Utilization of gluconate by Escherichia coli. Induction of gluconate kinase and 6-phosphogluconate dehydratase activities.
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ABSTRACT:
SUBMITTER: Kornberg HL
PROVIDER: S-EPMC1177835 | biostudies-other | 1973 Jun
REPOSITORIES: biostudies-other
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Project description:1. Cultures of Escherichia coli growing on gluconate use both gluconate and glucose when glucose is added. 2. Glycerol-grown cells adapt to gluconate utilization even in media containing glucose as well as gluconate. 3. The rates of gluconate utilization by cells growing on a mixture of glucose and gluconate, and the specific activities of the gluconate uptake system and of gluconate kinase, are greater if adenosine 3':5'-cyclic monophosphate (cyclic AMP) is present in the medium than in its absence. 4. Growth on media containing gluconate and cyclic AMP is accompanied by the formation of methyl glyoxal and pyruvate, and progressive inhibition of growth. 5. A mutant devoid of adenylate cyclase activity (cya) grew well on glucose in the absence of exogenous cyclic AMP but grew only poorly on gluconate; neither the gluconate uptake system nor gluconate kinase was adequately induced. The addition of cyclic AMP promoted growth on gluconate and facilitated the induction of proteins required for gluconate catabolism. 6. Phage Pl-mediated transduction of cya+ into the cya-mutant also restored the wild-type phenotype in its ability to adapt to gluconate utilization.
| S-EPMC1165711 | biostudies-other
Project description:1. From Escherichia coli strain K2.1.5(c).8.9, which is devoid of 6-phosphogluconate dehydrogenase (gnd) and 6-phosphogluconate dehydratase (edd) activities, a mutant R6 was isolated that was tolerant to gluconate though still edd(-), gnd(-). 2. Measurements of the fate of labelled gluconate, of the conversion of gluconate into 6-phosphogluconate, and of the induction of gluconate kinase by the two organisms show that, although both inducibly form a gluconate-transport system, strain R6 is impaired in its ability to convert the gluconate thus taken up into 6-phosphogluconate; it was therefore used for study of the kinetics and energetics of gluconate uptake. 3. Suspensions of strain R6 induced for gluconate uptake took up this substrate via a ;high affinity' transport process, with K(m) about 10mum and V(max.) about 25nmol/min per mg dry mass; a ;low affinity' system demonstrated to occur in certain E. coli mutants was not induced under the conditions used in this work. 4. The uptake of gluconate was inhibited by lack of oxygen and by inhibitors of electron transport; such inhibitors also promoted the efflux of gluconate taken up. 5. Membrane vesicles prepared from strain R6 also manifested these properties when incubated with suitable electron donors, at rates similar to those observed with whole cells. 6. The results indicate that the active transport of gluconate into the cells is the rate-limiting step in gluconate utilization by E. coli, and that the mechanism of this process can be validly studied with membrane vesicles.
| S-EPMC1167991 | biostudies-other
Project description:D-xylonate dehydratase YjhG from Escherichia coli can convert D-xylonate into 2-keto-3-deoxy- D-xylonate (KDX), and is a key enzyme in the biosynthesis of 1,2,4-butanetriol and other chemicals. However, the biochemical properties of YjhG still remain unknown. In this study, the activity assay method for YjhG was established based on semicarbazide method, in which KDX reacts with semicarbazide reagent, and is further quantified by high-resolution mass spectrometry. The effect of reaction conditions on YjhG activity was determined in vitro using purified His-tagged YjhG protein. This enzyme showed maximal activity at 30°C and pH 8.0. Bivalent metal ions such as Mg(2+) and Mn(2+) activated, whereas Ni(2+) and Zn(2+) inhibited the activity of YjhG. Under optimal conditions, the Km and Vmax values were 4.88 mM and 78.62 ?M l(-1)h(-1), respectively, when using D-xylonate as a substrate. Amino acids sequence alignments and catalytic properties analysis revealed that YjhG might be a member of IlvD/EDD family. Results obtained in this study may lay a foundation for further investigation on YjhG and will benefit its application in biosynthesis of related chemicals.
| S-EPMC4601382 | biostudies-literature
Project description:D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.
| S-EPMC7380123 | biostudies-literature
Project description:The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a V max of 61.6 U mg-1 and a K M of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses.
| S-EPMC7099567 | biostudies-literature
Project description:UnlabelledThe halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcaniiImportanceThe work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea.
| S-EPMC4966447 | biostudies-literature
Project description:The Sinorhizobium meliloti megaplasmid pSymA has previously been implicated in gluconate utilization. We report a locus on pSymA encoding a putative tripartite ATP-independent periplasmic (TRAP) transporter that is required for gluconate utilization. The expression of this locus is negatively regulated by a GntR family regulator encoded adjacent to the transporter operon.
| S-EPMC2631997 | biostudies-literature
Project description:The gntP gene, located between the fim and uxu loci in Escherichia coli K-12, has been cloned and characterized. Nucleotide sequencing of a region encompassing the gntP gene revealed an open reading frame of 447 codons with significant homology to the Bacillus subtilis gluconate permease. Northern (RNA) blotting indicated that the gntP gene was monocistronic and was transcribed as an mRNA with an apparent molecular size of 1.54 kb. The transcriptional start point was determined by primer extension analysis. The gntP gene was found to be under catabolite repression and was not induced by gluconate. Also, expression seemed to be stringently controlled. Several observations indicated that the GntP protein is an inner membrane protein; it contains characteristic membrane-spanning regions and was isolated predominantly from the inner-membrane fraction of fractionated host cells. A topology analysis predicted a protein with 14 membrane-spanning segments. The inability of a mutant strain to grow on gluconate minimal medium could be relieved by introduction of a plasmid encoding the gntP gene. Finally, the kinetics of GntP-mediated gluconate uptake were investigated, indicating an apparent Km for gluconate of 25 microM.
| S-EPMC177621 | biostudies-other
Project description:Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.
| S-EPMC10128022 | biostudies-literature
Project description:Cryptococcus neoformans is an environmental yeast and an opportunistic human pathogen. The ability to cause disease depends on the ability to adapt to the human host. Previous studies implicated infectivity-related kinase 3 (IRK3, CNAG_03048) as required for establishing an infection. We genetically and biochemically characterized IRK3 as a gluconate kinase and propose the name GNK1. This metabolic enzyme utilizes gluconate to produce 6-phosphogluconate as part of the alternative oxidative phase of the pentose phosphate pathway (AOXPPP). The presence of GNK1 confirms that the AOXPPP is present and able to compensate for loss of the traditional OXPPP, providing an explanation for its nonessentiality. C. neoformans can utilize gluconate as an alternative carbon source in a GNK1-dependent manner. In our efforts to understand the role of GNK1 in host adaptation and virulence, we found that GNK1-deficient mutants have variable virulence and carbon dioxide tolerance across multiple strains, suggesting that second site mutations frequently interact with GNK1 deletion mutations. In our effort to isolate these genetic loci by backcrossing experiments, we discovered that GNK1-deficient strains are unable to sporulate. These data suggest that gluconate metabolism is critical for sporulation of C. neoformans. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that contributes to nearly 180,000 deaths annually. We characterized a gene named GNK1 that appears to interact with other genetic loci involved with the ability of C. neoformans to act as a pathogen. While these interacting genetic loci remain elusive, we discovered that GNK1 plays roles in both metabolism and mating/sporulation. Further interrogation of the mechanistic role for GNK1 in sexual reproduction may uncover a larger network of genes that are important for host adaptation and virulence.
| S-EPMC9045243 | biostudies-literature