A5103179119- Antimicrobial Resistance in Staphylococcus
- RNA and protein synthesis mechanisms
- Photosynthetic Processes and Mechanisms
- Bacterial Genetics and Biotechnology
- Neurological diseases and metabolism
- Microbial Metabolism and Applications
- Melanoma and MAPK Pathways
- RNA regulation and disease
- Enzyme Structure and Function
- Metabolism and Genetic Disorders
- Mitochondrial Function and Pathology
- Advanced Fluorescence Microscopy Techniques
- Bacteriophages and microbial interactions
- Genomics and Phylogenetic Studies
- Light effects on plants
- Diphtheria, Corynebacterium, and Tetanus
- Probiotics and Fermented Foods
- Biotin and Related Studies
- Cancer Research and Treatments
- Bayesian Modeling and Causal Inference
- Syphilis Diagnosis and Treatment
- Salmonella and Campylobacter epidemiology
- Islanding Detection in Power Systems
- Antimicrobial agents and applications
- bioluminescence and chemiluminescence research
St. Jude Children's Research Hospital
2011-2024
IBM Research - Thomas J. Watson Research Center
2023
University of Tennessee at Knoxville
1988-2016
Wayne State University
2013
Vanderbilt University
2004
Post Graduate Institute of Medical Education and Research
2001
Institute of Plant Physiology and Genetics
2001
Central Leather Research Institute
1997
University of Wisconsin–Madison
1968
The rationale for the pursuit of bacterial type 2 fatty acid synthesis (FASII) as a target antibacterial drug discovery in Gram-positive organisms is being debated vigorously based on their ability to incorporate extracellular acids. regulation FASII by acids was examined Staphylococcus aureus and Streptococcus pneumoniae , representing two important groups pathogens. Both bacteria use same enzymatic tool kit conversion acyl-acyl carrier protein, elongation, incorporation into phospholipids....
Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP<sup>+</sup>-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and NADPH/CO<sub>2</sub>-dependent reductive carboxylation αKG ICT. Reductive by IDH1 was potently inhibited NADP<sup>+</sup> and, lesser extent, IDH2 with cancer-associated mutations at active site arginines were unable carry out αKG. These mutants also defective in ICT converted 2-hydroxyglutarate using NADPH. mutant...
Significance The enzymes required for the incorporation of host fatty acids into membrane phospholipids Gram-positive bacterial pathogens are unknown. Fatty acid kinase (Fak) is a new enzyme in lipid metabolism that requires two proteins to form acyl-phosphate: an ATP binding-domain protein (FakA) interacts with binding (FakB). analysis Staphylococcus aureus mutants reveals Fak essential phospholipid synthesis from extracellular and also impacts transcription numerous virulence factors. This...
FRET is a well established method for cellular and subcellular imaging of protein interactions. However, obligatorily necessitates fluorescence excitation with its concomitant problems photobleaching, autofluorescence, phototoxicity, undesirable stimulation photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past....
Bioluminescence resonance energy transfer (BRET) between Renilla luciferase and yellow fluorescent protein has been adapted to serve as a real-time reporter on protein-protein interactions in live plant cells by using the Arabidopsis Constitutive photomorphogenesis 1 (COP1) model system. COP1 is repressor of light signal transduction that functions part nuclear E3 ubiquitin ligase. possesses leucine-rich nuclear-exclusion resides domain implicated dimerization. BRET was applied conjunction...
Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved transit peptide recognition. The cytosolic region reveals 34% alpha-helical 26% beta-strand structure stabilized by intramolecular electrostatic interaction. binds both chloroplast preproteins isolated peptides guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that soluble, domain (Toc34deltaTM) functions as receptor vitro capable to compete with import...
Summary Bioluminescence resonance energy transfer (BRET) is a natural biophysical phenomenon that underlies an emerging technique to monitor protein–protein interactions in living cells real time. Here, we present series of technical advances enhance the utility BRET assay plants. A recombination cloning vectors was generated accelerate expression proteins tagged with Renilla luciferase or yellow fluorescent protein under transient conditions and stable transgenic Working stably transformed...
Abstract Pantothenate kinase generates 4′‐phosphopantothenate in the first and rate‐determining step of coenzyme A (CoA) biosynthesis. The human genome encodes three well‐characterized nearly identical pantothenate kinases (PANK1‐3) plus a putative bifunctional protein (PANK4) with predicted amino‐terminal domain fused to carboxy‐terminal phosphatase domain. Structural phylogenetic analyses show that all active, characterized PANKs contain key catalytic residues Glu138 Arg207 (HsPANK3...
Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) β-ketoacyl-ACP synthase III enzyme, FabH. E. ΔfabH knockout strains are viable because yiiD gene that allows FabH-independent initiation. However, molecular function product not known. Here, we show malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and...
Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC concentrations nonesterified coenzyme A (CoASH), thus compromising function and disrupting intermediary metabolism. Here, we use hypomorphic PA mouse model to test effectiveness BBP-671 correcting metabolic imbalances PA. high-affinity allosteric pantothenate kinase activator counteracts...
The fabA and fabB genes are responsible for anaerobic unsaturated fatty acid formation in Pseudomonas aeruginosa. Expression of the fabAB operon was repressed by exogenous acids, DNA sequences upstream translational start site were used to affinity purify DesT. single protein interaction with promoter detected wild-type cell extracts absent desT deletion strain, as repression expression acids. Thus, DesT senses overall composition acyl-coenzyme A pool coordinate operons (fabAB) aerobic...
Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but structure of protein conformation responsible exchange at membrane unknown. Staphylococcus aureus FakB1 a prototypical bacterial that binds palmitate within narrow, buried tunnel. Here, we define conformational change "closed" state an "open" associates with provides path entry...
Fatty acid kinase (Fak) is a two-component enzyme that generates acyl-phosphate for phospholipid synthesis. Fak consists of domain protein (FakA) phosphorylates fatty enveloped by binding (FakB). The structural basis FakB function has been established, but little known about FakA. Here, we used limited proteolysis to define three separate FakA domains: the amino terminal FakA_N, central FakA_L, and carboxy FakA_C. isolated domains lack activity, activity restored when FakA_N FakA_L are...
AbstractThe emerging technique of bioluminescence resonance energy transfer (BRET) allows us to detect protein interactions in live cells and real time, thus providing a new window into cellular signal transduction processes. We present experimental protocols for expressing fusion proteins between luciferase fluorescent that are the basis BRET measurement, as well measuring imaging variety cell types. Despite our focus on plant cells, techniques described here easily adaptable other systems...
Pantothenate kinase associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism and spasticity, leading to premature death. PKAN caused mutations in the <i>PANK2 </i>gene that result loss or reduction PANK2 protein function. one three kinases initiate regulate coenzyme A biosynthesis from vitamin B5 ability BBP-671, an allosteric activator pantothenate kinases, enter brain elevate was investigated. The metabolic stability, binding...
ABSTRACT Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σ M responds to diverse antibiotics cell wall synthesis. Here, we demonstrate wall-inhibiting drugs, such as bacitracin cefuroxime, induce -dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed generate putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), YtpB branchpoint enzyme for membrane-localized C 35 terpenoids....
Cells of Salmonella typhimurium grown in nutrient broth at 37 C were inoculated into samples sterile skimmilk (approximately 103 cells per ml) which then acidified with hydrochloric, lactic, or citric acids and incubated 22 C. Acids added 2-hr intervals uniform increments sufficient to reduce the pH from 6.70 4.0 (hydrochloric acid), 4.25 (lactic 4.48 (citric acid). Samples taken 4-hr plated, plates for 24 hr. At C, maximum numbers (109 attained after 12 hr, whereas (&gt;108 milks...
Abstract Background Pantothenate kinase (PANK) is the first and rate-controlling enzymatic step in only pathway for cellular coenzyme A (CoA) biosynthesis. PANK-associated neurodegeneration (PKAN), formerly known as Hallervorden–Spatz disease, a rare, life-threatening neurologic disorder that affects CNS arises from mutations human PANK2 gene. Pantazines, class of small molecules containing pantazine moiety, yield promising therapeutic effects an animal model brain CoA deficiency. reliable...