Noncovalent Inhibition and Covalent Inactivation of Proline Dehydrogenase by Analogs of -Propargylglycine.

The flavoenzyme proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the oxidation of l-proline to Δ-pyrroline-5-carboxylate. The enzyme is a target for chemical probe discovery because of its role in the metabolism of certain cancer cells. -propargylglycine is the first and best characterized mechanism-based covalent inactivator of PRODH.

ICP-MS Analysis of Iron from Biological Samples.

Elemental analysis can provide trace concentrations of iron and other transition elements at nanomolar (μg/L) concentrations in whole bacterial and mammalian cells, subcellular compartments, biological fluids, and tissues. The best method of analysis is by far Inductively Coupled Plasma Mass Spectrometry (ICP-MS). I describe here a very general method for the sample preparation, instrument settings, method development, and analysis.

Cobalt-Sulfur Coordination Chemistry Drives B Loading onto Methionine Synthase.

Cobalt-sulfur (Co-S) coordination is labile to both oxidation and reduction chemistry and is rarely seen in nature. Cobalamin (or vitamin B) is an essential cobalt-containing organometallic cofactor in mammals and is escorted via an intricate network of chaperones to a single cytoplasmic target, methionine synthase. In this study, we report that the human cobalamin trafficking protein, MMADHC, exploits the chemical lability of Co-S coordination for cofactor off-loading onto methionine synthase.

Cobalt-sulfur coordination chemistry drives B loading onto methionine synthase.

Cobalt-sulfur (Co-S) coordination is labile to both oxidation and reduction chemistry and is rarely seen in Nature. Cobalamin (or vitamin B ) is an essential cobalt-containing organometallic cofactor in mammals, and is escorted via an intricate network of chaperones to a single cytoplasmic target, methionine synthase. In this study, we report that the human cobalamin trafficking protein, MMADHC, exploits the chemical lability of Co-S coordination, for cofactor off-loading onto methionine synthase.

Putrescine Detected in Strains of .

Most forms of life, including the archaea, bacteria, and eukaryotes synthesize the polyamine putrescine. Although putrescine is widely distributed, several Gram-positive bacteria, including (), appear to be the exceptions. We report here that strains of can produce the polyamine putrescine, as well as the derivative -acetyl-putrescine.

Functional Impact of a Cancer-Related Variant in Human Δ-Pyrroline-5-Carboxylate Reductase 1.

Pyrroline-5-carboxylate reductase (PYCR) is a proline biosynthetic enzyme that catalyzes the NAD(P)H-dependent reduction of Δ-pyrroline-5-carboxylate (P5C) to proline. Humans have three PYCR isoforms, with PYCR1 often upregulated in different types of cancers. Here, we studied the biochemical and structural properties of the Thr171Met variant of PYCR1, which is found in patients with malignant melanoma and lung adenocarcinoma.

Coordination of metal center biogenesis in human cytochrome c oxidase.

Mitochondrial cytochrome c oxidase (CcO) or respiratory chain complex IV is a heme aa-copper oxygen reductase containing metal centers essential for holo-complex biogenesis and enzymatic function that are assembled by subunit-specific metallochaperones. The enzyme has two copper sites located in the catalytic core subunits. The COX1 subunit harbors the Cu site that tightly associates with heme a while the COX2 subunit contains the binuclear Cu site.

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted.

Evidence for Proline Catabolic Enzymes in the Metabolism of Thiazolidine Carboxylates.

Thiazolidine carboxylates such as thiazolidine-4-carboxylate (T4C) and thiazolidine-2-carboxylate (T2C) are naturally occurring sulfur analogues of proline. These compounds have been observed to have both beneficial and toxic effects in cells. Given that proline dehydrogenase has been proposed to be a key enzyme in the oxidative metabolism of thioprolines, we characterized T4C and T2C as substrates of proline catabolic enzymes using proline utilization A (PutA), which is a bifunctional enzyme with proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) activities.

Europium sulfide nanoprobes predict antiretroviral drug delivery into HIV-1 cell and tissue reservoirs.

Delivery of long-acting nanoformulated antiretroviral drugs (ARVs) to human immunodeficiency virus type one cell and tissue reservoirs underlies next generation antiretroviral therapeutics. Nanotheranostics, comprised of trackable nanoparticle adjuncts, can facilitate ARV delivery through real-time drug tracking made possible through bioimaging platforms. To model HIV-1 therapeutic delivery, europium sulfide (EuS) nanoprobes were developed, characterized and then deployed to cells, tissues, and rodents.

Disease variants of human Δ-pyrroline-5-carboxylate reductase 2 (PYCR2).

Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of l-proline biosynthesis by catalyzing the reduction of L-Δ-pyrroline-5-carboxylate (L-P5C) to l-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10.

Differential Defense Responses of Upland and Lowland Switchgrass Cultivars to a Cereal Aphid Pest.

Yellow sugarcane aphid (YSA) (, Forbes) is a damaging pest on many grasses. Switchgrass ( L.), a perennial C4 grass, has been selected as a bioenergy feedstock because of its perceived resilience to abiotic and biotic stresses.

Aphid-Responsive Defense Networks in Hybrid Switchgrass.

Aphid herbivory elicits plant defense-related networks that are influenced by host genetics. Plants of the upland switchgrass ( ) cultivar Summer can be a suitable host for greenbug aphids (; GB), and yellow sugarcane aphids (, YSA), whereas the lowland cultivar Kanlow exhibited multi-species resistance that curtails aphid reproduction. However, stabilized hybrids of Summer (♀) x Kanlow (♂) (SxK) with improved agronomics can be damaged by both aphids.

Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD-Dependent Enzymes.

Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD and the reducing agents TCEP and THPP.

Systematic age-, organ-, and diet-associated ionome remodeling and the development of ionomic aging clocks.

Aging involves coordinated yet distinct changes in organs and systems throughout life, including changes in essential trace elements. However, how aging affects tissue element composition (ionome) and how these changes lead to dysfunction and disease remain unclear. Here, we quantified changes in the ionome across eight organs and 16 age groups of mice.

Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations.

3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (HS), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms.

Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis.

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis.

Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4).

Overexpression of the Sorghum bicolor SbCCoAOMT alters cell wall associated hydroxycinnamoyl groups.

Sorghum (Sorghum bicolor) is a drought tolerant crop, which is being developed as a bioenergy feedstock. The monolignol biosynthesis pathway is a major focus for altering the abundance and composition of lignin. Caffeoyl coenzyme-A O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase that methylates caffeoyl-CoA to generate feruloyl-CoA, an intermediate required for the biosynthesis of both G- and S-lignin.

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine.

Hydrogen sulfide (HS) is a signaling molecule with many beneficial effects. However, its cellular concentration is strictly regulated to avoid toxicity. Persulfide dioxygenase (PDO or ETHE1) is a mononuclear non-heme iron-containing protein in the sulfide oxidation pathway catalyzing the conversion of GSH persulfide (GSSH) to sulfite and GSH.

Overexpression of SbMyb60 in Sorghum bicolor impacts both primary and secondary metabolism.

Few transcription factors have been identified in C grasses that either positively or negatively regulate monolignol biosynthesis. Previously, the overexpression of SbMyb60 in sorghum (Sorghum bicolor) has been shown to induce monolignol biosynthesis, which leads to elevated lignin deposition and altered cell wall composition. To determine how SbMyb60 overexpression impacts other metabolic pathways, RNA-Seq and metabolite profiling were performed on stalks and leaves.

Pseudomonas Quinolone Signal Induces Oxidative Stress and Inhibits Heme Oxygenase-1 Expression in Lung Epithelial Cells.

causes lung infections in patients with cystic fibrosis (CF). The quinolone signal (PQS) compound is a secreted virulence factor that contributes to the pathogenicity of We were able to detect PQS in sputum samples from CF patients infected with but not in samples from uninfected patients. We then tested the hypothesis that PQS induces oxidative stress in host cells by determining the ability of PQS to induce the production of reactive oxygen species (ROS) in lung epithelial cells (A549 and primary normal human bronchial epithelial [NHBE]) cells and macrophages (J774A.

Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast.

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized.

Structural and Mechanistic Insights into Hemoglobin-catalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products.

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize HS but, lacking organelles, cannot dispose of HS via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes HS to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates.

Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

Prostate epithelial cells control the potency and availability of androgen hormones in part by inactivation and elimination. UDP-glucose dehydrogenase (UGDH) catalyzes the NAD(+)-dependent oxidation of UDP-glucose to UDP-glucuronate, an essential precursor for androgen inactivation by the prostate glucuronidation enzymes UGT2B15 and UGT2B17. UGDH expression is androgen stimulated, which increases the production of UDP-glucuronate and fuels UGT-catalyzed glucuronidation.