Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Activities-Ribazole-Phosphate Phosphatase α Adenosylcobinamide Amidohydrolase and Bifunctional Enzyme with AV19 Encodes a Methanopyrus kandleri Gene of the Archaeon cbiS The
The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and... more
The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and ATP. Apart from EutT-type ACATs, two evolutionary unrelated types of ACATs have been identified, termed PduO and CobA. Although the three types of ACATs are nonhomologous, they all generate a four-coordinate cob(II)alamin (4C Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate capable of attacking the 5'-carbon of cosubstrate ATP. Previous spectroscopic studies of the EutT ACAT from Salmonella enterica ( SeEutT) revealed that this enzyme requires a divalent metal cofactor for the conversion of 5C Co(II)Cbl to a 4C species. Interestingly, LmEutT does not require a divalent metal cofactor for catalytic activity, which exemplifies an interesting phylogenetic divergence among the EutT enzymes. To explore if thi...
Research Interests:
ATP:Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of... more
ATP:Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of structurally and evolutionarily distinct proteins (i.e., CobA, PduO, and EutT). The structure of EutT is unknown, and an understanding of its mechanism is incomplete. The Salmonella enterica EutT ( SeEutT) enzyme is the best-characterized member of its class and is known to be a ferroprotein. Here, we report the identification and initial biochemical characterization of an enzyme representative of a new class of EutTs that does not require a metal ion for activity. In vivo and in vitro evidence shows that the metal-free EutT homologue from Listeria monocytogenes ( LmEutT) has ACAT activity and that, unlike other ACATs, the biologically active form of LmEutT is a tetramer. In vitro studies revealed that LmEutT was more efficient than SeEutT and disp...
Research Interests:
Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum... more
Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for s treptothricin a ce t yltransferase A , formerly YyaR) enzyme of Bacillus subtilis as the mechanism used by this soil bacterium to detoxify streptothricin. B. subtilis strains lacking satA were susceptible to streptothricin. Ectopic expression of satA + restored streptothricin resistance to B. subtilis satA strains. Purified Bs SatA acetylated streptothricin in vitro at the expense of acetyl-CoA. A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity ( K d = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin....
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Biochemistry and Molecular Genetics of Cobalamin Biosynthesis1 Michelle R. Rondon, Jodi R. Trzebiatowski and Jorge C. Escalante-Semerena2 Department of Bacteriology University of WisconsinMadison Madison, Wisconsin 53706-1567 I.... more
Biochemistry and Molecular Genetics of Cobalamin Biosynthesis1 Michelle R. Rondon, Jodi R. Trzebiatowski and Jorge C. Escalante-Semerena2 Department of Bacteriology University of WisconsinMadison Madison, Wisconsin 53706-1567 I. Nomenclature of Corrinoids 349 ...
Research Interests:
Research Interests:
Research Interests:
<p>A. The <i>C</i>-terminal domain of <i>Sl</i>Acs<sup>WT</sup> or <i>Se</i>Acs<sup>WT</sup> was incubated with [1-<sup>14</sup>C]-acetyl-CoA in the presence or... more
<p>A. The <i>C</i>-terminal domain of <i>Sl</i>Acs<sup>WT</sup> or <i>Se</i>Acs<sup>WT</sup> was incubated with [1-<sup>14</sup>C]-acetyl-CoA in the presence or absence of <i>Sl</i>PatA<sup>WT</sup>. Proteins were separated by SDS-PAGE and stained with Coomassie Blue R to visualize proteins. Acetylation was visualized by phosphor imaging. B. Alignment of the <i>C</i>-terminal domain of <i>Sl</i>Acs and <i>Se</i>Acs. “ * ” denotes conserved residues; “.” denotes similar residues; light gray boxes denote conserved loops of the AMP-forming CoA ligase family <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099817#pone.0099817-Gulick2" target="_blank">[39]</a>; dark gray box denotes catalytic lysine.</p
Research Interests:
<p>A. Acetyl-CoA synthetase activity of each chimera and <i>Sl</i>Acs<sup>WT</sup> relative to <i>Se</i>Acs<sup>WT</sup> (gray bars). Amount of acetylation in <a... more
<p>A. Acetyl-CoA synthetase activity of each chimera and <i>Sl</i>Acs<sup>WT</sup> relative to <i>Se</i>Acs<sup>WT</sup> (gray bars). Amount of acetylation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099817#pone-0099817-g003" target="_blank">figure 3C and 3F</a> was quantified and normalized to the total acetylation of <i>Se</i>Acs (black bars). <i>Sl</i>Acs-<i>Se</i>Acs chimera C3, the most efficiently acetylated, active chimera with the fewest <i>Se</i>Acs<sup>WT</sup>-derived residues, is noted with a star. Values are reported as the mean ± S.D. of three experiments. B. Sequence alignment of <i>Sl</i>Acs<sup>WT</sup>, <i>Se</i>Acs<sup>WT</sup>, chimera C3, <i>Rhodopseudomonas palustris</i> CGA009 Acs (<i>Rp</i>Acs), and <i>Mycobacterium smegmatis</i> mc<sup>2</sup>155 Acs (<i>Ms</i>Acs). Residues in chimera C3 that are derived from the <i>Se</i>Acs<sup>WT</sup> amino acid sequence are highlighted in black. <i>Sl</i>Acs residues conserved in the <i>Ms</i>Acs homologue are shown in bold typeface in the sequence of the latter. Black box indicates the target lysine.</p
Research Interests:
Table S1. Bacteria from which panD genes were amplified and cloned.
Research Interests:
Dataset S1. PanD list.
Research Interests:
SIR2 proteins have NAD -dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A... more
SIR2 proteins have NAD -dependent histone deacetylase activity, but no metabolic role has been assigned to any of these proteins. In Salmonella enterica, SIR2 function was required for activity of the acetyl-CoA synthetase (Acs) enzyme. A greater than two orders of magnitude increase in the specific activity of Acs enzyme synthesized by a sirtuin-deficient strain was measured after treatment with homogeneous S. enterica SIR2 protein. Human SIR2A and yeast SIR2 proteins restored growth of SIR2-deficient S. enterica on acetate and propionate, suggesting that eukaryotic cells may also use SIR2 proteins to control the synthesis of acetyl-CoA by the level of acetylation of acetyl-CoA synthetases. Consistent with this idea, growth of a quintuple sir2 hst1 hst2 hst3 hst4 mutant strain of the yeast Saccharomyces cerevisiae on acetate or propionate was severely impaired. The data suggest that the Hst3 and Hst4 proteins are the most important for allowing growth on these short-chain fatty aci...
We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism in Salmonella typhimurium . Results from 13 C-labeling experiments have identified the route of propionate breakdown and... more
We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism in Salmonella typhimurium . Results from 13 C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the α-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro. 1 H nuclear magnetic resonance spectroscopy and negative-io...
Research Interests:
The degradation of lignin-derived aromatic compounds such as benzoate has been extensively studied in , and the chemistry underpinning the conversion of benzoate to acetyl coenzyme A (acetyl-CoA) is well understood. Here we characterize... more
The degradation of lignin-derived aromatic compounds such as benzoate has been extensively studied in , and the chemistry underpinning the conversion of benzoate to acetyl coenzyme A (acetyl-CoA) is well understood. Here we characterize the last unknown gene of the (benzoic acid degradation) cluster. BadL function is required for growth under photoheterotrophic conditions with benzoate as the organic carbon source (i.e., light plus anoxia). On the basis of bioinformatics and and data, we show that BadL, a cn5-related cetylransferase (GNAT) (PF00583), acetylates aminobenzoates to yield acetamidobenzoates. The latter relieved repression of the operon by binding to BadM, triggering the synthesis of enzymes that activate and dearomatize the benzene ring. We also show that acetamidobenzoates are required for the expression of genes encoding the photosynthetic reaction center light-harvesting complexes through a BadM-independent mechanism. The effect of acetamidobenzoates on pigment synth...
Research Interests:
Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a... more
Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO....