Sesquiterpene α-Farnesene Synthase: Partial Purification, Characterization, and Activity in Relation to Superficial Scald Development in Apples

Abstract

<jats:p>To decipher the relation between α-farnesene metabolism and the development of superficial scald in apples, <jats:italic>trans,trans</jats:italic>-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-farnesene, was partially purified from skin tissue of `Delicious' apples (<jats:italic>Malus</jats:italic> ×<jats:italic>domestica</jats:italic> Borkh.) and characterized. Total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. α-Farnesene synthase was purified 70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as Mg<jats:sup>2+</jats:sup> or Mn<jats:sup>2+</jats:sup> for activity. It exhibited allosteric kinetics, S<jats:sub>(0.5)</jats:sub> for farnesyl pyrophosphate being 84±18 μmol·L<jats:sup>-1</jats:sup>, and a Hill coefficient (n<jats:sub>H</jats:sub>) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 °C, while 50% of the maximal activity was retained at 0 °C. In vivo α-farnesene synthase activity was minimal at harvest, then increased rapidly during 16 weeks storage in air at 0 °C, and decreased during further storage. Activity of α-farnesene synthase, α-farnesene content, and conjugated triene alcohol (the putative scald-causing oxidation product of α-farnesene) content in skin tissue were not correlated to the inherent nature of scald susceptibility or resistance in 11 apple cultivars tested.</jats:p>

Antioxidant enzyme activities in apple varieties and resistance to superficial scald development

“…In fact, earlier studies have shown that a-farnesene biosynthesis and its levels are higher on the non-scald developing side of apple fruit than the side showing symptoms of superficial scald (Rupasinghe et al, 1998). Rupasinghe et al (2000) have also reported that the a-farnesene contents among several apple cultivars varied, with Empire, Gala, Idared and McIntosh having low levels, while Cortland and Delicious having higher levels. As well, a comparison of scald-resistant and susceptible cultivars showed that afarnesene content in the skin of apple is not correlated with their inherent, relative scald susceptibility.…”

“…In addition, a-farnesene is synthesized in apple skin tissue by the direct conversion of trans, trans-farnesyl pyrophosphate (FPP) and the incorporation of radiolabel from FPP to a-farnesene was detected only in the skin tissue but not in the cortex tissues (Rupasinghe, Paliyath, & Murr, 1998. As well, a-farnesene levels are very low in the skin of apples at harvest and a-farnesene content and a-farnesene synthase activity increased rapidly during storage at 0°C (Rupasinghe et al, 1998, Rupasinghe, Paliyath, & Murr, 2000.…”

Modulation of carotenoid biosynthesis during tomato fruit ripening through phytochrome regulation of phytoene synthase activity

“…Other than the alcohol form of the substrate, the production of only one other radiolabled product indicates the absence of other non-specific interfering reactions. Production of the alcohol derivatives from pyrophosphate-isoprenoid substrates appears to be a common for in vitro reactions of isoprenoid enzymes [32,33]. GGOH formation was mostly stopped by enzyme inactivation (Fig.…”

Production of farnesene and santalene by Saccharomyces cerevisiae using fed‐batch cultivations with RQ‐controlled feed

“…The objective of this study was to express three farnesene synthase genes from plants in S. cerevisiae, i.e., a-farnesene synthase from Malus domestica and ß-farnesene synthase from Citrus junos and Artemisia annua, respectively, which have previously been characterized regarding their reaction conditions, co-factor requirements and product specificities (Crock et al, 1997;Green et al, 2007;Maruyama et al, 2001;Pechous and Whitaker, 2004;Picaud et al, 2005;Rupasinghe et al, 2000) and compare production of farnesene with production of another sesquiterpene. The objective of this study was to express three farnesene synthase genes from plants in S. cerevisiae, i.e., a-farnesene synthase from Malus domestica and ß-farnesene synthase from Citrus junos and Artemisia annua, respectively, which have previously been characterized regarding their reaction conditions, co-factor requirements and product specificities (Crock et al, 1997;Green et al, 2007;Maruyama et al, 2001;Pechous and Whitaker, 2004;Picaud et al, 2005;Rupasinghe et al, 2000) and compare production of farnesene with production of another sesquiterpene.…”

“…Farnesene synthases are widely spread in the plant kingdom and coding sequences are available for several organisms including Zea mays, Cucumis melo, Pyrus communis and Mentha arvensis (Nucleotide Database, National Center for Biotechnology Information). The objective of this study was to express three farnesene synthase genes from plants in S. cerevisiae, i.e., a-farnesene synthase from Malus domestica and ß-farnesene synthase from Citrus junos and Artemisia annua, respectively, which have previously been characterized regarding their reaction conditions, co-factor requirements and product specificities (Crock et al, 1997;Green et al, 2007;Maruyama et al, 2001;Pechous and Whitaker, 2004;Picaud et al, 2005;Rupasinghe et al, 2000) and compare production of farnesene with production of another sesquiterpene. For this purpose, an existing platform for sesquiterpene production was used to investigate production capacity in comparison to a-santalene.…”