title: Stereochemical Consequences of Vinylpyruvate Hydratase-Catalyzed Reactions.
abstract: 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.
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and ‡Department of Molecular Biosciences, University of Texas , Austin, Texas 78712, United States.
authors.forename: William H
authors.initials: WH
authors.lastname: Johnson
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and ‡Department of Molecular Biosciences, University of Texas , Austin, Texas 78712, United States.
authors.forename: Tyler M M
authors.initials: TM
authors.lastname: Stack
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and ‡Department of Molecular Biosciences, University of Texas , Austin, Texas 78712, United States.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Taylor
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and ‡Department of Molecular Biosciences, University of Texas , Austin, Texas 78712, United States.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Burks
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and ‡Department of Molecular Biosciences, University of Texas , Austin, Texas 78712, United States.
authors.forename: Christian P
authors.initials: CP
authors.lastname: Whitman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042743:Leptothrix / Q000201:enzymology / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; C061660:2-hydroxy-2,4-pentadienoic acid; C021917:4-oxalocrotonate decarboxylase; D002262:Carboxy-Lyases; C110907:2-hydroxypentadienoic acid hydratase; D006836:Hydro-Lyases