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Page 6 of 20Rai et al. • C. roseus Geranyl Diphosphate SynthasesFigure 4. Purification and Prenyltransferase Assay of Recombinant Heteromeric CrGPPS Expressed in E. coli.(A) SDS–PAGE analyses of co-expressed (His) 6 -tagged CrGGPPSs with non-(His) 6 -tagged CrGPPS.SSU. Samples contained purified proteins obtainedfrom the co-expression of (His) 6 –CrGGPPS1 and CrGPPS.SSU (lane 1), (His) 6 –CrGGPPS2 and CrGPPS.SSU (lane 2), (His) 6 –CrGGPPS3 and CrGPPS.SSU(lane 3), and (His) 6 –CrGGPPS4 and CrGPPS.SSU (lane 4). Co-purified CrGPPS.SSU is indicated by an arrow.(B) In vitro prenyltransferase assay of corresponding purified enzymes (as in (A)) in the presence of [1- 3 H]–IPP and DMAPP.(C) TLC analysis of products generated by recombinant heteromeric CrGPPS (CrGGPPS1+CrGPPS.SSU) in the presence of IPP and DMAPP.(D) SDS–PAGE profile of purified CrGPPS.LSU (lane 1) and CrGPPS.SSU (lane 2) and their corresponding prenyltransferase activity with DMAPP and[1- 3 H]-IPP (E). The cpm values were deducted from the background cpm values of empty vector control reaction.(F) TLC analysis of products from in vitro assays of purified CrGPPS.LSU (1) and CrGPPS.SSU (2), respectively, in the presence of allylic substratesDMAPP and IPP.(G) TLC analysis of products from in vitro assays of purified CrGPPS.LSU in the presence (+) or absence (–) of GPP/FPP with IPP. The product formedis indicated by a dashed box, while other spots are un-reacted substrates. Reaction products were hydrolyzed to their corresponding alcohols,extracted with hexane, and separated by reverse-phase thin layer chromatography. The products were visualized by exposing the TLC plates toiodine vapor and compared with authentic standards G (geranol), F (farnesol), and GG (geranylgeranol). Assays contained recombinant 5–20 μg ofpurified proteins. E, enzyme; B, boiled enzyme. Boiled protein is used as negative control.Downloaded from http://mplant.oxfordjournals.org/ at Central Inst. Of Medicinal & Aromatic Plants (Cimap) on July 17, 2013the exact mechanism of LSU and SSU interaction. SinceCrGGPPS1 interacted with CrGPPS.SSU, we hereafter refer tothis as CrGPPS.LSU.It has been reported in heteromeric GPPS that LSU alonecould be either inactive (Burke et al., 1999) or function as abona fide GGPPS enzyme on its own (Tholl et al., 2004; Wangand Dixon, 2009). Hence, to identify the function of CrGPPS.LSU, a truncated version starting from second methionine(M-27) was expressed in E. coli as a (His) 6 -tagged protein.The assay with soluble extracts containing recombinant(His) 6 –CrGPPS.LSU proteins using [1- 3 H]-IPP and DMAPP assubstrates and Mg 2+ as cofactor detected prenyltransferaseactivity. This activity was further confirmed using purified(His) 6 –CrGPPS.LSU proteins (Figure 4D, lane 1, and Figure 4E,panel 1). Product verification of (His) 6 –CrGPPS.LSU enzymeassay using IPP and DMAPP revealed the formation of both
Rai et al. • C. roseus Geranyl Diphosphate Synthases Page 7 of 20GPP and GGPP (~2:1) indicating the bifunctional nature ofthis enzyme (Figure 4F, panel 1). The bifunctional CrGPPS.LSU could provide both GPP and GGPP pool. GPP could beused as a precursor for terpene moiety formation, and GGPPcould be used for biosynthesis of diterpenes, chlorophylls,phylloquinone, and plastoquinone (Lange and Ghassemian,2003) or in protein prenylation like geranylgeranylation(Gerber et al., 2009). As GGPPSs are known to have an abilityto use other allylic diphosphates such as GPP and FPPin addition to DMAPP (Ogura and Koyama, 1998; Takayaet al., 2003; Schmidt et al., 2010), enzyme assays and productverification showed that CrGPPS.LSU produced GGPPby utilizing both GPP and FPP in the presence of IPP, suggestingthat reaction proceeds by sequential incorporationof IPP units (Figure 4G). The GGPPS activity of CrGPPS.LSUwas further confirmed by in vivo genetic complementation(Figure 5). The transformants carrying pACCAR25∆crtE andhuman GGPPS (HsGGPPS, positive control) are expectedto accumulate yellow pigmentation, confirming the substitutionof crtE, whereas cells harboring pACCAR25∆crtEand CrGPPS.SSU/pET28a empty vector were used as negativecontrols. Cotransformation of CrGPPS.LSU (which isthe same as CrGGPPS reported by Thabet et al., 2012) withpACCAR25∆crtE resulted in accumulation of yellow pigmentation(Figure 5B), confirming the in vitro assay data thatCrGPPS.LSU has a GGPPS activity. Although, M. piperitaheteromeric GPPS produced both GPP and GGPP in in vitroassays, the genetic complementation in E. coli did not showany detectable GGPPS activity (Chang et al., 2010).In contrast to CrGPPS.LSU forming GPP and GGPP, the LSU ofA. majus GPPS produced only GGPP (Tholl et al., 2004; Orlovaet al., 2009) whereas LSU from H. lupulus GPPS formed GPP, FPP,and GGPP (Wang and Dixon, 2009). It is known that most shortchainprenyltransferases produce only a single main product(GPP or FPP or GGPP) but some homomeric prenyltransferasesfound in Zea mays and P. abies, and heteromeric GPPS fromH. lupus and M. Piperita, make products with one more or onefewer C 5 units than the main product (Cervantes-Cervanteset al., 2006; Schmidt and Gershenzon, 2008; Wang and Dixon,2009; Chang et al., 2010). Unlike other bifunctional enzymesstated above, CrGPPS.LSU is functionally similar to a recentlyreported phylogenetically distant gymnosperm PaIDS1 fromP. abies, which showed bifunctional activity forming productsGPP and GGPP (9:1 ratio) that differ from each other in sizeby more than C 5 unit (Schmidt et al., 2010). In this context,PaIDS1 and CrGPPS.LSU belong to an unusual class of shortchainprenyltransferases that make two major products witha difference of more than C 5 unit (Schmidt et al., 2010). Thisis the first report demonstrating the ‘catalytic promiscuity’of an angiosperm prenyltransferase where the enzyme hasthe ability to catalyze an adventitious secondary activity(GPPS) at the active site responsible for the primary activity(GGPPS) (Copley, 2003; Schmidt et al., 2010). This ‘catalyticpromiscuity’ of CrGPPS.LSU suggests its possible involvement inprimary as well as secondary metabolism during the course ofgrowth and development of C. roseus. The overexpression ofsnapdragon GPPS.SSU changed the chain length specificityof endogenous GGPPSs into GPPS leading to enhancedDownloaded from http://mplant.oxfordjournals.org/ at Central Inst. Of Medicinal & Aromatic Plants (Cimap) on July 17, 2013Figure 5. In Vivo Genetic Complementation Assay for Detecting GGPPS Activity.(A) Schematic diagram for the carotenoid biosynthesis pathway, beginning with a coupled reaction of two C 20 –GGPP molecules and the constructpACCAR25ΔcrtE.(B) Genetic complementation assay for detecting yellow pigment production in E. coli harboring pACCAR25ΔcrtE and CrGPPSs expression vectors.Human GGPPS1 (pHsGGPPS1) and empty pET28a vector were used as positive and negative controls, respectively.
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