Functional characterization of OsPPT1, which encodes p-hydroxybenzoate polyprenyltransferase involved in ubiquinone biosynthesis in Oryza sativa.
Prenylation of the aromatic intermediate p-hydroxybenzoate (PHB) is a critical step in ubiquinone (UQ) biosynthesis. The enzyme that catalyzes this prenylation reaction is p-hydroxybenzoate polyprenyltransferase (PPT), which substitutes an aromatic proton at the m-position of PHB with a prenyl chain provided by polyprenyl diphosphate synthase. The rice genome contains three PPT candidates that share significant similarity with the yeast PPT (COQ2 gene), and the rice gene showing the highest similarity to COQ2 was isolated by reverse transcription-PCR and designated OsPPT1a. The deduced amino acid sequence of OsPPT1a contained a putative mitochondrial sorting signal at the N-terminus and conserved domains for putative substrate-binding sites typical of PPT protein family members. The subcellular localization of OsPPT1a protein was shown to be mainly in mitochondria based on studies using a green fluorescent protein-PPT fusion. A yeast complementation study revealed that OsPPT1a expression successfully recovered the growth defect of the coq2 mutant. A prenyltransferase assay using recombinant protein showed that OsPPT1a accepted prenyl diphosphates of various chain lengths as prenyl donors, whereas it showed strict substrate specificity for the aromatic substrate PHB as a prenyl acceptor. The apparent K (m) values for geranyl diphosphate and PHB were 59.7 and 6.04 microM, respectively. The requirement by OsPPT1a and COQ2 for divalent cations was also studied, with Mg2+ found to produce the highest enzyme activity. Northern analysis showed that OsPPT1a mRNA was accumulated in all tissues of O. sativa. These results suggest that OsPPT1a is a functional PPT involved in UQ biosynthesis in O. sativa.
Ohara
Kazuaki
K
Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji, Kyoto, 611-0011 Japan.
Yamamoto
Kyoko
K
Hamamoto
Masafumi
M
Sasaki
Kanako
K
Yazaki
Kazufumi
K
D019883:Alkyl and Aryl Transferases / Q000032:analysis / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D018744:DNA, Plant / Q000235:genetics / Q000502:physiology; D005091:Exons / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D017343:Genes, Plant / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D012275:Oryza / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010226:Parabens / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D018749:RNA, Plant / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014451:Ubiquinone / Q000096:biosynthesis*
D018744:DNA, Plant; D010226:Parabens; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D014451:Ubiquinone; D019883:Alkyl and Aryl Transferases; C021386:4-hydroxybenzoate polyprenyltransferase; C038193:4-hydroxybenzoic acid