Ring-opening mechanism revealed by crystal structures of NagB and its ES intermediate complex

Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme-substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme-substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring.     

The role of glucosamine-6-phosphate deaminase at the early stages of <Emphasis Type="Italic">Aspergillus niger</Emphasis> growth in a high-citric-acid-yielding medium

Glucosamine-6-phosphate (GlcN6P) deaminase seems to be the main enzyme in Aspergillus niger cells responsible for rapid glucosamine accumulation during the early stages of growth in a high-citric-acid-yielding medium. By determining basic kinetic parameters on the isolated enzyme, a high affinity toward fructose-6-phosphate (Fru6P) was measured, while in the reverse direction the K _m value for glucosamine-6-phosphate was lower than deaminases from other organisms measured so far. The enzyme characteristics of GlcN6P deaminase suggest it must compete with 6-phosphofructo-1-kinase (PFK1) for the common substrate—Fru6P in A. niger cells. Glucosamine accumulation seems therefore to remove an intermediate from the glycolytic flux, a situation which is reflected in slower citric acid accumulation and a specific growth rate after the germination of spores. When ammonium ions are depleted from the medium, one of the substrates for GlcN6P deaminase becomes limiting and Fru6P can be catabolised by PFK1 which enhances glycolytic flux. Other enzymatic features of GlcN6P deaminase such as pH optima for both aminating and deaminating reactions might play a significant role in rapid glucosamine accumulation during the early phase of fermentation and a slow consumption of aminosugar during the citric-acid-producing phase.     

Nutrient-specific proteomic analysis of the mucin degrading bacterium Akkermansia muciniphila

Akkermansia muciniphila is a prominent mucin-degrading bacterium that acts as a keystone species in regulating the human gut microbiota. Despite recently increasing research into this bacterium and its relevance to human health, a high-resolution database of its functional proteins remains scarce. Here, we provide a proteomic overview of A. muciniphila grown in different nutrient conditions ranging from defined to complex. Of 2318 protein-coding genes in the genome, we identified 841 (40%) that were expressed at the protein level. Overall, proteins involved in energy production and carbohydrate metabolism indicate that A. muciniphila relies mainly on the Embden-Meyerhof-Parnas pathway, and produces short-chain fatty acids through anaerobic fermentation in a nutrient-specific manner. Moreover, this bacterium possesses a broad repertoire of glycosyl hydrolases, together with putative peptidases and sulfatases, to cleave O-glycosylated mucin. Of them, putative mucin-degrading enzymes (Amuc_1220, Amuc_1120, Amuc_0052, Amuc_0480, and Amuc_0060) are highly abundant in the mucin-supplemented media. Furthermore, A. muciniphila uses mucin-derived monosaccharides as sources of energy and cell wall biogenesis. Our dataset provides nutrient-dependent global proteomes of A. muciniphila ATCC BAA-835 to offer insights into its metabolic functions that shape the composition of the human gut microbiota via mucin degradation.     

Comparative genetic analysis of,Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation

Induction and secretion of acid phosphatases （APases） is thought to be an adaptive mechanism that helps plants survive and grow under phosphate （Pi） deprivation, in Arabidopsis, there are 29 purple acid phosphatase （AtPAP） genes. To systematically investigate the roles of different AtPAPs, we first identified knockout or knock-down T-DNA lines for all 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAPlo is mainly a secreted APase. On Pi-deficient （P-） medium or P- medium supplemented with the organophosphates ADP and fructose-6-phosphate （Fru-6-P）, growth of atpaplo was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type （WT）. Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on P- or P- medium supplemented with ADP or Fru-6-P. Interest-ingly, Pi levels are essentially the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake.     

Characterization of a novel glucosamine-6-phosphate deaminase from a hyperthermophilic archaeon

A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD(Tk)) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD(Tk) was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD(Tk) clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD(Tk). In T. kodakaraensis cells, glmD(Tk) was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-beta-glucosaminidase gene (glmA(Tk)) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD(Tk) is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.     

嗜粘蛋白阿克曼菌在糖尿病和肥胖中的作用

肠道微生物在平衡健康与疾病的过程中起着重要作用。嗜粘蛋白阿克曼菌(Akkermansia muciniphila)是肠道微生物中的一种,其在降解肠道粘蛋白方面的特性使其成为肠腔与宿主细胞黏膜界面的关键生物。嗜粘蛋白阿克曼菌与肥胖、糖尿病、心血管代谢疾病和低度炎症呈负相关。口服嗜粘蛋白阿克曼菌可改善小鼠代谢疾病的相关症状,嗜粘蛋白阿克曼菌有望成为治疗2型糖尿病和肥胖的候选药物。本综述通过总结现有关于嗜粘蛋白阿克曼菌在糖尿病和肥胖症中发挥作用的研究,指出嗜粘蛋白阿克曼菌调节宿主和肠道微生物群之间相互作用可能存在的机制,为嗜粘蛋白阿克曼菌的进一步研究和糖尿病的新药研发提供思路。     

Why does Escherichia coli grow more slowly on glucosamine than on N-acetylglucosamine? Effects of enzyme levels and allosteric activation of GlcN6P deaminase (NagB) on growth rates

Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.     

De novo assembly of a wild pear (Pyrus betuleafolia) genome

China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia‐Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi‐C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein‐coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism's considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide‐binding site (NBS)‐type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR‐NBS‐LRR (TNL)‐type genes, which represented the greatest number of TNL‐type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism‐related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high‐quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding.     

pHmetrical determination of the glucosamine-6-phosphate isomerase deaminase reverse reaction

In the reverse direction, the reaction catalyzed by glucosamine 6-phosphate isomerase deaminase consumes ammonia and forms GlcN6P. As a consequence of the formation of a product with a lower pK than the substrates, a measurable pH drop in the reaction medium is produced. This property can be used to follow potentiometrically the course of the reaction. This property can be used to follow potentiometrically the course of the reaction. The usefulness of the method is demonstrated obtaining the inhibition pattern by GlcN6P when Fru6P is the varied substrate.     

Diet shapes the ability of human intestinal microbiota to degrade phytate – in vitro studies

Aims

Investigation of intestinal bacterial groups involved in phytate degradation and the impact of diets with different phytate contents on phytase activity.

Methods and Results

Faecal samples of adults on conventional (n = 8) or vegetarian (n = 8) diets and breastfed infants (n = 6) were used as an inoculum for modified media supplemented with phytate. Populations of Gram‐positive anaerobes (GPA), lactic acid bacteria (LAB), Proteobacteria–Bacteroides (P‐B), coliforms and anaerobes were studied. The PCR‐DGGE analysis revealed a random distribution of DGGE profiles in the dendrograms of GPA, P‐B and coliforms, and a partially diet‐specific distribution in the DGGE dendrograms of LAB and anaerobes. The degradation of phytic acid (PA) was determined with HPLC method in supernatants of the cultures. Regardless of the diet, the Gram‐positive anaerobes and LAB displayed the lowest ability to degrade phytate, whereas the coliforms and P‐B cultures produced higher amounts of intermediate myo‐inositol phosphates. Bacterial populations grown in a nonselective medium were the most effective ones in phytate degradation. It was the vegetarians' microbiota that particularly degraded up to 100% phytate to myo‐inositol phosphate products lower than InsP₃.

Conclusions

A diet rich in phytate increases the potential of intestinal microbiota to degrade phytate. The co‐operation of aerobic and anaerobic bacteria is essential for the complete phytate degradation.

Significance and Impact of the Study

This study provides insights on the effect of diet on specific metabolic activity of human intestinal microbiota.     

Biosynthesis of paromamine derivatives in engineered Escherichia coli by heterologous expression

Aims: Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6‐disubstituted 2‐deoxystreptamine (DOS)‐containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose‐6‐phosphate isomerase (pgi) of primary metabolic pathway to increase glucose‐6‐phosphate pool inside the host. Disruption was carried out by λ Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2‐deoxy‐scyllo‐inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Δpgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5‐ and 4,6‐disubtituted route of DOS‐containing aminoglycosides.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Akkermansia muciniphila alleviates high-fat-diet-related metabolic-associated fatty liver disease by modulating gut microbiota and bile acids

It has been reported that Akkermansia muciniphila improves host metabolism and reduces inflammation; however, its potential effects on bile acid metabolism and metabolic patterns in metabolic-associated fatty liver disease (MAFLD) are unknown. In this study, we have analysed C57BL/6 mice under three feeding conditions: (i) a low-fat diet group (LP), (ii) a high-fat diet group (HP) and (iii) a high-fat diet group supplemented with A. muciniphila (HA). The results found that A. muciniphila administration relieved weight gain, hepatic steatosis and liver injury induced by the high-fat diet. A. muciniphila altered the gut microbiota with a decrease in Alistipes, Lactobacilli, Tyzzerella, Butyricimonas and Blautia, and an enrichment of Ruminiclostridium, Osclibacter, Allobaculum, Anaeroplasma and Rikenella. The gut microbiota changes correlated significantly with bile acids. Meanwhile, A. muciniphila also improved glucose tolerance, gut barriers and adipokines dysbiosis. Akkermansia muciniphila regulated the intestinal FXR-FGF15 axis and reshaped the construction of bile acids, with reduced secondary bile acids in the caecum and liver, including DCA and LCA. These findings provide new insights into the relationships between probiotics, microflora and metabolic disorders, highlighting the potential role of A. muciniphila in the management of MAFLD.     

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation

Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady‐state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of ¹³C into metabolic intermediates and end products when cells were labelled with [1‐¹³C], [2‐¹³C], or [¹³C₆]glucose, in combination with ¹⁴C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid.     

A Preliminary Study Examining the Binding Capacity of Akkermansia muciniphila and Desulfovibrio spp., to Colonic Mucin in Health and Ulcerative Colitis

Background

Akkermansia muciniphila and Desulfovibrio spp. are commensal microbes colonising the mucus gel layer of the colon. Both species have the capacity to utilise colonic mucin as a substrate. A. muciniphila degrades colonic mucin, while Desulfovibrio spp. metabolise the sulfate moiety of sulfated mucins. Altered abundances of these microorganisms have been reported in ulcerative colitis (UC). However their capacity to bind to human colonic mucin, and whether this binding capacity is affected by changes in mucin associated with UC, remain to be defined.

Methods

Mucin was isolated from resected colon from control patients undergoing resection for colonic cancer (n = 7) and patients undergoing resection for UC (n = 5). Isolated mucin was purified and printed onto mucin microarrays. Binding of reference strains and three clinical isolates of A. muciniphila and Desulfovibrio spp. to purified mucin was investigated.

Results

Both A. muciniphila and Desulfovibro spp. bound to mucin. The reference strain and all clinical isolates of A. muciniphila showed increased binding capacity for UC mucin (p < .005). The Desulfovibrio reference strain showed increased affinity for UC mucin. The mucin binding profiles of clinical isolates of Desulfovibrio spp. were specific to each isolate. Two isolates showed no difference in binding. One UC isolate bound with increased affinity to UC mucin (p < .005).

Conclusion

These preliminary data suggest that differences exist in the mucin binding capacity of isolates of A. muciniphila and Desulfovibrio spp. This study highlights the mucin microarray platform as a means of studying the ability of bacteria to interact with colonic mucin in health and disease.