Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase

Citation
Tm. Stanley et al., Expression and stereochemical and isotope effect studies of active 4-oxalocrotonate decarboxylase, BIOCHEM, 39(4), 2000, pp. 718-726
Citations number
34
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
4
Year of publication
2000
Pages
718 - 726
Database
ISI
SICI code
0006-2960(20000201)39:4<718:EASAIE>2.0.ZU;2-N
Abstract
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.