title: Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase.
abstract: 4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes have been coexpressed and the complex has been purified to homogeneity. In addition, Glu-106, a potential catalytic residue in VPH, has been changed to glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-OD and purified to homogeneity. The 4-OD/E106QVPH complex retains full decarboxylase activity, with comparable kinetic parameters to those observed for 4-OD in the wild-type complex, but is devoid of any detectable hydratase activity. Decarboxylation of (5S)-2-oxo-3-[5-D]hexenedioate by either the 4-OD/VPH complex or the mutant complex generates 2-hydroxy-2,4E-[5-D]pentadienoate in D(2)O. Ketonization of 2-hydroxy-2,4-pentadienoate by the wild-type complex is highly stereoselective and results in the formation of 2-oxo-(3S)-[3-D]-4-pentenoate, while the mutant complex generates a racemic mixture. These results indicate that 2-hydroxy-2, 4-pentadienoate is the product of 4-OD and that 2-oxo-4-pentenoate results from a VPH-catalyzed process. On this basis, the previously proposed hypothesis for the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate has been revised [Lian, H., and Whitman, C. P. (1994) J. Am. Chem. Soc. 116, 10403-10411]. Finally, the observed (13)C kinetic isotope effect on the decarboxylation of 2-oxo-3-hexenedioate by the 4-OD/VPH complex suggests that the decarboxylation step is nearly rate-limiting. Because the value is not sensitive to either magnesium or manganese, it is likely that the transition state for carbon-carbon bond cleavage is late and that the metal positions the substrate and polarizes the carbonyl group, analogous to its role in oxalacetate decarboxylase.
authors.affiliation: Department of Chemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019-9425, USA.
authors.forename: T M
authors.initials: TM
authors.lastname: Stanley
authors.forename: W H
authors.initials: WH
authors.lastname: Johnson
authors.forename: E A
authors.initials: EA
authors.lastname: Burks
authors.forename: C P
authors.initials: CP
authors.lastname: Whitman
authors.forename: C C
authors.initials: CC
authors.lastname: Hwang
authors.forename: P F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D002247:Carbon Isotopes; D002262:Carboxy-Lyases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003903:Deuterium; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000302:isolation & purification; D013237:Stereoisomerism
chemical_list: D002247:Carbon Isotopes; D011522:Protons; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D003903:Deuterium; C021917:4-oxalocrotonate decarboxylase; D002262:Carboxy-Lyases; C110907:2-hydroxypentadienoic acid hydratase; D006836:Hydro-Lyases