Abstract:The oral microflora is composed of both health-promoting as well as disease-initiating bacteria. Many of the disease-initiating bacteria are anaerobic and include organisms such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. Here we investigated a novel therapeutic, amixicile, that targets pyruvate:ferredoxin oxidoreductase (PFOR), a major metabolic enzyme involved in energy generation through oxidative decarboxylation of pyruvate. PFOR is present in thes… Show more
“…The presence of a glutamine at this position has never been reported in other bacterial or plant DHDPS enzymes, and the crystal structure of PaDHDPS2-H56Q demonstrates that steric hindrance prevents lysine binding into the allosteric cleft. Pyruvate and ASA are used in multiple other biosynthetic pathways, including alanine and lactate synthesis [55][56][57], as well as threonine, isoleucine and methionine synthesis [58][59][60]. These findings not only further support the current dogma regarding the importance of position 56 as an allosteric determinant in bacterial DHDPS enzymes [20] but also expand it by adding glutamine to the list of residues that prevent allosteric inhibition.…”
Section: Discussionsupporting
confidence: 54%
“…Importantly, we speculate that the presence of two differentially regulated DHDPS enzymes in P. aeruginosa allows a means to control the flux of pyruvate and ASA into the DAP pathway as observed in plants [48,49,54]. Pyruvate and ASA are used in multiple other biosynthetic pathways, including alanine and lactate synthesis [55][56][57], as well as threonine, isoleucine and methionine synthesis [58][59][60]. Limiting ASA and pyruvate entry into the DAP pathway ensures substrate availability for other pathways.…”
Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital‐acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso‐DAP and lysine. The rate‐limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat = 29 s−1 and 44 s−1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50 = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen.
Databases
PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6P90.
“…The presence of a glutamine at this position has never been reported in other bacterial or plant DHDPS enzymes, and the crystal structure of PaDHDPS2-H56Q demonstrates that steric hindrance prevents lysine binding into the allosteric cleft. Pyruvate and ASA are used in multiple other biosynthetic pathways, including alanine and lactate synthesis [55][56][57], as well as threonine, isoleucine and methionine synthesis [58][59][60]. These findings not only further support the current dogma regarding the importance of position 56 as an allosteric determinant in bacterial DHDPS enzymes [20] but also expand it by adding glutamine to the list of residues that prevent allosteric inhibition.…”
Section: Discussionsupporting
confidence: 54%
“…Importantly, we speculate that the presence of two differentially regulated DHDPS enzymes in P. aeruginosa allows a means to control the flux of pyruvate and ASA into the DAP pathway as observed in plants [48,49,54]. Pyruvate and ASA are used in multiple other biosynthetic pathways, including alanine and lactate synthesis [55][56][57], as well as threonine, isoleucine and methionine synthesis [58][59][60]. Limiting ASA and pyruvate entry into the DAP pathway ensures substrate availability for other pathways.…”
Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital‐acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso‐DAP and lysine. The rate‐limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (kcat = 29 s−1 and 44 s−1 for PaDHDPS1 and PaDHDPS2, respectively), they are differentially allosterically regulated by lysine, with only PaDHDPS2 showing inhibition by the end product of the DAP pathway (IC50 = 130 μm). The differences in allostery are attributed to a single amino acid difference in the allosteric binding pocket at position 56. This is the first example of a bacterium that contains multiple bona fide DHDPS enzymes, which differ in allosteric regulation. We speculate that the presence of the two isoforms allows an increase in the metabolic flux through the DAP pathway when required in this clinically important pathogen.
Databases
PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6P90.
“…Features indicating potential anaerobic metabolic capabilities are also present, including full lactic acid and ethanol fermentation pathways, an incomplete Calvin-Benson-Bassham (CBB) cycle (glyceraldehyde-3P to ribulose-5P), an incomplete reductive TCA cycle (S-malate to citrate), and a complete acetyl-CoA pathway for potential carbon fixation and acetyl-CoA generation 62 . While Hybrid_5 possesses a full pyruvate dehydrogenase complex [EC 1.2.4.1; EC 2.3.1.12; EC 1.8.1.4] for pyruvate oxidation under aerobic conditions, its genome also has pyruvate:ferredoxin oxidoreductase [EC 1.7.2.1] for anaerobic oxidation of pyruvate 63 . Figure 5 Model of Hybrid_5 cellular systems based on genomic data.…”
Genome reconstruction from metagenomes enables detailed study of individual community members, their metabolisms, and their survival strategies. Obtaining high quality metagenome-assembled genomes (MAGs) is particularly valuable in extreme environments like sea ice cryoconites, where the native consortia are recalcitrant to culture and strong astrobiology analogues. We evaluated three separate approaches for MAG generation from Allen Bay, Nunavut sea ice cryoconites—HiSeq-only, MinION-only, and hybrid (HiSeq + MinION)—where field MinION sequencing yielded a reliable metagenome. The hybrid assembly produced longer contigs, more coding sequences, and more total MAGs, revealing a microbial community dominated by Bacteroidetes. The hybrid MAGs also had the highest completeness, lowest contamination, and highest N50. A putatively novel species of Octadecabacter is among the hybrid MAGs produced, containing the genus’s only known instances of genomic potential for nitrate reduction, denitrification, sulfate reduction, and fermentation. This study shows that the inclusion of MinION reads in traditional short read datasets leads to higher quality metagenomes and MAGs for more accurate descriptions of novel microorganisms in this extreme, transient habitat and has produced the first hybrid MAGs from an extreme environment.
“…As Hp lacks several essential genes of the glycolysis pathway, pyruvate formation through carbon dioxide fixation may be physiologically favored, because it is the single gluconeogenic pathway in this bacterium [46]. On the other hand, oxidative decarboxylation of pyruvate is a fundamental reaction catalyzed by the pyruvate dehydrogenase complex in most aerobic organisms or by POR in anaerobic ones [133,134]. Thus, Hp POR catalyzes the last step of carbohydrates fermentation as well as the inverse pyruvate oxidative decarboxylation [135].…”
Section: An Overview Of Flavodoxins and Of The Flavodoxin From Hpmentioning
Flavodoxins are small soluble electron transfer proteins widely present in bacteria and absent in vertebrates. Flavodoxins participate in different metabolic pathways and, in some bacteria, they have been shown to be essential proteins representing promising therapeutic targets to fight bacterial infections. Using purified flavodoxin and chemical libraries, leads can be identified that block flavodoxin function and act as bactericidal molecules, as it has been demonstrated for Helicobacter pylori (Hp), the most prevalent human gastric pathogen. Increasing antimicrobial resistance by this bacterium has led current therapies to lose effectiveness, so alternative treatments are urgently required. Here, we summarize, with a focus on flavodoxin, opportunities for pharmacological intervention offered by the potential protein targets described for this bacterium and provide information on other gastrointestinal pathogens and also on bacteria from the gut microbiota that contain flavodoxin. The process of discovery and development of novel antimicrobials specific for Hp flavodoxin that is being carried out in our group is explained, as it can be extrapolated to the discovery of inhibitors specific for other gastric pathogens. The high specificity for Hp of the antimicrobials developed may be of help to reduce damage to the gut microbiota and to slow down the development of resistant Hp mutants.
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