SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides
| Autor: | Steffen Hartwig, Thore Frister, Sascha Beutel, Thomas Scheper, Semra Alemdar, Zhaopeng Li |
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| Rok vydání: | 2015 |
| Předmět: |
molecular cloning
Dewey Decimal Classification::500 | Naturwissenschaften::540 | Chemie Dewey Decimal Classification::500 | Naturwissenschaften::570 | Biowissenschaften Biologie transferase khusimol protein binding Biochemistry gel electrophoresis Polyisoprenyl Phosphates Peptide mass fingerprinting Chrysopogon protein purification protein cleavage Protein purification genetics isoelectric point substrate concentration Cloning Molecular Dewey Decimal Classification::500 | Naturwissenschaften Gel electrophoresis Chemistry protein domain Zizaene farnesyl diphosphate Directed evolution Terpene synthase mass fragmentography unclassified drug cold shock response priority journal ddc:540 enzyme synthesis Sesquiterpene synthase activity plant extract ddc:500 SUMO protein gene expression system Sesquiterpenes Khusimene sesquiterpene enzymology Recombinant Fusion Proteins SUMO-1 Protein Biophysics Michaelis–Menten kinetics zizaene synthase Chrysopogon zizanioides extract Michaelis Menten kinetics ddc:570 Escherichia coli enzyme mechanism Enzyme kinetics Molecular Biology Polycyclic Sesquiterpenes hybrid protein nonhuman Alkyl and Aryl Transferases Molecular mass isolation and purification molecular weight Cell Biology isoprenoid phosphate SUMO protein analysis Vetiveria metabolism SUMO 1 protein |
| Zdroj: | Biochemical and Biophysical Research Communications 458 (2015), Nr. 4 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2015.02.053 |
| Popis: | An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L−1 were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni2+-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg2+ containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of KM = 1.111 μM (±0.113), vmax = 0.3245 μM min−1 (±0.0035), kcat = 2.95 min−1, as well as a catalytic efficiency kcat/KM = 4.43 × 104 M−1 s−1 were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. EU/EFRE/ZW-8-80130940 |
| Databáze: | OpenAIRE |
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