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Kinetic, Crystallographic, and Mechanistic Characterization of TomN: Elucidation of a Function for a 4-Oxalocrotonate Tautomerase Homologue in the Tomaymycin Biosynthetic Pathwayâ€
- Publication Year :
- 2011
-
Abstract
- The biosynthesis of the C ring of the anti-tumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from L-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from the one processed by 4-OT. In order to establish the function and mechanism of TomN and its relationship to 4-OT, kinetic, mutagenic, and structural studies have been carried out. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutant of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with an RMS deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.
- Subjects :
- Staphylococcus aureus
Stereochemistry
Mutant
Isomerase
Crystallography, X-Ray
Biochemistry
Article
Substrate Specificity
Protein structure
Bacterial Proteins
Escherichia coli
Isomerases
Alanine
chemistry.chemical_classification
Benzodiazepinones
biology
Pseudomonas putida
Escherichia coli Proteins
Active site
Amino acid
Biosynthetic Pathways
Protein Structure, Tertiary
Kinetics
Enzyme
chemistry
Structural Homology, Protein
4-Oxalocrotonate tautomerase
biology.protein
Signal Transduction
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....1e19d562f901e128b1706013c295fc1a