title: Cloning, expression and characterization of lepidopteran isopentenyl diphosphate isomerase.
abstract: Isopentenyl diphosphate isomerase (IPPI) of the spruce budworm, Choristoneura fumiferana, and of the tobacco hornworm, Manduca sexta, was cloned and its catalytic properties assessed. In the presence of Mg(2+) or Mn(2+), the recombinant protein from C. fumiferana (CfIPPI) efficiently isomerized IPP to dimethylallyl diphosphate (DMAPP). While C. fumiferana IPPI transcript levels were evenly distributed in a wide variety of tissues, they were highly abundant in the corpora allata. Because IPPI plays an alternate role in lepidopteran juvenile hormone (JH) biosynthesis by catalyzing the isomerization of the homologous substrate, homoisopentenyl diphosphate (HIPP), the ability of CfIPPI to convert HIPP to homodimethylallyl diphosphate (HDMAPP) was also studied. As expected, HIPP isomerization was efficient and the formation of HDMAPP occurred, but the regiospecificity of the reaction was lower than previously found in M. sexta corpora allata homogenates and with purified Bombyx mori IPPI. Differences in inhibitory potency for several alkylated ammonium diphosphates and higher homologs of DMAPP were noted between CfIPPI and a vertebrate IPPI, suggesting that the lepidopteran enzyme has a larger active site cavity. To determine the structural factors responsible for homologous substrate coupling, site directed mutagenesis of several residues identified through sequence alignment and homology modeling analysis was performed. The results suggest that unlike other IPPIs, W216 (C. fumiferana numbering) works in concert with a tyrosine residue (Y105) to allow binding of larger substrates and to stabilize the high-energy intermediate formed during substrate isomerization.
authors: Department of Chemistry, The College of New Jersey, 2000 Pennington Road, Ewing, NJ 08628, USA. sen@tcnj.edu
authors: Stephanie E
authors: SE
authors: Sen
authors: Ashley
authors: A
authors: Tomasello
authors: Michael
authors: M
authors: Grasso
authors: Ryan
authors: R
authors: Denton
authors: Joseph
authors: J
authors: Macor
authors: Catherine
authors: C
authors: Béliveau
authors: Michel
authors: M
authors: Cusson
authors: Dring N
authors: DN
authors: Crowell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D045782:Hemiterpenes; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D018402:Manduca / Q000201:enzymology*; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D045782:Hemiterpenes; D019476:Insect Proteins; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase