Reisinger, Bernd and Bocola, Marco and List, Felix and Claren, Joerg and Rajendran, Chitra and Sterner, Reinhard (2012) A sugar isomerization reaction established on various (beta alpha)(8)-barrel scaffolds is based on substrate-assisted catalysis. PROTEIN ENGINEERING DESIGN & SELECTION, 25 (11). pp. 751-760. ISSN 1741-0126, 1741-0134
Full text not available from this repository. (Request a copy)Abstract
In the course of tryptophan biosynthesis, the isomerization of phosphoribosylanthranilate (PRA) is catalyzed by the (beta alpha)(8)-barrel enzyme TrpF. The reaction occurs via a general acidbase mechanism with an aspartate and a cysteine residue acting as acid and base, respectively. PRA isomerase activity could be established on two (beta alpha)(8)-barrel enzymes involved in histidine biosynthesis, namely HisA and HisF, and on a HisAF chimera, by introducing two aspartate-to-valine substitutions. We have analyzed the reaction mechanism underlying this engineered activity by measuring its pH dependence, solving the crystal structure of a HisF variant with bound product analogue, and applying molecular dynamics simulations and mixed quantum and molecular mechanics calculations. The results suggest that PRA is anchored by the C-terminal phosphate-binding sites of HisA, HisF and HisAF. As a consequence, a conserved aspartate residue, which is equivalent to Cys7 from TrpF, is properly positioned to act as catalytic base. However, no obvious catalytic acid corresponding to Asp126 from TrpF could be identified in the three proteins. Instead, this role appears to be carried out by the carboxylate group of the anthranilate moiety of PRA. Thus, the engineered PRA isomerization activity is based on a reaction mechanism including substrate-assisted catalysis and thus differs substantially from the naturally evolved reaction mechanism used by TrpF.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PHOSPHORIBOSYL ANTHRANILATE ISOMERASE; DIFFERENT METABOLIC PATHWAYS; SITE-DIRECTED MUTAGENESIS; ESCHERICHIA-COLI; TRYPTOPHAN BIOSYNTHESIS; MOLECULAR-GRAPHICS; KEMP ELIMINATION; PK(A) VALUES; FORCE-FIELD; ENZYME; (beta alpha)(8)-barrel; enzyme design; histidine biosynthesis; substrate-assisted catalysis; tryptophan biosynthesis |
| Subjects: | 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: | 04 May 2020 10:30 |
| Last Modified: | 04 May 2020 10:30 |
| URI: | https://pred.uni-regensburg.de/id/eprint/17881 |
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