A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif.
Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg(2+) and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.
Hsiao
Yu-Yun
YY
Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan.
Jeng
Mei-Fen
MF
Tsai
Wen-Chieh
WC
Chuang
Yu-Chen
YC
Li
Chia-Ying
CY
Wu
Tian-Shung
TS
Kuoh
Chang-Sheng
CS
Chen
Wen-Huei
WH
Chen
Hong-Hwa
HH
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004122:Dimethylallyltranstransferase / Q000235:genetics*; D035264:Flowers / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009812:Odorants; D029595:Orchidaceae / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018744:DNA, Plant; D039821:Monoterpenes; D010940:Plant Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; D004122:Dimethylallyltranstransferase