Expression and Stereochemical and Isotope Effect Studies of Active 4-Oxalocrotonate Decarboxylase
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 h...
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Published in | Biochemistry (Easton) Vol. 39; no. 4; pp. 718 - 726 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
01.02.2000
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Subjects | |
Online Access | Get full text |
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Summary: | 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 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 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 13C 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. |
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Bibliography: | istex:6E4EAEA1BB7A590AD56D604E3CC28062E92B9B60 ark:/67375/TPS-Z3XKZWFD-V This research was supported by the National Institutes of Health to C.P.W. (GM-41239) and P.F.C. (GM 36799). ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi9918902 |