Enzymes are categorized into superfamilies by sequence, structural, and mechanistic similarities. The evolutionary implications can be profound. Until the mid-1990s, the approach was fragmented largely due to limited sequence and structural data.
View Article and Find Full Text PDFDihydroxy phenanthrene, fluoranthene, and pyrene derivatives are intermediates in the bacterial catabolism of the corresponding parent polycyclic aromatic hydrocarbon (PAH). Ring-opening of the dihydroxy species followed by a series of enzyme-catalyzed reactions generates metabolites that funnel into the Krebs Cycle with the eventual production of carbon dioxide and water. One complication in delineating these pathways and harnessing them for useful purposes is that the initial enzymatic processing produces multiple dihydroxy PAHs with multiple ring opening possibilities and products.
View Article and Find Full Text PDFA 4-oxalocrotonate tautomerase (4-OT) trimer has been isolated from Burkholderia lata, and a kinetic, mechanistic, and structural analysis has been performed. The enzyme is the third described oligomer state for 4-OT along with a homo- and heterohexamer. The 4-OT trimer is part of a small subset of sequences (133 sequences) within the 4-OT subgroup of the tautomerase superfamily (TSF).
View Article and Find Full Text PDFLaccases were studied for their ability to remove two compounds, 2-chlorophenol and sulfamethoxazole, in batch studies, both in buffered solutions and in wastewater samples from different points in a municipal water resource recovery facility. Two enzymes with and without a mediator (acetosyringone) were investigated: a commercial product derived from Myceliphthora thermophile and a laboratory-generated enzyme mix derived from Tramates versicolor. The chlorophenol was removed rapidly by the commercial enzyme in the presence of acetosyringone, but the primary products were coupling complexes of the reactants.
View Article and Find Full Text PDFNahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways.
View Article and Find Full Text PDF5-Halo-2-hydroxy-2,4-pentadienoates (5-halo-HPDs) are reportedly generated in the bacterial catabolism of halogenated aromatic hydrocarbons by the meta-fission pathway. The 5-halo-HPDs, where the halogen can be bromide, chloride, or fluoride, result in the irreversible inactivation of 4-oxalocrotonate tautomerase (4-OT), which precedes the enzyme that generates them. The loss of activity is due to the covalent modification of the nucleophilic amino-terminal proline.
View Article and Find Full Text PDFThe tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown.
View Article and Find Full Text PDFA Pseudomonas sp. UW4 protein (UniProt K9NIA5) of unknown function was identified as similar to 4-oxalocrotonate tautomerase (4-OT)-like and cis-3-chloroacrylic acid dehalogenase (cis-CaaD)-like subgroups of the tautomerase superfamily (TSF). This protein lacks only Tyr-103 of the amino acids critical for cis-CaaD activity (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114).
View Article and Find Full Text PDF5-Halo-2-hydroxymuconates and 5-halo-2-hydroxy-2,4-pentadienoates are stable dienols that are proposed intermediates in bacterial -fission pathways for the degradation of halogenated aromatic compounds. The presence of the halogen raises questions about how the bulk and/or electronegativity of these substrates would affect enzyme catalysis or whether some pathway enzymes have evolved to accommodate it. To address these questions, 5-halo-2-hydroxymuconates and 5-halo-2-hydroxy-2,4-pentadienoates (5-halo = Cl, Br, F) were synthesized and a preliminary analysis of their enzymatic properties carried out.
View Article and Find Full Text PDFtrans- and cis-3-Chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively) catalyze the hydrolytic dehalogenation of their respective isomers and represent key steps in the bacterial conversion of 1,3-dichloropropene to acetaldehyde. In prior work, a kinetic mechanism for the CaaD-catalyzed reaction could not be unequivocally determined because (1) the order of product release could not be determined and (2) the fluorescence factor for the enzyme species, E*PQ (where P = bromide and Q = malonate semialdehyde, the two products of the reaction) could not be assigned. The ambiguities in the model have now been resolved by stopped-flow experiments following the reaction using an active site fluorescent probe, αY60W-CaaD and 3-bromopropiolate, previously shown to be a mechanism-based inhibitor of CaaD, coupled with the rate of bromide release in the course of CaaD inactivation.
View Article and Find Full Text PDFA stereochemical analysis has been carried out on two vinylpyruvate hydratases (VPH), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate in meta-fission pathways. Bacterial strains with this pathway can use aromatic compounds as sole sources of energy and carbon. The analysis was carried out using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate with the enzymes from Pseudomonas putida mt-2 (Pp) and Leptothrix cholodnii SP-6 (Lc).
View Article and Find Full Text PDFThe enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process.
View Article and Find Full Text PDFThe first enzyme in the oxalocrotonate branch of the naphthalene-degradation lower pathway in Pseudomonas putida G7 is NahI, a 2-hydroxymuconate semialdehyde dehydrogenase which converts 2-hydroxymuconate semialdehyde to 2-hydroxymuconate in the presence of NAD(+). NahI is in family 8 (ALDH8) of the NAD(P)(+)-dependent aldehyde dehydrogenase superfamily. In this work, we report the cloning, expression, purification and preliminary structural and kinetic characterization of the recombinant NahI.
View Article and Find Full Text PDFCg10062 is a cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue from Corynebacterium glutamicum with an unknown function and an uninformative genomic context. It shares 53% pairwise sequence similarity with cis-CaaD including the six active site amino acids (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) that are critical for cis-CaaD activity. However, Cg10062 is a poor cis-CaaD: it lacks catalytic efficiency and isomer specificity.
View Article and Find Full Text PDFTautomerase superfamily members are characterized by a β-α-β building block and a catalytic amino terminal proline. 4-Oxalocrotonate tautomerase (4-OT) and malonate semialdehyde decarboxylase (MSAD) are the title enzymes of two of the five known families in the superfamily. Two recent developments in these families indicate that there might be more metabolic diversity in the tautomerase superfamily than previously thought.
View Article and Find Full Text PDFtrans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored.
View Article and Find Full Text PDFMethylibium petroleiphilum strain PM1 uses various petroleum products including the fuel additive methyl tert-butyl ether and straight chain and aromatic hydrocarbons as sole carbon and energy sources. It has two operons, dmpI and dmpII, that code for the enzymes in a pair of parallel meta-fission pathways. In order to understand the roles of the pathways, the 4-oxalocrotonate tautomerase (4-OT) isozyme from each pathway was characterized.
View Article and Find Full Text PDFBiochemistry
July 2013
Malonate semialdehyde decarboxylase from Pseudomonas pavonaceae 170 (designated Pp MSAD) is in a bacterial catabolic pathway for the nematicide 1,3-dichloropropene. MSAD has two known activities: it catalyzes the metal ion-independent decarboxylation of malonate semialdehyde to produce acetaldehyde and carbon dioxide and a low-level hydration of 2-oxo-3-pentynoate to yield acetopyruvate. The latter activity is not known to be biologically relevant.
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