Tm. Stanley et al., Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase, BIOCHEM, 39(4), 2000, pp. 718-726
4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) fro
m 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 faci
litate mechanistic and structural studies of the complex, the two enzymes h
ave been coexpressed and the complex has been purified to homogeneity. In a
ddition, Glu-106, a potential catalytic residue in VPH, has been changed to
glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-O
D and purified to homogeneity. The 4-OD/E106QVPH complex retains full decar
boxylase activity, with comparable kinetic parameters to those observed for
4-OD in the wild-type complex, but is devoid of any detectable hydratase a
ctivity. 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]pentadie
noate in D2O. 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 t
his 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, Sec. 116, 10403-10411], Finally, the obs
erved C-13 kinetic isotope effect on the decarboxylation of 2-oxo-3-hexened
ioate by the 4-OD/VPH complex suggests that the decarboxylation step is nea
rly rate-limiting. Because the value is not sensitive to either magnesium o
r manganese, it is likely that the transition state for carbon-carbon bond
cleavage is late and that the metal positions the substrate and polarizes t
he carbonyl group, analogous to its role in oxalacetate decarboxylase.