Geranyl diphosphate synthase from Abies grandis: cDNA isolation, functional expression, and characterization

Area of Science:

• Plant Biochemistry
• Enzyme Catalysis
• Molecular Biology

Background:

• Geranyl diphosphate (GPP) is a crucial precursor in monoterpene biosynthesis.
• Geranyl diphosphate synthase (GPPS) catalyzes the formation of GPP from dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP).

Purpose of the Study:

• To isolate and characterize novel GPPS genes from grand fir (Abies grandis).
• To investigate the enzymatic activity and quaternary structure of recombinant grand fir GPPS enzymes.

Main Methods:

• cDNA library screening using a hybridization probe from Taxus canadensis geranylgeranyl diphosphate synthase.
• Expression of isolated cDNA clones in Escherichia coli.
• Enzymatic assays with DMAPP and IPP as cosubstrates.
• Gel filtration to determine enzyme quaternary structure.

Main Results:

• Four full-length cDNA clones encoding putative GPPS were isolated from Abies grandis.
• Three recombinant enzymes produced GPP, and one produced geranylgeranyl diphosphate (GGPP).
• AgGPPS2 was confirmed as a specific GPP synthase, exhibiting high catalytic efficiency (kcat = 1.8s⁻¹).
• Recombinant GPPS enzymes, lacking plastidial targeting sequences, exist as homodimers.

Conclusions:

• Novel GPPS genes have been identified in Abies grandis, expanding the known diversity of monoterpene biosynthesis enzymes.
• AgGPPS2 represents a highly specific and efficient geranyl diphosphate synthase.
• The homodimeric structure of grand fir GPPS contrasts with the heterotetrameric structure found in some other plant species.