-Asp8 is avoided during the time that the active site must remain open to solvent to allow the exchange of the bound product G6P with the substrate betaG1P. On the basis of structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 portion of the domain-domain linker to a functional position where it forms a hydrogen bond to the substrate leaving group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH group is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of Asp10 with Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by betaG1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert betaG1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray crystal structure of the T16P mutant at 2.7 A resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core domain active site. The His20 and Lys76 site-directed mutants exhibit reduced activity in catalysis of the Asp8-mediated phosphoryl transfer between betaG1,6bisP and betaG1P but no reduction in the rate of phospho-Asp8 hydrolysis. Taken together, the results support a substrate induced-fit model of catalysis in which betaG1P binding to the core domain facilitates recruitment of the general acid/base Asp10 to the catalytic site and induces cap closure.

Activated Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzes the conversion of beta-d-glucose 1-phosphate (betaG1P) derived from maltose to beta-d-glucose 6-phosphate (G6P). Activation requires Mg(2+) binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-)(1), K(m) = 49 +/- 4 microM for the substrate betaG1P and K(m) = 6.5 +/- 0.7 microM for the activator beta-d-glucose 1,6-bisphosphate (betaG1,6bisP). The observed transient accumulation of [(14)C]betaG1,6bisP (12% at approximately 0.1 s) in the single turnover reaction carried out with excess betaPGM (40 microM) and limiting [(14)C]betaG1P (5 microM) and betaG1,6bisP (5 microM) supported the role of betaG1,6bisP as a reaction intermediate in the conversion of the betaG1P to G6P. Single turnover reactions of [(14)C]betaG1,6bisP with excess betaPGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the betaG1,6bisP binds to the active site in two different orientations (one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with betaPGM, [(14)C]betaG1P, and unlabeled betaG1,6bisP demonstrated complete exchange of label to the betaG1,6bisP during the catalytic cycle. Thus, the reorientation of the betaG1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of betaG1P suggest that the reorientation and phosphoryl transfer from betaG1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of betaG1,6bisP to betaG1P plus G6P was examined to evidence a significant stabilization of betaPGM aspartyl phosphate.

Congenital disorder of glycosylation type 1a (CDG-1a) is a congenital disease characterized by severe defects in nervous system development. It is caused by mutations in alpha-phosphomannomutase (of which there are two isozymes, alpha-PMM1 and alpha-PPM2). Here we report the x-ray crystal structures of human alpha-PMM1 in the open conformation, with and without the bound substrate, alpha-D-mannose 1-phosphate. Alpha-PMM1, like most haloalkanoic acid dehalogenase superfamily (HADSF) members, consists of two domains, the cap and core, which open to bind substrate and then close to provide a solvent-exclusive environment for catalysis. The substrate phosphate group is observed at a positively charged site of the cap domain, rather than at the core domain phosphoryl-transfer site defined by the Asp(19) nucleophile and Mg(2+) cofactor. This suggests that substrate binds first to the cap and then is swept into the active site upon cap closure. The orientation of the acid/base residue Asp(21) suggests that alpha-phosphomannomutase (alpha-PMM) uses a different method of protecting the aspartylphosphate from hydrolysis than the HADSF member beta-phosphoglucomutase. It is hypothesized that the electrostatic repulsion of positive charges at the interface of the cap and core domains stabilizes alpha-PMM1 in the open conformation and that the negatively charged substrate binds to the cap, thereby facilitating its closure over the core domain. The two isozymes, alpha-PMM1 and alpha-PMM2, are shown to have a conserved active-site structure and to display similar kinetic properties. Analysis of the known mutation sites in the context of the structures reveals the genotype-phenotype relationship underlying CDG-1a.

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.

This communication reports the X-ray crystal structure of the alpha-d-galactose-1-phosphate complex with that of Lactococcus lactis beta-phosphoglucomutase (beta-PGM) crystallized in the presence of Mg2+ cofactor and the enzyme-to-phosphorus ratio determined by protein and phosphate analyses of the crystalline complex. The 1:1 ratio determined for this complex was compared to the 1:2 ratio determined for the crystals of beta-PGM grown in the presence of substrate and Mg2+ cofactor. This result verifies the published structure assignment of this latter complex as the phosphorane adduct formed by covalent bonding between the active site Asp8 carboxylate to the C(1)phosphorus of the beta-glucose 1,6-bisphosphate ligand and rules out the proposal of a beta-PGM-glucose-6-phosphate-1-MgF3- complex.