SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides
Creators: name: Hartwig, Steffen
name: Frister, Thore
name: Alemdar, Semra
name: Li, Zhaopeng
name: Scheper, Thomas
name: Beutel, Sascha
Publisher: name: Cambridge, MA : Elsevier Academic Press
Year: 2015
Subjects: subject: Enzyme kinetics
subject: Khusimene
subject: Sesquiterpenes
subject: SUMO
subject: Terpene synthase
subject: Zizaene
subject: Chrysopogon zizanioides extract
subject: farnesyl diphosphate
subject: plant extract
subject: SUMO protein
subject: unclassified drug
subject: zizaene synthase
subject: hybrid protein
subject: isoprenoid phosphate
subject: khusimol
subject: sesquiterpene
subject: SUMO 1 protein
subject: terpene synthase
subject: transferase
subject: cold shock response
subject: enzyme mechanism
subject: enzyme synthesis
subject: Escherichia coli
subject: gel electrophoresis
subject: gene expression system
subject: isoelectric point
subject: mass fragmentography
subject: Michaelis Menten kinetics
subject: molecular weight
subject: nonhuman
subject: priority journal
subject: protein analysis
subject: protein binding
subject: protein cleavage
subject: protein domain
subject: protein purification
subject: substrate concentration
subject: enzymology
subject: genetics
subject: isolation and purification
subject: metabolism
subject: molecular cloning
subject: Vetiveria
subject: Alkyl and Aryl Transferases
subject: Cloning, Molecular
subject: Polyisoprenyl Phosphates
subject: Recombinant Fusion Proteins
subject: Dewey Decimal Classification::500 | Naturwissenschaften
subject: Dewey Decimal Classification::500 | Naturwissenschaften::570 | Biowissenschaften, Biologie
subject: Dewey Decimal Classification::500 | Naturwissenschaften::540 | Chemie

description: 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.