46 research outputs found
From L-Dopa to Dihydroxyphenylacetaldehyde: A Toxic Biochemical Pathway Plays a Vital Physiological Function in Insects
One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects
Reclaiming the child left behind: the case for corporate cultural responsibility
Although a reasonable understanding of corporate social responsibility (CSR) exists, one dimension remains largely ignored. That is, the cultural impacts of corporations, or the bearing, at various levels of their business models, activities, and outcomes on the value systems and enduring beliefs of affected people. We introduce the notion of corporate cultural responsibility (CCR). The way corporations address CCR concerns can be reflected according to three stances: cultural destructiveness, cultural carelessness, and cultural prowess. Taken sequentially, they reflect a growing comprehension and increasingly active consideration of CCR concerns by corporations. In turn, we explicitly address issues related to the complex question of determining the cultural responsibilities of corporate actors; specify key CCR-related conceptualizations; and lay a foundation for discussions, debates, and research efforts centered on CCR concerns and rationales
Identification and Characterization of a Tyramine–Glutamate Ligase (MfnD) Involved in Methanofuran Biosynthesis
Methanofuran
is the first in a series of coenzymes involved in
the reduction of carbon dioxide to methane. All methanofuran structural
variants contain a basic core structure of 4-[<i>N</i>-(γ-l-glutamyl-γ-l-glutamyl)-<i>p</i>-(β-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan
(APMF-(Glu)<sub>2</sub>) with different attached side chains depending
on the source organism. Recently, we discovered the biosynthetic route
for the production of 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P),
a precursor to the furan moiety of methanofuran. However, how the
γ-linked glutamates are incorporated into methanofuran’s
structure remains unknown. Here, we report the identification of an
ATP-grasp enzyme encoded by the gene Mefer_1180 in Methanocaldococcus fervens (the homologue of MJ0815
in Methanocaldococcus jannaschii, annotated
as MfnD) that catalyzes the ATP-dependent addition of one glutamate
to tyramine via a γ-linked amide bond. The occurrence of this
reaction is consistent with the presence of γ-glutamyltyramine
in cell extracts of M. jannaschii.
Our steady-state kinetic analysis of the recombinant enzyme showed
that MfnD exhibits a catalytic ability comparable to other ATP-grasp
enzymes such as the Escherichia coli glutathione synthetase (GS), with a similar apparent <i>k</i><sub>cat</sub> and <i>K</i><sub>M</sub>. In addition, its
activity is divalent metal-dependent, with the highest activity observed
with Mn<sup>2+</sup>. The previously solved crystal structure of MfnD
from Archaeoglobus fulgidus exhibits
a classical ATP-grasp fold with three structural domains; the ATP-binding
and metal-binding motifs are conserved in MfnD as seen in other ATP-grasp
enzymes. We used site-directed mutagenesis and kinetic analysis to
demonstrate that Arg251 is an important residue for both catalysis
and glutamate binding. By comparing the active site of MfnD with GS
and by molecular docking substrates to the MfnD active site, we predicted
the possible glutamate- and tyramine-binding pocket. This is the first
report describing the enzymology of the incorporation of the initial l-glutamate molecule into the methanofuran structure. It also
provides the first example of an ATP-grasp enzyme activating the γ-carboxylate
of glutamate as substrate
Promiscuity of Methionine Salvage Pathway Enzymes in \u3ci\u3eMethanocaldococcus jannaschii\u3c/i\u3e
A weakness of the life sciences is our inability to predict enzymatic function of genes. Our work aims to establish gene function by discovering new biosynthetic pathways and enzyme functions within the pathway. Using the well-studied oxygen dependent methionine salvage pathway as a guide, we searched for new pathways in methanogens. Here we established the first three predicted methanogenic enzymes have the same function as in the aerobic pathway but with promiscuous activities, implying additional roles. However, how methanogens are able to circumvent the use of oxygen has yet to be discovered, but will likely lead to new biochemistry
Nonchromatographic âStir and Filter Approachâ (SAFA) for Isolating Sc\u3csub\u3e3\u3c/sub\u3eN@C\u3csub\u3e80\u3c/sub\u3e Metallofullerenes
Separation difficulties have led to a paucity of purified metallic nitride fullerenes (MNFs). Fundamental research and application development has been hampered with limited sample availability. Separation techniques designed to remove contaminant empty-cage fullerenes (e.g., C60, C70...C2n) and classical metallofullerenes (e.g., non-MNFs) traditionally require expensive and tedious chromatographic methods. Our motivation is an alternative purification approach to minimize dependence on HPLC. Herein we report the use of cyclopentadienyl (CPD) and amino functionalized silica to selectively bind contaminant fullerenes. This “Stir and Filter Approach” (SAFA) provides purified MNF samples at ambient and reflux conditions. Under reflux conditions, purified MNFs (80% recovery, 41 h) are obtained using CPD silica. However, at room temperature, there is an equilibrium established between fullerenes and CPD silica, and no purified MNF samples are obtained using SAFA. In contrast, purified MNF samples (99+%) are readily obtained at room temperature using amino, diamino, and triamino silica at recoveries of 93% (11 h), 76% (9 h), and 50% (6 h), respectively
Biosynthesis of the 5‑(Aminomethyl)-3-furanmethanol Moiety of Methanofuran
We have established the biosynthetic
pathway and the associated
genes for the biosynthesis of the 5-(aminomethyl)-3-furanmethanol
(F1) moiety of methanofuran in the methanogenic archaeon <i>Methanocaldococcus
jannaschii</i>. The recombinant enzyme, derived from the MJ1099
gene, was shown to readily condense glyceraldehyde 3-phosphate (Ga-3P)
and dihydroxyacetone-P (DHAP) to form 4-(hydroxymethyl)-2-furancarboxaldehyde
phosphate (4-HFC-P). The recombinant purified pyridoxal 5′-phosphate-dependent
aminotransferase, derived from the MJ0684 gene, was found to be specific
for catalyzing the transamination reaction between 4-HFC-P and [<sup>15</sup>N]alanine to produce [<sup>15</sup>N] 5-(aminomethyl)-3-furanmethanol-P
(F1-P) and pyruvate. To confirm these results in cell extracts, we
developed sensitive analytical methods for the liquid chromatography–ultraviolet–electrospray
ionization mass spectrometry analysis of F1 as a 7-nitrobenzofurazan
derivative. This method has allowed for the quantitation of trace
amounts of F1 and F1-P in cell extracts and the measurement of the
incorporation of stable isotopically labeled precursors into F1. After
incubation of cell extracts with [1,2,3-<sup>13</sup>C<sub>3</sub>]pyruvate and DHAP, 4-([<sup>2</sup>H<sub>2</sub>]hydroxymethyl)-2-furancarboxylic
acid phosphate (4-HFCA-P) or 4-([<sup>2</sup>H<sub>2</sub>]hydroxymethyl)-2-furancarboxaldehyde
phosphate (4-HFC-P) was found to be incorporated into F1-P. 4-HFCA-P
and 4-HFC-P were confirmed in cell extracts after removal of the phosphate.
The low level of incorporation of [1,2,3-<sup>13</sup>C<sub>3</sub>]pyruvate into F1-P in these experiments is explained by the fact
that the labeled pyruvate must first be converted into Ga-3-P through
gluconeogenesis before being incorporated into 4-HFC-P. Cell extracts
incubated with 4-HFC-P and a mixture of [<sup>15</sup>N]aspartate,
[<sup>15</sup>N]glutamate, and [<sup>15</sup>N]alanine produced [<sup>15</sup>N]F1-P. We also demonstrated that aqueous solutions of methylglyoxal
or pyruvate heated with dihydroxyacetone led to the formation of 4-HFC
and 4-HFCA, suggesting a possible prebiotic route to this moiety of
methanofuran
Overexpression of AtLOV1 in Switchgrass alters plant architecture, lignin content, and flowering time.
BACKGROUND: Switchgrass (Panicum virgatum L.) is a prime candidate crop for biofuel feedstock production in the United States. As it is a self-incompatible polyploid perennial species, breeding elite and stable switchgrass cultivars with traditional breeding methods is very challenging. Translational genomics may contribute significantly to the genetic improvement of switchgrass, especially for the incorporation of elite traits that are absent in natural switchgrass populations. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we constitutively expressed an Arabidopsis NAC transcriptional factor gene, LONG VEGETATIVE PHASE ONE (AtLOV1), in switchgrass. Overexpression of AtLOV1 in switchgrass caused the plants to have a smaller leaf angle by changing the morphology and organization of epidermal cells in the leaf collar region. Also, overexpression of AtLOV1 altered the lignin content and the monolignol composition of cell walls, and caused delayed flowering time. Global gene-expression analysis of the transgenic plants revealed an array of responding genes with predicted functions in plant development, cell wall biosynthesis, and flowering. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first report of a single ectopically expressed transcription factor altering the leaf angle, cell wall composition, and flowering time of switchgrass, therefore demonstrating the potential advantage of translational genomics for the genetic improvement of this crop
Inhibition of the Flavin-Dependent Monooxygenase Siderophore A (SidA) Blocks Siderophore Biosynthesis and <i>Aspergillus fumigatus</i> Growth
<i>Aspergillus
fumigatus</i> is an opportunistic fungal
pathogen and the most common causative agent of fatal invasive mycoses.
The flavin-dependent monooxygenase siderophore A (SidA) catalyzes
the oxygen and NADPH dependent hydroxylation of l-ornithine
(l-Orn) to <i>N</i><sup>5</sup>-l-hydroxyornithine
in the biosynthetic pathway of hydroxamate-containing siderophores
in <i>A. fumigatus</i>. Deletion of the gene that codes
for SidA has shown that it is essential in establishing infection
in mice models. Here, a fluorescence polarization high-throughput
assay was used to screen a 2320 compound library for inhibitors of
SidA. Celastrol, a natural quinone methide, was identified as a noncompetitive
inhibitor of SidA with a MIC value of 2 μM. Docking experiments
suggest that celastrol binds across the NADPH and l-Orn pocket.
Celastrol prevents <i>A. fumigatus</i> growth in blood agar.
The addition of purified ferric-siderophore abolished the inhibitory
effect of celastrol. Thus, celastrol inhibits <i>A. fumigatus</i> growth by blocking siderophore biosynthesis through SidA inhibiton