Attempts to develop an enzyme converting DHIV to KIV
| Autor: | Stephen L. Mayo, Kenji Oki, Frederick S Lee |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Models
Molecular Protein Conformation Bioengineering Substrate analog Protein Engineering Biochemistry Substrate Specificity chemistry.chemical_compound Hemiterpenes Valerates Amino Acid Sequence Phosphorylation Molecular Biology Magnesium ion Hydro-Lyases chemistry.chemical_classification biology Picrophilus torridus Enolase superfamily Mandelate racemase biology.organism_classification Keto Acids Enzyme chemistry Dehydratase Mutation Biocatalysis biology.protein Computer-Aided Design Adenosine triphosphate Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection. 32:261-270 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/gzz042 |
| Popis: | Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained. |
| Databáze: | OpenAIRE |
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