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A tale of two enzymes : probing the aromatic hydrocarbon metabolism of Methylibium petroleiphilum, a degrader of petroleum groundwater pollutants, with the 4-oxalocrotonate tautomerase enzymesTerrell, Cassidy Renee 13 November 2013 (has links)
The cleanup of petroleum groundwater contamination is a major concern for the United States and as microbial bioremediation gains popularity the improvement of this method requires further research into how these microbes degrade water-soluble petroleum pollutants. Methylibium petroleiphilum (M. petroleiphilum) is one such bacterium of special interest, because it can metabolize a wide range of gasoline contaminants. The primary goal of this research is to investigate M. petroleiphilum's capacity for metabolizing different aromatic hydrocarbon compounds from petroleum pollution. The source of this unusual ability is hypothesized to stem from two seemingly redundant aromatic hydrocarbon-degrading operons, wherein the two operons would catabolize different petroleum contaminants and thus offer versatility to the bacteria. In this study, in vitro biochemical techniques probe two homologous proteins, specifically the 4-oxalocrotonate tautomerase (4OT) enzymes, within each operon. The 4OT family is well characterized and these proteins are especially good candidates for this research due to their ease of expression, purification, and robust catalytic properties. Furthermore, the 4OT family is known to be promiscuous with respect to substrate preference and catalysis. Also, the sequence identity between Tautomerase I and II is the lowest of all redundant aromatic hydrocarbon degrading enzymes, which suggests that these enzymes may show the most significant distinctions in substrate preference. As such, each enzyme has been recombinantly expressed and purified to enable detailed molecular-level characterization. The structure of each enzyme has been solved, using x-ray crystallography, and comparison of which provides insight into their potential functional differences. Kinetic analysis of two petroleum metabolites, specifically those of benzene and toluene, were measured for each enzyme. These results also suggest substrate preferences. The results of this research provide insight into the metabolism of petroleum groundwater pollutants, specifically the aromatic hydrocarbons, by Methylibium petroleiphilum. / text
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Random and rational evolution of tautomerase superfamily members : analysis and implicationsDarty, Joseph Edward 10 April 2014 (has links)
P[Kappa]a is not responsible for the improved activity. Hence, stabilization of an enediolate intermediate may be important for catalysis. In the second part of this work, the Chloroflexus aurantiacus J-10-fl heterohexameric 4-OT tautomerase was employed in random and rational directed evolution studies to introduce a CaaD activity. Genetic selection and a high throughput screening assay were used to identify mutants. Genetic selection was unsuccessful due to plasmid instability in the host strain. A small mutant library in the screening assay precluded the identification of any mutants with CaaD activity. Finally, rational design using structure-function relationships was investigated and a single mutant was discovered for hh4-OT that incorporated CaaD activity into the enzyme, the [alpha]L9R hh4-OT, this mutant has been characterized kinetically and the evolutionary implications for the tautomerase superfamily are described. / text
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A snapshot of the unity and diversity of biological systems at the level of chemistry : structural and mechanistic studies of Cg10062, a homologue of cis-3-chloroacrylic acid dehalogenase, FG41 malonate semialdehyde decarboxylase and the catalytic domain of pyruvate dehydrogenase phosphatase 1Guo, Youzhong, 1974- 15 September 2010 (has links)
The tautomerase superfamily is composed of a group of proteins characterized by two key features: the N-terminal proline and a beta-alpha-beta-motif. This superfamily has been divided into five families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF). Cg10062 is a homologue of cis-CaaD, but has several distinct biochemical properties from cis-CaaD. For example, Cg10062 can be irreversibly inhibited by (R)- or (S)-oxirane-2-carboxylate, whereas cis-CaaD can only be irreversibly inhibited by (R)-oxirane-2-carboxylate. FG41MSAD is a homologue of MSAD, with comparable decarboxylase activity but missing Arg-73 known to be crucial for the MSAD activity. In order to understand the unique biochemical characteristics of Cg10062 and FG41MSAD, we have solved five crystal structures. These crystal structures have established a solid structural basis for understanding the mechanisms of their activities.
The eukaryotic protein phosphatases are composed of a group of proteins that are responsible for reversible phosphorylation. The eukaryotic protein phosphatases have been divided into three families, the phosphoprotein phosphatase (PPP) family, the protein phosphatase Mg2+- or Mn2+-dependent (PPM) family and the protein Tyr phosphatase (PTP) family. PDP1 is a member of PPM family. PDP1 is also an important component of the large pyruvate dehydrogenase complex (PDC) which catalyzes the decarboxylation of pyruvate to yield acetyl-CoA with the accompanying reduction of NAD+. In order to understand the mechanism in which it dephosphorylates its target protein we have solved the structure of the catalytic domain of PDP1.
Analysis of these structures in the light of their evolutionary contexts enables us to appreciate the unity and diversity of the biological systems at the chemical level and help us solve interesting problems, such as the possible physiological functions for some members within the tautomerase superfamily. / text
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Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implicationsSerrano, Hector, doctor of pharmacy 05 September 2012 (has links)
Members of the tautomerase superfamily are characterized by a [beta-alpha-beta] structural fold motif as well as a catalytic N-terminal proline (Pro-1). Three members of the superfamily are involved in the degradation of the nematocide 1,3-dichloropopene; trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and malonate semialdehyde decarboxylase (MSAD). CaaD and cis-CaaD are involved in the hydration of their respective 3-chloroacrylic acid isomers to generate malonate semialdehyde. Subsequently, MSAD is responsible for catalyzing the decarboxylation of malonate semialdehyde to generate acetaldehyde. All three of these enzymes contain an N-terminal proline (Pro-1) that functions as a general acid, in contrast to other tautomerase superfamily members, such as 4-oxalocrotonate tautomerase (4-OT) and macrophage migration inhibitory factor (MIF), where Pro-1 acts as a catalytic base. Two new members of the tautomerase superfamily have been cloned and characterized; FG41 MSAD, a homologue of MSAD from Coryneform Bacterium strain FG41, and Cg10062, a homologue of cis-CaaD from Corynebacterium glutamicum, with low-level cis-CaaD and CaaD activities. As part of an effort to delineate the mechanisms of CaaD, cis-CaaD and Cg10062, secondary activities for all three enzymes were characterized. The three enzymes function as efficient phenylpyruvate tautomerases (PPT), converting phenylenolpyruvate to phenylpyruvate. The activity also indicates that the active site of these three enzymes can ketonize enol compounds, thereby providing evidence for the presence of an enediolate intermediate. The characterization of FG41 MSAD uncovered an activity it shares with MSAD. FG41 MSAD catalyzes the hydration of 2-oxo-3-pentynoate, but at a rate that is 50-fold less efficient than that of MSAD (as assessed by kcat/Km values). Mutagenesis studies of FG41 MSAD revealed that a single mutation resulted in a 8-fold increase in the activity. The characterization of Cg10062 and attempts to enhance the low-level cis-CaaD activity demonstrated the need for a bacterial screen that could screen a library of mutants. The resulting bacterial screen could be used to screen other members of the superfamily for dehalogenase activity. An in-depth exploration of the Cg10062 and FG41 MSAD activities may lead to a better understanding of the mechanism of cis-CaaD and MSAD and further delineate the evolutionary pathway for the tautomerase superfamily. / text
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Prostaglandin-E2 is produced by adult human epidermal melanocytes in response to UVB in a melanogenesis-independent manner.Gledhill, Karl, Rhodes, L.E., Brownrigg, M., Haylett, A.K., Masoodi, Mojgan, Thody, Anthony J., Nicolaou, Anna, Tobin, Desmond J. January 2010 (has links)
No / Erythema occurs in human skin following excessive exposure to ultraviolet radiation (UVR), and this is in part mediated by the vasodilator prostaglandin E2 (PGE2). While keratinocytes are a major source of this pro-inflammatory eicosanoid, epidermal melanocytes (EM) also express some of the cellular machinery required for PGE2 production. The primary aim of this study is to determine whether EM can produce PGE2 and so potentially also contribute to UVR-induced skin inflammation. Furthermore, we investigate the likely pathway by which this PGE2 production is achieved and investigate whether PGE2 production by EM is correlated with melanogenic capacity. Primary cultures of EM were established from nine normal healthy individuals with skin phototype-1 (n=4) and 4 (n=5), and PGE2 production and melanogenic status were assessed. EM produced PGE2 under baseline conditions and this was increased further upon stimulation with arachidonic acid. Moreover, EM expressed cytoplasmic phospholipase A2, cyclooxygenase-1 and cytoplasmic prostaglandin E synthase. However, no EM culture expressed cyclooxygenase-2 under baseline conditions or following arachidonic acid, UVB- or H2O2 treatments. PGE2 production in response to UVB was highly variable in EM cultures derived from different donors but when pooled for skin phototype exhibited a positive correlation only with SPT-1 derived EM. Interestingly, PGE2 production by EM in response to UVB showed no correlation with baseline levels of melanin, tyrosinase expression/activity or tyrosinase-related protein-1 expression. However, there was an apparent negative correlation with baseline expression of dopachrome tautomerase (DCT), a melanogenic enzyme with reported anti-oxidant potential. These findings suggest that EM have the potential to contribute to UVR-induced erythema via PGE2 production, but that this response may be more related to oxidative stress than to their melanogenesis status. / The Wellcome Trust
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The role of eicosanoids in the human skin's response to ultraviolet radiationGledhill, Karl January 2009 (has links)
Erythema is a hallmark skin response to excessive ultraviolet radiation (UVR) and is associated with cutaneous inflammation. Both are mediated by inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2) and chemoattractants such as 12-hydroxyeicosatetraenoic acid (12-HETE) leading to vasodilation and increased leukocyte infiltration. The erythematous response is more pronounced in individuals with low basal melanin levels or who fail to respond to UVR with a robust up-regulation of melanogenesis. While melanin production is a key function of melanocytes, these cells can also produce NO and PGE2, and are located in close proximity to the dermal vasculature. It has been hypothesized that melanocytes with poor melanogenic capacity may participate in the inflammatory response to UVR. The aim of this project was to investigate the inflammatory response in the skin of individuals with either skin phototype (SPT) 1 or 4 to UVR. Sixteen normal healthy individuals were selected for study (8 SPT-1 & 8 SPT-4). Buttock skin was investigated by immunohistochemistry for leukocyte subtypes, eicosanoid producing enzymes and NO synthases under basal and UVR-stimulated conditions. In addition primary cultures of epidermal melanocytes (EM) were established from 16 individuals (8 SPT-1 & 8 SPT-4) and assessed for the presence of eicosanoid-producing enzymes, melanogenic enzymes and NO synthases, by immunocytochemistry, Polymerase Chain Reaction and Western Blotting and for the production of the main pro-inflammatory eicosanoid PGE2 by ELISA and Mass Spectrometry. Moreover, the fatty acid composition of cultured melanocytes was assessed by Gas Chromatography. Results showed that individuals with SPT-1 had significantly greater neutrophil infiltration into the epidermis than those with SPT-4 at 24 hrs post-UVR. Moreover, CD3+ lymphocyte infiltration into the dermis was significantly greater in individuals with SPT-4 than those with SPT-1 at 24 and 72 hrs post-UVR. NOS-1, NOS-3, 12-LOX and COX-2 expression were significantly increased in SPT-1 skin, while NOS-2 and 15-LOX were significantly increased in SPT-4 skin. As 12-LOX and COX-2 products are chemoattractive (for neutrophils) and pro-inflammatory respectively these data could explain the greater observed neutrophil infiltration in SPT-1. The 15-LOX product (15-HETE) is anti-inflammatory and may suggest that 15-LOX up-regulation in SPT-4 skin may aid resolution of the sunburn response, which in part may be mediated by CD3+ lymphocytes and a class-switch in eicosanoid production from COX to LOX products. Melanocyte primary cultures surprisingly showed that SPT was not correlated with melanin content or melanogenic enzyme expression/activity suggesting that all melanocytes in vitro contained the necessary cellular machinery to produce melanin. This finding may reflect also their equal treatment under these enriched culture conditions, which may or may not be available to these cells in situ. Moreover, all melanocytes expressed the necessary machinery (PLA2, COX-1, cPGES) to produce PGE2. However, only some cultures did so at baseline and in response to UVR, and this was not correlated with SPT. A positive correlation was found however between expression level of dopachrome tautomerase (DCT) and protection against PGE2 production in response to UVR, which may suggest a novel role for DCT unrelated to melanogenesis. In summary this research project has generated data that highlights differences between the skin of individuals with SPT-1 and those with SPT-4, and may provide evidence that the keratinocyte partner contributes significantly to the SPT-associated response. This research may also suggest DCT as a novel therapeutic target to protect EM from participation in the UVR-associated inflammatory response in skin.
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The role of eicosanoids in the human skin's response to ultraviolet radiation.Gledhill, Karl January 2009 (has links)
Erythema is a hallmark skin response to excessive ultraviolet radiation (UVR) and is associated with cutaneous inflammation. Both are mediated by inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2) and chemoattractants such as 12-hydroxyeicosatetraenoic acid (12-HETE) leading to vasodilation and increased leukocyte infiltration. The erythematous response is more pronounced in individuals with low basal melanin levels or who fail to respond to UVR with a robust up-regulation of melanogenesis. While melanin production is a key function of melanocytes, these cells can also produce NO and PGE2, and are located in close proximity to the dermal vasculature. It has been hypothesized that melanocytes with poor melanogenic capacity may participate in the inflammatory response to UVR.
The aim of this project was to investigate the inflammatory response in the skin of individuals with either skin phototype (SPT) 1 or 4 to UVR. Sixteen normal healthy individuals were selected for study (8 SPT-1 & 8 SPT-4). Buttock skin was investigated by immunohistochemistry for leukocyte subtypes, eicosanoid producing enzymes and NO synthases under basal and UVR-stimulated conditions. In addition primary cultures of epidermal melanocytes (EM) were established from 16 individuals (8 SPT-1 & 8 SPT-4) and assessed for the presence of eicosanoid-producing enzymes, melanogenic enzymes and NO synthases, by immunocytochemistry, Polymerase Chain Reaction and Western Blotting and for the production of the main pro-inflammatory eicosanoid PGE2 by ELISA and Mass Spectrometry. Moreover, the fatty acid composition of cultured melanocytes was assessed by Gas Chromatography.
Results showed that individuals with SPT-1 had significantly greater neutrophil infiltration into the epidermis than those with SPT-4 at 24 hrs post-UVR. Moreover,
CD3+ lymphocyte infiltration into the dermis was significantly greater in individuals with SPT-4 than those with SPT-1 at 24 and 72 hrs post-UVR. NOS-1, NOS-3, 12-LOX and COX-2 expression were significantly increased in SPT-1 skin, while NOS-2 and 15-LOX were significantly increased in SPT-4 skin. As 12-LOX and COX-2 products are chemoattractive (for neutrophils) and pro-inflammatory respectively these data could explain the greater observed neutrophil infiltration in SPT-1. The 15-LOX product (15-HETE) is anti-inflammatory and may suggest that 15-LOX up-regulation in SPT-4 skin may aid resolution of the sunburn response, which in part may be mediated by CD3+ lymphocytes and a class-switch in eicosanoid production from COX to LOX products.
Melanocyte primary cultures surprisingly showed that SPT was not correlated with melanin content or melanogenic enzyme expression/activity suggesting that all melanocytes in vitro contained the necessary cellular machinery to produce melanin. This finding may reflect also their equal treatment under these enriched culture conditions, which may or may not be available to these cells in situ. Moreover, all melanocytes expressed the necessary machinery (PLA2, COX-1, cPGES) to produce PGE2. However, only some cultures did so at baseline and in response to UVR, and this was not correlated with SPT. A positive correlation was found however between expression level of dopachrome tautomerase (DCT) and protection against PGE2 production in response to UVR, which may suggest a novel role for DCT unrelated to melanogenesis.
In summary this research project has generated data that highlights differences between the skin of individuals with SPT-1 and those with SPT-4, and may provide evidence that the keratinocyte partner contributes significantly to the SPT-associated response. This research may also suggest DCT as a novel therapeutic target to protect EM from participation in the UVR-associated inflammatory response in skin. / Wellcome Trust
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