The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane α-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.
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Research Article|
June 26 2009
Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid
Kazuaki Ohara;
Kazuaki Ohara
1
*Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji 611-0011, Japan
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Ayumu Muroya;
Ayumu Muroya
†Science & Technology Systems, Inc., 1-20-1 Shibuya, Tokyo 150-0002, Japan
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Nobuhiro Fukushima;
Nobuhiro Fukushima
†Science & Technology Systems, Inc., 1-20-1 Shibuya, Tokyo 150-0002, Japan
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Kazufumi Yazaki
Kazufumi Yazaki
2
*Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji 611-0011, Japan
2To whom correspondence should be addressed (email yazaki@rish.kyoto-u.ac.jp).
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Publisher: Portland Press Ltd
Received:
September 30 2008
Revision Received:
April 21 2009
Accepted:
April 24 2009
Accepted Manuscript online:
April 24 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 421 (2): 231–241.
Article history
Received:
September 30 2008
Revision Received:
April 21 2009
Accepted:
April 24 2009
Accepted Manuscript online:
April 24 2009
Citation
Kazuaki Ohara, Ayumu Muroya, Nobuhiro Fukushima, Kazufumi Yazaki; Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid. Biochem J 15 July 2009; 421 (2): 231–241. doi: https://doi.org/10.1042/BJ20081968
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