Busch, Markus R. and Rajendran, Chitra and Sterner, Reinhard (2023) Structural and Functional Characterization of the Ureidoacrylate Amidohydrolase RutB from Escherichia coli. BIOCHEMISTRY, 62 (3). pp. 863-872. ISSN 0006-2960
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We present a detailed structure-function analysis of the ureidoacrylate amidohydrolase RutB from Eschericha coli, which is an essential enzyme of the Rut pathway for pyrimidine utilization. Crystals of selenomethionine-labeled RutB were produced, which allowed us to determine the first structure of the enzyme at a resolution of 1.9 angstrom and to identify it as a new member of the isochorismatase-like hydrolase family. RutB was co crystallized with the substrate analogue ureidopropionate, revealing the mode of substrate binding. Mutation of residues constituting the catalytic triad (D24A, D24N, K133A, C166A, C166S, C166T, C166Y) resulted in complete inactivation of RutB, whereas mutation of other residues close to the active site (Y29F, Y35F, N72A, W74A, W74F, E80A, E80D, S92A, S92T, S92Y, Q105A, Y136A, Y136F) leads to distinct changes of the turnover number (kcat) and/or the Michaelis constant (KM). The results of our structural and mutational studies allowed us to assign specific functions to individual residues and to formulate a plausible reaction mechanism for RutB.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PYRIMIDINE CATABOLIC PATHWAY; DEGRADATION; FAMILY; (+)-GAMMA-LACTAMASE; ISOCHORISMATASE; BARBITURASE; SOFTWARE; ENZYME; MEMBER; PHENIX |
| Subjects: | 500 Science > 540 Chemistry & allied sciences 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 22 Feb 2024 05:25 |
| Last Modified: | 22 Feb 2024 06:02 |
| URI: | https://pred.uni-regensburg.de/id/eprint/58948 |
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