Cloning, expression and characterization of lepidopteran isopentenyl diphosphate isomerase.
Insect Biochem Mol Biol
; 42(10): 739-50, 2012 Oct.
Article
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| MEDLINE
| ID: mdl-22820710
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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Clonación Molecular
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Manduca
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Proteínas de Insectos
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Isomerasas de Doble Vínculo Carbono-Carbono
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Mariposas Nocturnas
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Insect Biochem Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Año:
2012
Tipo del documento:
Article
País de afiliación:
Estados Unidos