scholarly journals Characterization of an L-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

2019 ◽  
Author(s):  
Tyler Stack ◽  
Katelyn Morrison ◽  
Thomas Dettmer ◽  
Brendan Wille ◽  
Chan Kim ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Sequence Similarity ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Benzilic Acid ◽  
Amidohydrolase Superfamily ◽  
Sequence Similarity Networks

<p>L-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. <i>Ralstonia eutropha </i>H16 (<i>Cupriavidus necator </i>ATCC 17699) uses L-ascorbate as sole carbon source but lacks the genes encoding the known catabolic pathways. RNAseq identified eight candidate catabolic genes. Sequence similarity networks and genome neighborhood networks guided predictions for function of the encoded proteins; the predictions were confirmed by <i>in vitro</i> assays and <i>in vivo</i> growth phenotypes of gene deletion mutants. L-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b<sub>561</sub> and gluconolactonase families, respectively, to form 2,3-diketo-L-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-L-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-L-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD<sup>+</sup> catalytically. The product, L-lyxonate, is catabolized to alpha-ketoglutarate by a previously characterized pathway.</p>

scholarly journals Characterization of an L-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations

2019 ◽  
Author(s):  
Tyler Stack ◽  
Katelyn Morrison ◽  
Thomas Dettmer ◽  
Brendan Wille ◽  
Chan Kim ◽  
...  
Keyword(s):  
Ralstonia Eutropha ◽  
Sequence Similarity ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Benzilic Acid ◽  
Amidohydrolase Superfamily ◽  
Sequence Similarity Networks

<p>L-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. <i>Ralstonia eutropha </i>H16 (<i>Cupriavidus necator </i>ATCC 17699) uses L-ascorbate as sole carbon source but lacks the genes encoding the known catabolic pathways. RNAseq identified eight candidate catabolic genes. Sequence similarity networks and genome neighborhood networks guided predictions for function of the encoded proteins; the predictions were confirmed by <i>in vitro</i> assays and <i>in vivo</i> growth phenotypes of gene deletion mutants. L-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b<sub>561</sub> and gluconolactonase families, respectively, to form 2,3-diketo-L-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-L-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-L-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD<sup>+</sup> catalytically. The product, L-lyxonate, is catabolized to alpha-ketoglutarate by a previously characterized pathway.</p>

scholarly journals Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Suwen Zhao ◽  
Ayano Sakai ◽  
Xinshuai Zhang ◽  
Matthew W Vetting ◽  
Ritesh Kumar ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Large Scale ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Enzymatic Activities ◽  
Or Gene ◽  
Enzyme Functions

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ~85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.

scholarly journals Regulation of σB by an Anti- and an Anti-Anti-Sigma Factor in Streptomyces coelicolor in Response to Osmotic Stress

2004 ◽  
Vol 186 (24) ◽  
pp. 8490-8498 ◽  
Author(s):  
Eun-Jin Lee ◽  
You-Hee Cho ◽  
Hyo-Sub Kim ◽  
Bo-Eun Ahn ◽  
Jung-Hye Roe
Keyword(s):  
Osmotic Stress ◽  
Sigma Factor ◽  
Target Genes ◽  
Sequence Similarity ◽  
Streptomyces Coelicolor ◽  
Dependent Manner ◽  
Its Gene ◽  
Genes Encoding

ABSTRACT σB, a homolog of stress-responsive σB of Bacillus subtilis, controls both osmoprotection and differentiation in Streptomyces coelicolor A3 (2). Its gene is preceded by rsbA and rsbB genes encoding homologs of an anti-sigma factor, RsbW, and its antagonist, RsbV, of B. subtilis, respectively. Purified RsbA bound to σB and prevented σB-directed transcription from the sigBp1 promoter in vitro. An rsbA-null mutant exhibited contrasting behavior to the sigB mutant, with elevated sigBp1 transcription, no actinorhodin production, and precocious aerial mycelial formation, reflecting enhanced activity of σB in vivo. Despite sequence similarity to RsbV, RsbB lacks the conserved phosphorylatable serine residue and its gene disruption produced no distinct phenotype. RsbV (SCO7325) from a putative six-gene operon (rsbV-rsbR-rsbS-rsbT-rsbU1-rsbU) was strongly induced by osmotic stress in a σB-dependent manner. It antagonized the inhibitory action of RsbA on σB-directed transcription and was phosphorylated by RsbA in vitro. These results support the hypothesis that the rapid induction of σB target genes by osmotic stress results from modulation of σB activity by the kinase-anti-sigma factor RsbA and its phosphorylatable antagonist RsbV, which function by a partner-switching mechanism. Amplified induction could result from a rapid increase in the synthesis of both σB and its inhibitor antagonist.

scholarly journals N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

2002 ◽  
Vol 184 (18) ◽  
pp. 4952-4961 ◽  
Author(s):  
S. H. Willis ◽  
K. M. Kazmierczak ◽  
R. H. Carter ◽  
L. B. Rothman-Denes
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sequence Similarity ◽  
Rna Polymerases ◽  
Transcriptional Analysis ◽  
Amino Acid Residues ◽  
Genes Encoding ◽  
Single Subunit

ABSTRACT Bacteriophage N4 middle genes are transcribed by a phage-coded, heterodimeric, rifampin-resistant RNA polymerase, N4 RNA polymerase II (N4 RNAPII). Sequencing and transcriptional analysis revealed that the genes encoding the two subunits comprising N4 RNAPII are translated from a common transcript initiating at the N4 early promoter Pe3. These genes code for proteins of 269 and 404 amino acid residues with sequence similarity to the single-subunit, phage-like RNA polymerases. The genes encoding the N4 RNAPII subunits, as well as a synthetic construct encoding a fusion polypeptide, have been cloned and expressed. Both the individually expressed subunits and the fusion polypeptide reconstitute functional enzymes in vivo and in vitro.

scholarly journals Discovery and Characterization of Three New Escherichia coli Septal Ring Proteins That Contain a SPOR Domain: DamX, DedD, and RlpA

2009 ◽  
Vol 192 (1) ◽  
pp. 242-255 ◽  
Author(s):  
S. J. Ryan Arends ◽  
Kyle Williams ◽  
Renada J. Scott ◽  
Silvana Rolong ◽  
David L. Popham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Caulobacter Crescentus ◽  
Negative Regulator ◽  
Aquifex Aeolicus ◽  
E Coli ◽  
Division Site ◽  
Genes Encoding

ABSTRACT SPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus, localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.

scholarly journals l-Rhamnose Transport Is Sugar Kinase (RhaK) Dependent in Rhizobium leguminosarum bv. trifolii

2007 ◽  
Vol 189 (23) ◽  
pp. 8437-8446 ◽  
Author(s):  
Jason S. Richardson ◽  
Ivan J. Oresnik
Keyword(s):  
Abc Transporter ◽  
Rhizobium Leguminosarum ◽  
Nodule Occupancy ◽  
Vitro Assay ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Pentose Sugar ◽  
Sugar Kinase

ABSTRACT Strains of Rhizobium leguminosarum which are unable to catabolize l-rhamnose, a methyl-pentose sugar, are compromised in the ability to compete for nodule occupancy versus wild-type strains. Previous characterization of the 11-kb region necessary for the utilization of rhamnose identified a locus carrying catabolic genes and genes encoding the components of an ABC transporter. Genetic evidence suggested that the putative kinase RhaK carried out the first step in the catabolism of rhamnose. Characterization of this kinase led to the observation that strains carrying rhamnose kinase mutations were unable to transport rhamnose into the cell. The absence of a functional rhamnose kinase did not stop the transcription and translation of the ABC transporter components. By developing an in vitro assay for RhaK activity, we have been able to show that (i) RhaK activity is consistent with RhaK phosphorylating rhamnose and (ii) biochemical activity of RhaK is necessary for rhamnose transport.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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