Crystal structure of (+)-delta-cadinene synthase from Gossypium arboreum and evolutionary divergence of metal binding motifs for catalysis.
(+)-Delta-cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 A resolution and the structure of its complex with three putative Mg(2+) ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 A resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich D(307)DTYD(311) motif on helix D that interacts with Mg(2+)(A) and Mg(2+)(C). However, DCS appears to be unique among terpenoid cyclases in that it does not contain the "NSE/DTE" motif on helix H that specifically chelates Mg(2+)(B), which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg(2+)(B) ligands). Instead, DCS contains a second aspartate-rich motif, D(451)DVAE(455), that interacts with Mg(2+)(B). In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg(2+)(B) binding motif. Analyses of DCS mutants with alanine substitutions in the D(307)DTYD(311) and D(451)DVAE(455) segments reveal the contributions of these segments to catalysis.
Gennadios
Heather A
HA
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
Gonzalez
Veronica
V
Di Costanzo
Luigi
L
Li
Amang
A
Yu
Fanglei
F
Miller
David J
DJ
Allemann
Rudolf K
RK
Christianson
David W
DW
D019943:Amino Acid Substitution; D001464:Barium / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D019143:Evolution, Molecular; D003368:Gossypium / Q000201:enzymology*; D006860:Hydrogen Bonding; D020128:Inhibitory Concentration 50; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D011106:Polyisoprenyl Phosphates / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
C525013:2-fluorofarnesyl diphosphate; D004791:Enzyme Inhibitors; D008670:Metals; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D001464:Barium; D007535:Isomerases; C097608:delta-cadinene synthase; D008274:Magnesium