Directed evolution of D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars
| Autor: | Chi Ching Mak, Chi-Huey Wong, Sun Fong, Timothy D. Machajewski |
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| Jazyk: | angličtina |
| Předmět: |
Models
Molecular Magnetic Resonance Spectroscopy Stereochemistry Molecular Sequence Data Clinical Biochemistry Carbohydrates Fructose-bisphosphate aldolase Protein Engineering 010402 general chemistry Polymerase Chain Reaction 01 natural sciences Biochemistry Catalysis Substrate Specificity chemistry.chemical_compound Aldol reaction Enzyme Stability Drug Discovery Aldolase Escherichia coli Amino Acid Sequence Molecular Biology Aldehyde-Lyases Pharmacology Molecular Structure biology Pseudomonas putida 010405 organic chemistry Circular Dichroism Aldolase A Temperature Enantioselective synthesis Substrate (chemistry) D- and L-Sugars Stereoisomerism General Medicine Directed evolution 0104 chemical sciences Kinetics chemistry Mutagenesis Biocatalysis biology.protein Molecular Medicine Organic synthesis Directed Molecular Evolution Sequence Alignment |
| Zdroj: | Chemistry & Biology. (11):873-883 |
| ISSN: | 1074-5521 |
| DOI: | 10.1016/S1074-5521(00)00035-1 |
| Popis: | Background: Exploitation and improvement of enzymes as catalysts for organic synthesis is of current interest in biocatalysis. A representative enzyme for investigation is the Escherichia coli D-2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes the highly specific reversible aldol reaction using the D-configurated KDPG as substrate. Results: Using in vitro evolution, the aldolase has been converted into aldolases with improved catalytic efficiency, altered substrate specificity and stereoselectivity. In particular, some evolved aldolases capable of accepting both D- and L- glyceraldehyde in the non-phosphorylated form as substrates for reversible aldol reaction have been obtained, providing a new direction to the enzymatic synthesis of both D- and L-sugars. Conclusions: This research has demonstrated the effectiveness of using in vitro evolution to rapidly alter the properties of an aldolase to improve its utility in asymmetric synthesis. The evolved aldolases, differing from the native enzyme which is highly phosphate- and D-sugar-dependent, catalyze the efficient synthesis of both D- and L-sugars from non-phosphorylated aldehydes and pyruvate. The principles and strategies described in this study should be applicable to other aldolases to further expand the scope of their synthetic utility. |
| Databáze: | OpenAIRE |
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