Oliynyk, Z., Briseno-Roa, L., Janowitz, T., Sondergeld, P., Fersht, A. R. (April 2004) Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS. Protein Eng Des Sel, 17 (4). pp. 383-90. ISSN 1741-0126 (Print)1741-0126
Abstract
KDO8PS (3-deoxy-d-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased k(cat) of the enzymes in the presence of activating Mn(2+) ions. The M25P N26C mutant of E.coli KDO8PS had a value of k(cat)/K(M) in the presence of Mn(2+) ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn(2+) but no longer required Mn(2+) to function and eventually lost one of metal-binding residues.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics > genomics and proteomics > design > protein design |
| CSHL Authors: | |
| Communities: | CSHL labs > Janowitz lab |
| Highlight: | Janowitz, Tobias |
| Depositing User: | Matthew Dunn |
| Date: | April 2004 |
| Date Deposited: | 10 Oct 2018 16:20 |
| Last Modified: | 10 Oct 2018 16:20 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/37239 |
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