TY - JOUR
T1 - Purification and kinetic properties of elisabethatriene synthase from the coral Pseudopterogorgia elisabethae
AU - Brück, Thomas B.
AU - Kerr, Russell G.
N1 - Funding Information:
Financial support for this research was provided by the National Science Foundation (grant no: MCB-0119011) and the American Cancer Society (grant no: RSG-97-170-04-CDD) which are gratefully acknowledged. We also would like to thank the Florida Institute of Oceanography for providing ship time aboard the R/V Bellows. This is contribution number P200522 from the Centre of Excellence in Biomedical and Marine Biotechnology.
PY - 2006/3
Y1 - 2006/3
N2 - The Bahamian octocoral Pseudopterogorgia elisabethae is the source of pseudopterosins, diterpene glycosides with potent anti-inflammatory activity. The first committed step in pseudopterosin biosynthesis comprises the cyclisation of the universal diterpene precursor geranylgeranyl diphosphate to elisabethatriene. This reaction is catalysed by elisabethatriene synthase, which was purified to homogeneity from a crude coral extract. This represents the first purification to apparent homogeneity of a terpene cyclase from any marine source. The reaction kinetics of elisabethatriene synthase was examined using a steady state approach with 3H-labelled isoprenyldiphosphates varying in carbon chain length (C10, C15, C20). For the reaction of elisabethatriene synthase with its natural substrate geranylgeranyl diphosphate, values of Km (2.3 × 10- 6 M), V max (3.4 × 104 nM elisabethatriene* s - 1) and the specificity constant (kcat / Km = 1.8 × 10- 10 M- 1* s- 1) were comparable with diterpene cyclases from terrestrial plants. Elisabethatriene synthase also catalysed the conversion of C15 and C10 isoprenyldiphosphate analogues to monoterpene and sesquiterpene olefins, respectively. Kinetic parameters indicated that substrate specificity and K m of elisabethatriene synthase decreased with decreasing isoprenoid carbon chain length. Furthermore, GC-MS analysis showed increased product diversity with decreasing isoprenoid carbon chain length.
AB - The Bahamian octocoral Pseudopterogorgia elisabethae is the source of pseudopterosins, diterpene glycosides with potent anti-inflammatory activity. The first committed step in pseudopterosin biosynthesis comprises the cyclisation of the universal diterpene precursor geranylgeranyl diphosphate to elisabethatriene. This reaction is catalysed by elisabethatriene synthase, which was purified to homogeneity from a crude coral extract. This represents the first purification to apparent homogeneity of a terpene cyclase from any marine source. The reaction kinetics of elisabethatriene synthase was examined using a steady state approach with 3H-labelled isoprenyldiphosphates varying in carbon chain length (C10, C15, C20). For the reaction of elisabethatriene synthase with its natural substrate geranylgeranyl diphosphate, values of Km (2.3 × 10- 6 M), V max (3.4 × 104 nM elisabethatriene* s - 1) and the specificity constant (kcat / Km = 1.8 × 10- 10 M- 1* s- 1) were comparable with diterpene cyclases from terrestrial plants. Elisabethatriene synthase also catalysed the conversion of C15 and C10 isoprenyldiphosphate analogues to monoterpene and sesquiterpene olefins, respectively. Kinetic parameters indicated that substrate specificity and K m of elisabethatriene synthase decreased with decreasing isoprenoid carbon chain length. Furthermore, GC-MS analysis showed increased product diversity with decreasing isoprenoid carbon chain length.
KW - Elisabethatriene synthase
KW - Kinetic properties
KW - Octocoral
KW - Protein purification
KW - Terpene
UR - http://www.scopus.com/inward/record.url?scp=33344476624&partnerID=8YFLogxK
U2 - 10.1016/j.cbpb.2005.11.016
DO - 10.1016/j.cbpb.2005.11.016
M3 - Article
C2 - 16423548
AN - SCOPUS:33344476624
SN - 1096-4959
VL - 143
SP - 269
EP - 278
JO - Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
JF - Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
IS - 3
ER -