Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways
| Autor: | Catharina Jürgens, Reinhard Sterner, Stefan Hettwer, Matthias Wilmanns, D. Wegener, Alexander Strom |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Ribonucleotide
Stereochemistry Molecular Sequence Data Isomerase Biology 01 natural sciences Catalysis Protein Structure Secondary Evolution Molecular 03 medical and health sciences chemistry.chemical_compound Biosynthesis Escherichia coli Amino Acid Sequence Cloning Molecular Histidine Aldose-Ketose Isomerases 030304 developmental biology Gene Library chemistry.chemical_classification 0303 health sciences Multidisciplinary 010405 organic chemistry Biological Sciences Directed evolution Recombinant Proteins 0104 chemical sciences Amino acid Metabolic pathway Kinetics Enzyme Biochemistry chemistry Amino Acid Substitution Mutagenesis Directed Molecular Evolution Sequence Alignment Plasmids |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America. 97(18) |
| ISSN: | 0027-8424 |
| Popis: | Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N ′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC 5.3.1.16 ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC 5.3.1.24 ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (βα) 8 -barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro , and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (βα) 8 -barrel enzymes are very suitable for the design of new catalytic activities. |
| Databáze: | OpenAIRE |
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