4-Oxalocrotonate Tautomerase, Its Homologue YwhB, and Active Vinylpyruvate Hydratase:  Synthesis and Evaluation of 2-Fluoro Substrate Analogues

Johnson, William H.; Wang, Susan C.; Stanley, Thanuja M.; Czerwiński, Robert; Almrud, J.J.; Poelarends, Gerrit J.; Murzin, and Alexey G.; Whitman, Christian P.

doi:10.1021/bi049489p

Cited by 12 publications

(13 citation statements)

References 34 publications

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“…Various groups have proposed that the water would be activated by the metal ion. 4,5,[10][11][12] The strongest support for this mechanism comes from the crystallographic analysis of OHED hydratase carried out by Izumi et al 12 Based on this analysis, the mechanism shown in Figure 3 was formulated. The authors suggested that the metal ion (Mg 2+ ) is coordinated by the C-2 carbonyl group and the C-1 carboxylate group (of 20) based on modeling the substrate into the crystal structure of the enzyme-oxalate complex.…”

Section: Discussionmentioning

confidence: 99%

Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions

et al. 2016

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A 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). In both organisms, VPH is in a complex with the preceding enzyme in the pathway, 4-oxalocrotonate decarboxylase (4-OD). In D2O, a deuteron is incorporated stereospecifically at the C-3 and C-5 positions of product by both Pp and Lc enzymes. Accordingly, the complexes generate (3S,5S)-3,5-[di-D]-2-keto-4S-hydroxyhexanoate and (3S,5R)-3,5-[di-D]-2-keto-4R-hydroxy-5-chloropentanoate (4R and 5R due to a priority numbering change). The substitution at C-5 (CH3 or Cl) or the source of the enzyme (Pp or Lc) does not change the stereochemical outcome. One mechanism that can account for the results is the ketonization of the 5-substituted dienol to the α,β-unsaturated ketone (placing a deuteron at C-5 in D2O), followed by the conjugate addition of water (placing a deuteron at C-3). The stereochemical outcome for VPH (from Pp and Lc) is the same as that reported for a related enzyme, 2-oxo-hept-4-ene-1,7-dioate hydratase, from Escherichia coli C. The combined observations suggest similar mechanisms for these three enzymes that could possibly be common to this group of enzymes.

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Section: Discussionmentioning

confidence: 99%

Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions

et al. 2016

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“…4-OD is a metal-dependent enzyme requiring magnesium ion for activity. Johnson and colleagues identified conserved and potential catalytic residues in XylI and homologues, and constructed XylI mutants that were expressed and purified as a complex with XylJ (VPH) . Two mutants (E109Q and E111Q) lacked the decarboxylase activity of 4-OD, but retained the hydratase activity of VPH.…”

Section: Resultsmentioning

confidence: 99%

“…Attempts to address these questions in the 4-OD/VPH complex from P. putida mt-2 have been limited by the fact that XylI is unstable in the absence of XylJ (6, 12, 13) . By using NahK from P. putida G7, we have now produced a stable 4-OD in large quantities that has similar kinetic parameters to those of 4-OD in the XylI/XylJ native complex.…”

Section: Introductionmentioning

confidence: 99%

“…12 Attempts to address these questions in the 4-OD/VPH complex from P. putida mt-2 have been limited by the fact that XylI is unstable in the absence of XylJ. 6,12,13 By using NahK from P. putida G7, we have now produced a stable 4-OD in large quantities that has kinetic parameters similar to those of 4-OD in the XylI/XylJ native complex. Crystallographic analysis of the apo form and substrate analogue complexes has uncovered the structural basis for the metal-assisted decarboxylation.…”

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confidence: 99%

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Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid

et al. 2016

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The 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. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg2+ as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystalline structures implicates a lid domain in substrate binding, and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.

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“…18 There are also some examples of HWE reactions of aromatic or short-chain aliphatic aldehydes with 2-fluorophosphonoacetates. Depending on the reaction conditions, 2-fluoro-or 2,4-difluorodienes were provided in good 2E- [19][20][21] or 2Z-selectivity. 20,22 HWE reactions, leading to analogues of retinoic acid bearing one vinylic fluorine atom in the 2-and/or 4-position on the polyene side chain, have also been described.…”

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confidence: 99%