Cloning, expression and characterization of lepidopteran isopentenyl diphosphate isomerase

• Published in: Insect biochemistry and molecular biology Vol. 42; no. 10; pp. 739 - 750
• Main Authors: Sen, Stephanie E, Tomasello, Ashley, Grasso, Michael, Denton, Ryan, Macor, Joseph, Béliveau, Catherine, Cusson, Michel, Crowell, Dring N
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-10-2012
• Subjects: Amino Acid Sequence; Ammonium; Animals; Bombyx mori; Carbon-Carbon Double Bond Isomerases - chemistry; Carbon-Carbon Double Bond Isomerases - genetics; Carbon-Carbon Double Bond Isomerases - metabolism; Cavities; Choristoneura fumiferana; Cloning, Molecular; Corpora allata; Dimethylallyl diphosphate; Enzymes; Homology; Homology model; Inhibitor; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; Isomerization; Isopentenyl diphosphate; Isopentenyl diphosphate isomerase; Juvenile hormone; Juvenile hormones; Kinetics; Lepidoptera; Magnesium; Manduca - enzymology; Manduca sexta; Molecular Sequence Data; Moths - chemistry; Moths - enzymology; Moths - genetics; Mutagenesis; Nucleotide sequence; Sequence Alignment; site-directed mutagenesis; Transcription; Tyrosine; Homology model; Inhibitor; Isopentenyl diphosphate isomerase; Dimethylallyl diphosphate; Lepidoptera; Juvenile hormone
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2012.07.001

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 Mg2+ or Mn2+, 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.