ChemInform Abstract: Chemical and Enzymatic Ketonization of 2‐Hydroxymuconate, a Conjugated Enol.

Whitman, Christian P.; Aird, Bruce A.; Gillespie, William R.; Stolowich, Neal J.

doi:10.1002/chin.199130095

Cited by 12 publications

(43 citation statements)

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“…Stock solutions (20 mM) of 2-hydroxymuconate ( 1 ) and phenylenolpyruvate ( 3 ) were made up in ethanol, and diluted (with ethanol) to 10 mM. The ketonization of 1 to 2 was measured by following the increase in absorbance at 236 nm (ε = 6580 M −1 cm −1 ) using substrate concentrations ranging from 10–200 μM (4). The ketonization of 3 to 4 was measured by following the decrease in absorbance at 283 nm (ε = 18,000 M −1 cm −1 ) using substrate concentrations ranging from 10–140 μM (5).…”

Section: Methodsmentioning

confidence: 99%

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily,

et al. 2010

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Abstract4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of substrate to the C-5 position of product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. In order to characterize † This research was supported by the National Institutes of Health Grants GM-41239 (CPW) and AI-60915 (SDP), the Robert A.Welch Foundation grant F-1334 (CPW), and the Department of Defense Grant W81XWH0710445 USAMRAA (SDP). Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract N. W-31-109-Eng-38. The Analytical Instrumentation Facility Core (College of Pharmacy, The University of Texas at Austin) is supported by an NIH Center grant ES07784. * Corresponding authors (CPW) Tel: 512-471-6198; Fax: 512-232-2606; whitman@mail.utexas.edu. (SDP) Tel: 303-871-2533; Fax: 303-871-2254; scott.pegan@du.edu. The atomic coordinates and structure factors have been deposited with the Brookhaven Protein Data Bank (PDB codes 3MB2). 1 Abbreviations: Ap, ampicillin; BSA, bovine serum albumin; dNTPs, deoxyribose nucleotide triphosphates; CHMI, 5-(carboxymethyl)-2-hydroxymuconate isomerase; CaaD, trans-3-chloroacrylic acid dehalogenase; cis-CaaD, cis-3-chloroacrylic acid dehalogenase; DSC, differential scanning calorimetry; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane sulfonate; hh4-OT, heterohexamer 4-oxalocrotonate tautomerase; IPTG, isopropyl-β-D-thiogalactoside; Kn, kanamycin; LB, Luria-Bertani; MIF, macrophage migration inhibitory factor; MSAD, malonate semialdehyde decarboxylase; NCBI, National Center for Biotechnology Information; NMR, nuclear magnetic resonance; 4-OT, 4-oxalocrotonate tautomerase; PEG, polyethylene glycol; PPT, phenylpyruvate tautomerase; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SUPPORTING INFORMATION AVAILABLEThe expression, overproduction, and purification protocols for the hh4-OT are provided in the Supporting Information. The experimental procedures used for the construction, expression, overproduction, purification, and mass spectral analysis of the hh4-OT mutants and the construction and expression of the separate subunits of the hh4-OT are also provided in the Supporting Information. Finally, the molecular modeling studies are described. This material is available free of charge via the Internet at http://pubs.acs.org....

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Section: Methodsmentioning

confidence: 99%

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily,

et al. 2010

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“…The syntheses of 2-hydroxymuconate ( 4 ) (4), 5-(methyl)-2-hydroxymuconate ( 6 ) (16), 5-(carboxymethyl)-2-hydroxymuconate ( 8 ) (17), 2-hydroxy-2,4-pentadienoate ( 9 ) (18), 2- hydroxy-2,4-heptadiene-1,7-dioate ( 12 ) (19), and 2-oxo-3-pentynoate ( 17 ) (20) are reported in the indicated references. Recombinant 4-OT was purified by previously reported procedures (15,21).…”

Section: Methodsmentioning

confidence: 99%

Kinetic, Crystallographic, and Mechanistic Characterization of TomN: Elucidation of a Function for a 4-Oxalocrotonate Tautomerase Homologue in the Tomaymycin Biosynthetic Pathway

Burks¹,

Yan²,

Johnson³

et al. 2011

Biochemistry

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The biosynthesis of the C ring of the anti-tumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from L-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from the one processed by 4-OT. In order to establish the function and mechanism of TomN and its relationship to 4-OT, kinetic, mutagenic, and structural studies have been carried out. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutant of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with an RMS deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.

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Section: Introductionmentioning

confidence: 99%

“…It exists as a hexamer where each monomer consists of 62 amino acids [18–21]. Initially, it was thought that 4-OT catalyzed a 1,3-allylic rearrangement of 2-oxo-4-hexenedioate ( 1 , Scheme 1) to 2-oxo-3-hexenedioate ( 3 ) through the dienol intermediate known commonly as 2-hydroxymuconate ( 2 ) [18, 22, 23]. Recent stereochemical findings suggest that 4-OT more likely catalyzes a simple 1,5-keto-enol tautomerization of 2 to 3 where Pro-1 functions as a general base and abstracts the 2-hydroxyl proton and delivers it stereoselectively to the C-5 position [19, 24].…”

Section: Introductionmentioning

confidence: 99%

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Poelarends

Veetil

Whitman

2008

Cell. Mol. Life Sci.

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163

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Tautomerase superfamily members have an amino-terminal proline and a β–α–β fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF), which exhibits a phenylpyruvate tautomerase (PPT) activity. Pro-1 is a base (4-OT, CHMI, the PPT activity of MIF) or an acid (CaaD, cis-CaaD, MSAD). Components of the catalytic machinery have been identified and mechanistic hypotheses formulated. Characterization of new homologues shows that these mechanisms are incomplete. 4-OT, CaaD, cis-CaaD, and MSAD also have promiscuous activities with a hydratase activity in CaaD, cis-CaaD, and MSAD, PPT activity in CaaD and cis-CaaD, and CaaD and cis-CaaD activities in 4-OT. The shared promiscuous activities provide evidence for divergent evolution from a common ancestor, give hints about mechanistic relationships, and implicate catalytic promiscuity in the emergence of new enzymes.

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ChemInform Abstract: Chemical and Enzymatic Ketonization of 2‐Hydroxymuconate, a Conjugated Enol.

Cited by 12 publications

References 1 publication

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily,

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily,

Kinetic, Crystallographic, and Mechanistic Characterization of TomN: Elucidation of a Function for a 4-Oxalocrotonate Tautomerase Homologue in the Tomaymycin Biosynthetic Pathway

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

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