TY - JOUR

T1 - SUMO-fusion, purification, and characterization of a (þ)-zizaene synthase from Chrysopogon zizanioides

AU - Hartwig, S.

AU - Frister, T.

AU - Alemdar, S.

AU - Li, Z.

AU - Scheper, T.

AU - Beutel, S.

N1 - Funding information: The authors would like to thank Ralf Günter Berger and Ulrich Krings from the Institute of Food Chemistry in Hannover for kind support with GC–MS analytics. We also thank Ursula Rinas and Manfred Nimtz from the Helmholtz Centre for Infection Research in Braunschweig for providing the MALDI-PMF analysis. This study was funded by the European Union as part of the EFRE (European Regional Development Fund) project “Refinement of plant resources” (ZW 8-80130940).

PY - 2015/2/19

Y1 - 2015/2/19

N2 - Abstract 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-1s-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.

AB - Abstract 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-1s-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.

KW - Enzyme kinetics

KW - Khusimene

KW - Sesquiterpenes

KW - SUMO

KW - Terpene synthase

KW - Zizaene

UR - http://www.scopus.com/inward/record.url?scp=84925506231&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2015.02.053

DO - 10.1016/j.bbrc.2015.02.053

M3 - Article

C2 - 25701786

AN - SCOPUS:84925506231

VL - 458

SP - 883

EP - 889

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 4

ER -