Geranyl diphosphate (GPP) is the direct precursor of all monoterpenoids and is the prenyl source of many meroterpenoids, such as geranylated coumarins. GPP synthase (GPPS) localized in plastids is responsible for providing the substrate for monoterpene synthases and prenyltransferases for synthesis of aromatic substances that are also present in plastids, but GPPS activity in Lithospermum erythrorhizon localizes to the cytosol, in which GPP is utilized for the biosynthesis of naphthoquinone pigments, which are shikonin derivatives. This study describes the identification of the cytosol-localized GPPS gene, LeGPPS, through expressed sequence tag (EST)- and homology-based approaches followed by functional analyses. The deduced amino acid sequence of the unique LeGPPS showed greater similarity to that of farnesyl diphosphate synthase (FPPS), which generally localizes to the cytosol, than to plastid-localized conventional GPPS. Biochemical characterization revealed that recombinant LeGPPS predominantly produces GPP along with a trace amount of FPP. LeGPPS expression was mainly detected in root bark, in which shikonin derivatives are produced, and in shikonin-producing cultured cells. The GFP fusion protein in onion (Allium cepa) cells localized to the cytosol. Site-directed mutagenesis of LeGPPS and another FPPS homolog identified in this study, LeFPPS1, showed that the His residue at position 100 of LeGPPS, adjacent to the first aspartate rich motif, contributes to substrate preference and product specificity, leading to GPP formation. These results suggest that LeGPPS, which is involved in shikonin biosynthesis, is recruited from cytosolic FPPS and that point mutation(s) result in the acquisition of GPPS activity.

中文翻译：

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红藻紫草的胞浆本地化的香叶基二磷酸香叶酯二磷酸合酶及其分子进化。

Geranyl diphosphate（GPP）是所有单萜类化合物的直接前体，并且是许多类萜类化合物（例如，geranylated香豆素）的异戊二烯基来源。位于质体中的GPP合酶（GPPS）负责为单萜合酶和异戊二烯基转移酶提供底物，以合成质体中也存在的芳香物质，但紫草中的GPPS活性定位于胞质溶胶，其中GPP用于生物合成萘醌颜料是紫草素的衍生物。这项研究描述了通过表达序列标签（EST）和基于同源性的方法，然后进行功能分析，来鉴定胞浆定位的GPPS基因LeGPPS。推论出的独特LeGPPS氨基酸序列与法呢基二磷酸合酶（FPPS）的相似性更高，通常定位于细胞质，而不是定位于质体的常规GPPS。生化特征表明重组LeGPPS主要产生GPP和痕量FPP。LeGPPS表达主要在产生紫草素衍生物的根皮和产生紫草素的培养细胞中检测到。洋葱（葱属）细胞中的GFP融合蛋白位于细胞质中。LeGPPS和本研究中鉴定的另一个FPPS同源物LeFPPS1的定点诱变表明，LeGPPS 100位的His残基与第一个富含天冬氨酸的基序相邻，有助于底物偏好和产物特异性，导致GPP形成。这些结果表明，参与紫草素生物合成的LeGPPS