Isolation and Functional Analysis of Homogentisate Phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis

Isolation and Functional Analysis of HomogentisatePhytyltransferase from Synechocystis sp. PCC 6803and Arabidopsis1

Eva Collakova and Dean DellaPenna*

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing,Michigan 48824

Tocopherols, collectively known as vitamin E, are lipid-soluble antioxidants synthesized exclusively by photosyntheticorganisms and are required components of mammalian diets. The committed step in tocopherol biosynthesis involvescondensation of homogentisic acid and phytyl diphosphate (PDP) catalyzed by a membrane-bound homogentisate phytyl-transferase (HPT). HPTs were identified from Synechocystis sp. PCC 6803 and Arabidopsis based on their sequence similarityto chlorophyll synthases, which utilize PDP in a similar prenylation reaction. HPTs from both organisms used homogentisicacid and PDP as their preferred substrates in vitro but only Synechocystis sp. PCC 6803 HPT was active with geranylgeranyldiphosphate as a substrate. Neither enzyme could utilize solanesyl diphosphate, the prenyl substrate for plastoquinone-9synthesis. In addition, disruption of Synechocystis sp. PCC 6803 HPT function causes an absence of tocopherols withoutaffecting plastoquinone-9 levels, indicating that separate polyprenyltransferases exist for tocopherol and plastoquinonesynthesis in Synechocystis sp. PCC 6803. It is surprising that the absence of tocopherols in this mutant had no discernibleeffect on cell growth and photosynthesis.

Tocopherols are a group of amphipathic com-pounds synthesized only by photosynthetic organ-isms. The best characterized and probably most im-portant function of tocopherols is to act as recyclablechain reaction terminators of polyunsaturated fattyacid free radicals generated by lipid oxidation. Toco-pherols have a well-documented role in mammalsboth as an essential nutrient (vitamin E) and generalantioxidant (Fryer, 1993; Liebler, 1998; Brigelius-Flohe and Traber, 1999). A similar though less well-documented antioxidant role is also proposed fortocopherols in photosynthetic organisms (Fryer,1992; Niyogi, 1999).

From a biosynthetic perspective, tocopherols aremembers of a large, multifunctional family of lipid-soluble compounds called prenylquinones that alsoinclude tocotrienols, plastoquinones, and phylloqui-nones (vitamin K1). Structural features shared by allprenylquinones include hydrophobic prenyl tails ofvarious lengths attached to aromatic head groupsthat can reversibly change their redox states. Toco-pherols contain a chromanol head-group and li-pophillic tail derived from the 20-carbon alcohol phy-tol, whereas plastoquinones contain a quinone headgroup and isoprenoid tails of 40, 45, or 50 carbons.Such structural features are essential for the diverse

biochemical and physiological roles fulfilled by var-ious prenylquinones.

The committed step in the synthesis of all pre-nylquinones is the condensation of various aromaticprecursors and prenyl-diphosphate (DP) substratesin reactions catalyzed by a small family of relatedpolyprenyltransferases (Lopez et al., 1996). Most ar-omatic and prenyl-DP substrates are utilized by morethan one polyprenyltransferase (Fig. 1). For example,the aromatic compound homogentisic acid (HGA) isused for condensation with phytyl DP (PDP), gera-nylgeranyl DP (GGDP), or solanesyl DP (SDP) intocopherol, tocotrienol, and plastoquinone synthesis,respectively, whereas PDP is used as the isoprenoid-derived tail in the synthesis of tocopherols, phyllo-quinones, and chlorophylls (Threlfall and Whistance,1971; Schulze-Siebert et al., 1987; Oster et al., 1997).Thus, polyprenyltransferases act at biosyntheticbranch points and are potential key regulatory en-zymes for the synthesis of many essential com-pounds in photosynthetic organisms.

In plant chloroplasts, the synthesis of tocopherolsand plastoquinones is closely related. Biochemicalstudies have shown that condensation of HGA withPDP or SDP yields 2-methyl-6-phytyl-1,4-benzoquinol(2-Me-6-Ph-1,4-BQ) and 2-demethylplastoquinol-9,the first prenylquinol intermediates in tocopherol andplastoquinone-9 (PQ-9) synthesis, respectively (Hut-son and Threlfall, 1980; Soll et al., 1980; Marshall et al.,1985). Although these studies could not distinguishwhether one or more polyprenyltransferases catalyzedthese reactions, it was suggested that separate en-zymes might be involved (Schulze-Siebert et al., 1987).

1 This work was supported by a grant from Pioneer Hi-Bred, Inc.* Corresponding author; e-mail [email protected]; fax 517–

353–9334.Article, publication date, and citation information can be found

at www.plantphysiol.org/cgi/doi/10.1104/pp.010421.

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In contrast, recent genetic data from Arabidopsissuggested involvement of a single polyprenyltrans-ferase activity in tocopherol and PQ-9 synthesis. Twoloci were identified, PDS1 and PDS2 (phytoene de-saturation), which when mutated, decreased the lev-els of both tocopherols and plastoquinones belowdetection (Norris et al., 1995), consistent with thedisruption of enzymes shared in their synthesis. ThePDS1 locus has been cloned and encodes p-hydroxy-phenylpyruvate dioxygenase (HPPD; Norris et al.,1998), which catalyzes formation of HGA. The pds2mutation was proposed to disrupt another sharedpathway enzyme, most likely a polyprenyltrans-ferase, which could utilize either PDP or SDP assubstrates for condensation with HGA (Norris et al.,1995). Unlike PDS1, the PDS2 locus has not yet beencloned.

As an alternative to purifying the membrane-boundPDS2 gene product or walking to the PDS2 locus, weattempted to clone an orthologous gene from the cya-nobacterium Synechocystis sp. PCC 6803, which alsosynthesizes �-tocopherol. In this paper, we report thecloning and functional analysis of gene products fromSynechocystis sp. PCC 6803 and Arabidopsis encodingpolyprenyltransferases specific to tocopherol biosyn-thesis. We also present biochemical and physiologicalcharacterization of the corresponding Synechocystis sp.PCC 6803 polyprenyltransferase knockout mutant,which completely lacks tocopherols.

RESULTS

Identification and Disruption of aPolyprenyltransferase Involved in TocopherolBiosynthesis in Synechocystis sp. PCC 6803

Due to the metabolic synteny observed for the pre-nyllipid biosynthetic pathways in photosynthetic or-ganisms, we decided to utilize a genomics-basedapproach to identify the gene encoding the homogen-tisate polyprenyltransferase involved in tocopherolsynthesis, first from cyanobacteria, and subsequentlyfrom plants. We hypothesized that this polyprenyl-transferase would show some similarity to previouslycharacterized polyprenyltransferases from cyanobac-teria and plants that utilize similar prenyl-DPs assubstrates.

Chlorophyll synthase is a polyprenyltransferasethat attaches PDP or GGDP to the tetrapyrrole core ofchlorophyllide during chlorophyll biosynthesis(Lopez et al., 1996; Oster et al., 1997). The Synecho-cystis sp. PCC 6803 chlorophyll synthase open read-ing frame (ORF; ChlG, GenBank accession no.BAA10281) was used to query CyanoBase, whichcontains the complete Synechocystis sp. PCC 6803 ge-nome sequence (Kaneko et al., 1996). Several ORFsshowing varying degrees of similarity were identi-fied and SLR1736 was selected as a putative HPTbased on its protein identity to ChlG (21%) and thepresence of prenyl-DP- and divalent cation-binding

motifs characteristic of polyprenyltransferases (Lopezet al., 1996; Fig. 2A). SLR1736 is also a highly hydro-phobic protein (Fig. 2B), as would be expected for amembrane-bound HPT (Soll et al., 1980, 1984).

To test the hypothesis that SLR1736 is involved intocopherol biosynthesis, a disruption mutant(SLR1736::Kmr) was generated by homologous recom-bination of the kanamycin cassette-disrupted SLR1736gene into the wild-type SLR1736 locus (Fig. 3). If theSLR1736::Kmr mutation disrupted HPT activity, onewould expect a complete absence of tocopherols andtheir prenylquinol intermediates. HPLC analysisshows that wild-type Synechocystis sp. PCC 6803 lipidextracts contain predominantly �-tocopherol (Fig. 4,Table I). In contrast, �-tocopherol and its prenylchro-manol and quinol precursors are absent fromSLR1736::Kmr lipid extracts (Fig. 4, Table I, and datanot shown), consistent with the hypothesis thatSLR1736 encodes a polyprenyltransferase involved intocopherol synthesis.

As shown in Figure 1, the various polyprenyltrans-ferases in photosynthetic organisms utilize many ofthe same aromatic and prenyl-DP substrates. HGA isthe aromatic precursor in both tocopherol and plas-toquinone synthesis and PDP is a substrate for to-copherol, phylloquinone, and chlorophyll polypre-nyltransferases (Threlfall and Whistance, 1971;Schulze-Siebert et al., 1987; Oster et al., 1997). Giventhis biosynthetic relationship, disrupting SLR1736 ac-tivity could directly or indirectly affect the synthesisof other prenylated compounds in pathways that alsoutilize these substrates. To determine the effect of theSLR1736 gene disruption on the synthesis of otherprenylated compounds, we analyzed plastoquinone,phylloquinone, and chlorophyll levels in theSLR1736::Kmr mutant relative to wild type. No sig-nificant differences were observed in the levels ofthese compounds (Table I).

Biochemical Characterization of the SLR1736Gene Product

The SLR1736::Kmr phenotype strongly suggeststhat SLR1736 encodes a polyprenyltransferase spe-cific to tocopherol synthesis. To determine the activ-ity and substrate specificity of the SLR1736 geneproduct, HGA polyprenyltransferase assays wereperformed using SLR1736 protein expressed in Esch-erichia coli. These assays are based on thin-layer chro-matography (TLC) separation and subsequent auto-radiography or HPLC separation of prenylatedquinones formed from radioactive HGA and variousunlabeled prenyl-DPs in the presence of a putativepolyprenyltransferase.

When various prenyl-DPs at the same molar con-centrations were tested as potential substrates for theSLR1736 protein, PDP was used most efficiently,though GGDP could also be utilized (Figs. 5 and 6, Band D). The amount of geranylgeranylated benzoqui-

Collakova and DellaPenna

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none product formed was approximately 18% that ofthe phytylated product. No products were observedwhen SDP, the prenyl-DP substrate for PQ-9 synthe-sis, was used (Fig. 5). In the case of PDP, the mainreaction product comigrated with 2�-trans-2-Me-6-Ph-1,4-BQ in both TLC and HPLC analyses (Figs. 5and 6B).

We also observed a couple of minor products onTLC and HPLC (Figs. 5 and 6). On TLC, a band (Fig.5, lane 6, RF approximately 0.3) likely corresponds tothe quinol form of 2-Me-6-Ph-1,4-BQ because inten-sity of this band increases when the samples are notoxidized with AgO prior to TLC (data not shown). Asmall radioactive peak eluting before the major 2�-trans-2-Me-6-Ph-1,4-BQ peak was also observed inHPLC analysis (Fig. 6B). This peak probably corre-sponds to 2�-cis-2-Me-6-Ph-1,4-BQ formed by isomer-

ization of the trans-isomer as previously reported(Hutson and Threlfall, 1980; Henry et al., 1987). It isunlikely that this peak represents the correspondingquinol because quinols are eluted much later thanquinones in the HPLC system used. Due to their lowabundance, further analyses of these minor peakscould not be performed.

When GGDP was used as a substrate the SLR1736enzyme also produced a major and minor product.It is unfortunate that 2-methyl-6-geranylgeranyl-1,4-benzoquinone (2-Me-6-GG-1,4-BQ), the expectedproduct of HGA and GGDP condensation, was notavailable. However, indirect evidence suggests thatthe major GGDP reaction product is 2-Me-6-GG-1,4-BQ. First, consistent with previous reports (Soll andSchultz, 1979; Hutson and Threlfall, 1980), this prod-uct migrates slightly slower than its phytylated coun-

Figure 1. Generalized overview of prenylquinone biosynthetic pathways in photosynthetic organisms. Prenylation steps andsubstrates in tocopherol and plastoquinone synthesis are shown in detail, whereas those for other prenyllipids areincomplete for clarity. Aromatic and prenyl-DP substrates are shared among the various polyprenyltransferases (see text fordetails). Enzymes are depicted as numbers in black circles: 1, homogentisate phytyltransferase (HPT); 2, homogentisatesolanesyltransferase; 3, chlorophyll synthase; 4, 1,4-dihydroxy-2-naphthoate phytyltransferase; 5, GGDP reductase; 6,p-HPPD; and 7, SDP synthase. Compounds in parentheses indicate where GGDP may be used in place of PDP by HPTresulting in a tocotrienol product.

Synechocystis and Arabidopsis Homogentisate Phytyltransferases

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terpart in the TLC system used (Compare lane 6 withlane 7, Fig. 5). Moreover, the chromatographic prop-erties of the major product in normal-phase HPLCare consistent with those previously published for2-Me-6-GG-1,4-BQ (Hutson and Threlfall, 1980). Tobe specific, it elutes 9 min after the internal control2�-trans-2-Me-6-Ph-1,4-BQ (Fig. 6C), which is in goodagreement with the previously reported relative elu-tion difference between 2-Me-6-Ph-1,4-BQ and 2-Me-6-GG-1,4-BQ (Hutson and Threlfall, 1980). As withPDP, an unknown minor GGDP product migratingprior to the major peak is also present in HPLCanalysis. It is unfortunate that neither GGPD prod-ucts are produced in sufficient quantity to allowfurther analytical characterization. Based on the com-bined results of these polyprenyltransferase assays

and the tocopherol-specific phenotype of theSLR1736::Kmr mutant, the protein encoded by theSLR1736 gene was named SynHPT, for Synechocystissp. PCC 6803 HPT.

Identification and Characterization of an HPT Homologfrom Arabidopsis

To identify an HPT homolog from plants, we usedthe SynHPT protein sequence as a database query forBLAST searches (Altschul et al., 1990). A single pre-dicted Arabidopsis gene on chromosome 2 (bacteriaartificial chromosome clone F19F24) containing re-gions of significant similarity to SynHPT was identi-fied. The corresponding cDNA subsequently was iso-lated from an Arabidopsis seed cDNA library and

Figure 2. A, Alignment of Synechocystis sp. PCC 6803 and Arabidopsis polyprenyltransferases. HPTs from Synechocystis sp.PCC 6803 (SynHPT, GenBank accession no. S74813) and Arabidopsis (AtHPT, accession no. AF324344) share 41% proteinidentity, whereas SynHPT and ChlG (Synechocystis sp. PCC 6803 chlorophyll synthase, accession no. BAA10281) share22% protein identity. Residues conserved in at least two of three sequences are shaded in gray, whereas residues identicalin all three proteins are labeled by black dots. The conserved prenyl-DP and divalent cation binding domains are indicatedby dashed and black boxes, respectively. The predicted AtHPT chloroplast-targeting domain cleavage site is indicated by ablack arrow. B, Kyte/Doolittle hydrophillicity profiles of AtHPT and SynHPT. The two profiles are nearly identical. Negativevalues indicate hydrophobicity.





fully sequenced. The predicted protein encoded bythis cDNA (GenBank accession no. AF324344) shares41% identity with SynHPT. In addition, both proteinshave remarkably similar hydrophobicity profiles andcontain prenyl-DP and divalent cation binding motifsconserved in both location and sequence (Fig. 2). TheArabidopsis protein also contains an additional 95-amino acid N-terminal extension that is not presentin SynHPT. The first 36 amino acids of this domainexhibit features of a chloroplast targeting sequence(Emanuelsson et al., 1999), consistent with the re-ported chloroplast envelope localization of HPT ac-tivity in plants (Soll et al., 1980, 1984). The Arabidop-sis protein was tentatively named AtHPT forArabidopsis HPT.

To determine the activity and substrate specificityof the putative AtHPT and compare it with SynHPT,AtHPT was expressed in E. coli and HGA polypre-nyltransferase assays were performed. Like SynHPT,AtHPT catalyzed condensation of HGA and PDP toform 2�-trans-2-Me-6-Ph-1,4-BQ as a major productand was not active with the substrates HGA andSDP. Unlike SynHPT, no products were observedwhen HGA and GGDP were used as substrates (Figs.5 and 6D). To test whether this difference betweenthe two enzymes was due to the presence of

chloroplast-targeting sequences in AtHPT, we alsotested two truncated versions of the protein. Onetruncation removed the predicted 36-amino acidchloroplast transit peptide, whereas the second re-moved 95 N-terminal amino acids not present inSynHPT. Neither truncation altered the specific ac-tivity or substrate specificity of AtHPT (results notshown).

The specific activity of AtHPT expressed in E. coliwas approximately 3% that of SynHPT expressedfrom the same vector. Several explanations are plau-sible for this difference, including decreased proteinstability, poor protein expression in E. coli due tocodon bias, or an improper lipid environment rela-tive to that of chloroplasts. Neither AtHPT nor Syn-HPT could be visualized on Coomassie Blue-stainedgels following induction, indicating both are ex-pressed at low levels in E. coli. Addition of lipidsextracted from Arabidopsis leaves or seeds to reac-tions had no discernible effect on AtHPT activity(data not shown). Finally, addition of Tween 80 orCHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid} to final concentrations of0.2%, 0.5%, 1%, or 2% (w/v) moderately stimulatedboth AtHPT and SynHPT activities without appre-ciably changing their specific activities relative toeach other (data not shown). It appears that a com-bination of lower expression and/or lower stabilityof AtHPT relative to SynHPT may be the cause oflimited AtHPT activity in E. coli.

Physiological Consequences of Tocopherol Deficiencyin Synechocystis sp. PCC 6803

Given the importance of tocopherols in free radicalscavenging, a photosynthetic organism lacking toco-

Figure 4. HPLC traces of tocopherol standards and lipids extractedfrom wild-type Synechocystis sp. PCC 6803 and SLR1736::Kmr.Equivalent weights of fresh cells were extracted for the analysisshown. Tocopherol analysis was performed on a normal phase col-umn using 8% (v/v) di-isopropyl ether in hexane as a solvent. A,Separation of �-, �-, �-, and �-tocopherol (Toc, tocopherol) and tocolstandards. B, Wild-type cells accumulate predominantly�-tocopherol (gray trace). No tocopherols were detected in theSLR1736::Kmr mutant (black trace). Tocol was used as an internalstandard.

Figure 3. Construction of the Synechocystis sp. PCC 6803SLR1736::Kmr mutant. A, Simplified scheme of the wild-typeSLR1736 ORF in the Synechocystis sp. PCC 6803 genome. Insertionof a kanamycin (Kmr) cassette into MfeI site of the SLR1736 ORF andthe SLR1736F and R PCR primers (F and R, small arrows) are indi-cated. B, Autoradiograph of the PCR products amplified from wildtype (lane 1) and the SLR1736::Kmr mutant (lane 2) genomic DNA.No wild-type copies of SLR1736 were detected in the SLR1736::Kmr

mutant.





pherols might be expected to be compromised ingrowth or exhibit increased sensitivity to high lightstress. To address this question, we comparedgrowth of the tocopherol deficient SLR1736::Kmr mu-tant and wild-type Synechocystis sp. PCC 6803 underlow-light or high-light conditions (approximately 30and 110 �E m�2 s�1, respectively). It is surprisingthat the doubling times of both strains under photo-autotrophic or heterotrophic conditions in both lowand high light were comparable (Table II and datanot shown). Whole-chain oxygen evolution mea-sured at 0.75, 2, and 5 mE m�2 s�1 was also found tobe similar in both strains, indicating that the initialrates of photosynthesis in SLR1736::Kmr and wildtype are comparable (Table II).

DISCUSSION

Due to the conserved evolution of photosyntheticorganisms, many biosynthetic pathways in cyanobac-teria and plants are often quite similar (Whistance andThrelfall, 1970; Marechal et al., 1997). The rapidgrowth of expressed sequence tag and genome data-bases in a wide variety of organisms allows plantbiochemists to utilize such inter-kingdom conserva-tion in their research more effectively. In particular,the availability of the fully sequenced Synechocystis sp.PCC 6803 genome in a searchable online database,CyanoBase (Kaneko et al., 1996), coupled withstraightforward gene disruption methods for analysisof gene function in this organism (Williams, 1988)makes Synechocystis sp. PCC 6803 an attractive systemto complement studies of tocopherol synthesis inplants. Two tocopherol biosynthetic enzymes, GGDPreductase (Addlesee et al., 1996; Keller et al., 1998) and�-tocopherol methyltransferase (Shintani and Della-Penna, 1998), have already been cloned and character-ized from Synechocystis sp. PCC 6803 and usedsuccessfully as database probes to identify orthologsfrom Arabidopsis. The �-tocopherol methyltrans-ferases from both organisms were shown to havenearly identical activities (Shintani and DellaPenna,1998). We have employed a similar genomics-basedapproach to identify and characterize a third enzymeof the tocopherol pathway from Synechocystis sp. PCC

6803 and Arabidopsis, HGA phytyltransferase, andassess whether this step in tocopherol synthesis is alsoconserved between cyanobacteria and plants.

In photosynthetic organisms, condensation of HGAwith either a 20- or 45-carbon prenyl-DP is the branchpoint in tocopherol and plastoquinone synthesis, re-spectively. Early biochemical studies established thatthe tocopherol and plastoquinone pathways are re-markably similar in oxygenic cyanobacteria, algae,and plants (Whistance and Threlfall, 1970). Althoughthese studies could not distinguish separate polypre-nyltransferase activities for tocopherol and plasto-quinone synthesis, it was suggested that separateprenylation enzymes might be involved (Schulze-Siebert et al., 1987). Genetic analysis of the pathwaysin Arabidopsis more recently identified two lociwhose mutant phenotypes are consistent with thedisruption of enzymes shared in the synthesis oftocopherols and plastoquinones (Norris et al., 1995).This was found to be the case for the PDS1 locus,which encodes HPPD, the enzyme that produces thearomatic head group HGA in both the plastoquinoneand tocopherol pathways (Norris et al., 1998). ThePDS2 locus was suggested to encode a similarlyshared polyprenyltransferase that could utilize eitherPDP or SDP for tocopherol and plastoquinone syn-thesis, respectively (Norris et al., 1995). The cloningof plant and cyanobacterial HPTs now allows us todirectly address the nature of polyprenyltransferasesinvolved in tocopherol and plastoquinone synthesisin oxygenic photosynthetic organisms.

The Synechocystis sp. PCC 6803 SLR1736::Kmr mu-tant lacks tocopherols but accumulates wild-type lev-

Figure 5. Homogentisate polyprenyltransferase assays. Individual re-actions contained the indicated prenyl-DP and protein extracts fromE. coli expressing empty vector or the indicated phytyltransferases.Radiolabeled prenylquinol reaction products were extracted, oxi-dized to corresponding quinones, separated by TLC, and subjected toautoradiography. SynHPT can utilize both PDP and GGDP asprenyl-DP substrates (lanes 6 and 7), whereas AtHPT can only usePDP (lane 10). Neither enzyme could catalyze condensation of HGAand SDP (lanes 8 and 12). No prenylquinone products were detectedin control reactions (lanes 1–5 and 9). The arrow indicates the origin.

Table I. Prenyllipid contents of photoautotrophically grown wild-type Synechocystis sp. PCC 6803 and the SLR1736::Kmr mutant

Other than the absence of tocopherols in the SLR1736::Kmr mu-tant, no significant differences were observed between wild-type andmutant cells for the presented parameters. Each value is the mean �SD of at least five separate measurements per experiment. Eachexperiment was repeated at least three times.

Prenyllipid Wild Type SLR1736::Kmr

�-Tocopherola 18.7 � 1.4 Not detectedPlastoquinone-9a 31.6 � 4.3 28.4 � 1.7Phylloquinonea 13.8 � 1.1 12.5 � 1.2Chlorophyll ab 4.3 � 0.3 4.6 � 0.4

a mmol mol�1 chlorophyll a. b mmol mg�1 cells.





els of PQ-9, consistent with the existence of separateHGA polyprenyltransferases in tocopherol and plas-toquinone synthesis. This conclusion is also sup-

ported by enzymatic studies showing that SynHPTcan utilize the 20-carbon tocopherol substrates, PDPor GGDP, but is inactive with the 45-carbon PQ-9substrate, SDP. Thus, it appears that Synechocystis sp.PCC 6803 contains a single polyprenyltransferasespecific to tocopherol synthesis, SynHPT, and a sep-arate, yet-to-be characterized polyprenyltransferasespecific to plastoquinone synthesis.

In Arabidopsis, the prenyltransferase reaction in-volving HGA and PDP substrates appears to benearly identical to that in Synechocystis sp. PCC 6803.The AtHPT and SynHPT enzymes share 61% proteinsimilarity and both enzymes use PDP as their pre-ferred prenyl-DP substrate in vitro to form 2�-trans-2-Me-6-Ph-1,4-BQ as a major product. This is inagreement with previous biochemical studies of HPTactivity in isolated spinach, lettuce, and pea chloro-plasts where 2-Me-6-Ph-1,4-BQ was the only productdetected (Hutson and Threlfall, 1980; Soll et al., 1980;Marshall et al., 1985). Like SynHPT, AtHPT did notgenerate detectable prenylquinone products withSDP as a substrate, suggesting Arabidopsis likelycontains separate polyprenyltransferases for tocoph-erol and plastoquinone synthesis. An ArabidopsisHPT knockout mutant is needed to rigorously ad-dress this question.

In considering the nature of homogentisate poly-prenyltransferase reactions in plants, it is importantto note that our original goal of cloning the Arabi-dopsis PDS2 locus has not been achieved. Tocopheroland plastoquinone levels are reduced below detec-tion in pds2, leading to the hypothesis that PDS2encodes a polyprenyltransferase shared in tocoph-erol and plastoquinone synthesis (Norris et al., 1995).However, given that AtHPT is a phytyltransferaseencoded by a single-copy gene on chromosome 2,whereas PDS2 maps to chromosome 3, this proposalnow seems unlikely. If PDS2 is not a polyprenyl-transferase shared in tocopherol and plastoquinonesynthesis, what does it encode? One explanation isthat PDS2 encodes an enzyme specific to plastoqui-none synthesis (i.e. HGA solanesyltransferase or SDP

Table II. Growth and O2 evolution rates of wild-typeSynechocystis sp. PCC 6803 and SLR1736::Kmr

Doubling times and photosynthetic activity of wild-type and mu-tant cells are similar. Each value is the mean � SD of three indepen-dent measurements in a representative experiment. Each experimentwas repeated at least three times.

Parameter Wild Type SLR1736::Kmr

Growth, doubling time (h)a

Photoautotrophic 22 � 3 19 � 3Photoheterotrophic 12 � 1 13 � 1

Oxygen evolution ratesb

Whole chain 156 � 6 163 � 8a The cells were grown at high light (105–110 �E m�2

s�1). b The cells were grown photoautotrophically; O2 evolutionrates are in mmol O2 mg�1 chlorophyll h�1 and measured at 5 mEm�2 s�1.

Figure 6. Normal phase HPLC separation of radiolabeled prenylqui-nones produced from HGA and prenyl-DP substrates by HPTs. Ho-mogentisate polyprenyltransferase reactions were performed in atotal volume of 0.5 mL for SynHPT with PDP (B) and 5 mL forSynHPT with GGDP, and AtHPT with PDP or GGDP (C–E, respec-tively) as described in “Materials and Methods.” Elution of the inter-nal standard 2�-trans-2-Me-6-Ph-1,4-BQ was monitored at 252 nm,whereas that of the prenylquinones formed during the assay wasmonitored by scintillation counting of collected fractions. The UVtraces are not shown for B through D for clarity, but when alignedwith the radioelution profiles shown, the major radiolabeled phyty-lated products co-chromatographed with authentic 2�-trans-2-Me-6-Ph-1,4-BQ standard (indicated by arrows). A, Elution of 2�-trans-2-Me-6-Ph-1,4-BQ; B and C, SynHPT and AtHPT catalyzed formationof 2�-trans-2-Me-6-Ph-1,4-BQ from HGA and PDP; D, SynHPT cat-alyzed formation of 2-Me-6-GG-1,4-BQ from HGA and GGDP (cir-cles). AtHPT did not produce a product with HGA and GGDP assubstrates (triangles).





synthase) and that the absence of tocopherols in pds2is a pleiotropic effect of this mutation. In this sce-nario, the absence of plastoquinone, the main lipidsoluble electron carrier in plastids, results in suchhigh levels of oxidative stress in pds2 that any toco-pherols produced are rapidly oxidized and de-graded, and hence undetectable. In an alternate man-ner, plastoquinone may be a cofactor required for thesynthesis of tocopherols and its absence arrests to-copherol synthesis. Regardless of mechanism, it ap-pears likely that the tocopherol deficiency in pds2 isan indirect, rather than a direct effect of the pds2mutation.

Though SynHPT and AtHPT are similar in theirsubstrate specificities, there is one notable exception:SynHPT can use both PDP and GGDP as substrates,whereas AtHPT only uses PDP. The utilization ofboth PDP and GGDP as substrates by a polyprenyl-transferase is not unprecedented. An analogous re-action occurs in chlorophyll biosynthesis where chlo-rophyll synthase can attach either PDP or GGDP tothe tetrapyrrole moiety, and in cyanobacteria, PDP isthe preferred substrate (Oster et al., 1997). We ob-served a similarly strong preference of SynHPT forPDP over GGDP. The use of GGDP by SynHPT invivo would yield tocotrienol intermediates and endproducts that only differ from their tocopherol coun-terparts in having an unsaturated rather than satu-rated hydrophobic tail. This would necessitate sub-sequent enzymes in the pathway being active towardgeranylgeranylated substrates. At least one other to-copherol biosynthetic enzyme from cyanobacteriahas been shown to utilize both phytylated and gera-nylgeranylated intermediates in vitro, tocopherol cy-clase from Anabaena variabilis (Stocker et al., 1996).However, Synechocystis sp. PCC 6803 does not accu-

mulate tocotrienols, suggesting that any geranylgera-nylated intermediate produced by SynHPT is eitherefficiently reduced (most likely by GGDP reductase),or that GGDP is not a substrate in vivo. Additionalwork is required to delineate the in vivo substrate(s)and product(s) of SynHPT.

Within the limits of our assay sensitivity (approx-imately 3% of PDP product levels), AtHPT did notutilize GGDP as a substrate. Other researchers alsofailed to demonstrate condensation of HGA andGGDP using isolated spinach, lettuce, and pea chlo-roplasts (Hutson and Threlfall, 1980; Soll et al., 1980).These data are consistent with the general observa-tion that dicots do not produce tocotrienols (Piironenet al., 1986; Franzen and Haas, 1991). However, manymonocots and gymnosperms do produce both toco-pherols and tocotrienols (Piironen et al., 1986; Fran-zen and Haas, 1991; Franzen et al., 1991) and wespeculate that HPTs from such organisms would uti-lize GGDP and PDP as substrates, analogous to Syn-HPT. Phylogenetic analysis of polyprenyltrans-ferases from various photosynthetic organismsshows that HPTs from cyanobacteria, monocots, anddicots form separate groups (Fig. 7), which probablyrepresents taxonomic differences, although it may inpart reflect differences in the substrate specificities ofthese enzymes. As with Synechocystis sp. PCC 6803,we would also anticipate that pathway enzymes afterHPT in monocots would be active toward both gera-nylgeranylated and phytylated intermediates. Thesubstrate specificity of tocopherol biosynthetic en-zymes from monocots has not been characterized;however, in spinach (a dicot), where enzymology ofthe pathway has been best studied, later methyltrans-ferases of the pathway are active toward variousgeranylgeranyl intermediates (Soll and Schultz,

Figure 7. Phylogenetic analysis of various prenyllipid polyprenyltransferases. Sequence alignment and phylogenetic anal-ysis were performed using MacVector software (Genetic Computer Group, Madison, WI). Numbers indicate distancesbetween protein sequences estimated by the uncorrected p distance method. Numbers in parentheses indicate percentagesof the tree confidence calculated by bootstrapping. Chlorophyll synthases form a separate clade from HPTs. Within HPTs,cyanobacterial, monocot, and dicot HPTs also form distinct subgroups.





1979). It appears that at some point in evolution,dicots, like monocots, could likely produce tocotrien-ols but have lost this ability as their HPTs haveevolved substrate specificity for PDP over GGDP.

Numerous studies suggest that tocopherols are im-portant antioxidants involved in photoprotection ofplants. Tocopherol levels correlate well with the de-gree of oxidative stress in numerous plant speciesgrown under various stress conditions, includinghigh light, drought, and low temperatures (Wildi andLutz, 1996; Streb et al., 1998; Bartoli et al., 1999;Havaux et al., 2000; Munne-Bosch and Alegre, 2000).Given this suggestive role of tocopherols in antioxi-dant and photoprotective function, a mutation thateliminates tocopherol synthesis in a photosyntheticorganism would be anticipated to increase sensitivityto oxidative stress, reducing growth or viability ofthis organism under stressful conditions. Variousmutants and transgenic plants with decreased to-copherol levels have been reported to exhibit photo-bleaching phenotypes and compromised growth(Henry et al., 1986; Norris et al., 1995; Tanaka et al.,1999). However, in all these cases, other prenyllipidssuch as phylloquinone, chlorophylls, carotenoids, orplastoquinone were affected in addition to toco-pherols (Henry et al., 1986; Norris et al., 1995; Tanakaet al., 1999). Therefore, it was impossible to specifi-cally attribute these phenotypes to tocopherol defi-ciency. The tocopherol-specific phenotype ofSLR1736::Kmr provides a unique tool to begin tospecifically address the question of tocopherol func-tion in photosynthetic organisms.

It is surprising that SLR1736::Kmr growth ratesunder photoautotrophic and photoheterotrophic con-ditions in low and high light were indistinguishablefrom wild type. Mutant and wild-type whole-chainoxygen evolution rates were also similar. The obser-vation that the absence of tocopherols did not appre-ciably affect growth, photosynthetic electron trans-port, and tolerance to high-light stress seeminglycontradicts the concept that tocopherols are essentiallipid soluble antioxidants. However, there are severalpossible explanations for these apparently incongru-ous results. First, �-tocopherol is not the only antiox-idant present in photosynthetic membranes. Photo-synthetic organisms have evolved multiplemechanisms for protection from oxidative stresses(carotenoids, ascorbate, superoxide dismutases, etc.)that if up-regulated, could partially or fully compen-sate for the absence of tocopherols in SLR1736::Kmr

under certain conditions. In an alternate manner,tocopherols may protect from a specific type of lipidperoxidation or at a particular site. Finally, the lim-ited oxidative treatments used in this report may notbe sufficient to produce detectable differences be-tween wild-type Synechocystis sp. PCC 6803 andSLR1736::Kmr at the level of culture growth rates. Adetailed analysis of membrane lipids, lipid peroxida-tion products, and other component of oxidative

stress compensation and adaptation are needed todiscern any effects of tocopherol deficiency inSLR1736::Kmr.

Finally, one potential application of the describedwork would be to allow engineering of elevated to-copherol levels in food crops for nutritional pur-poses. Given the central position of HPT in tocoph-erol synthesis, it seems likely that the enzyme may bean important step for controling flux into the path-way. A crucial observation is that eliminating HPTactivity in Synechocystis sp. PCC 6803 does not affectthe levels of other biosynthetically related com-pounds (plastoquinone, phylloquinone, and chloro-phylls). This suggests that altering HPT enzyme lev-els in plants may also be tocopherol specific and havelittle effect on the synthesis of other prenylquinonecompounds in the plastid. Experiments are underway to positively modify AtHPT expression in Ara-bidopsis to test whether the activity is a target forengineering tocopherol levels in plants.

MATERIALS AND METHODS

Chemicals and Bacterial Strains

Prenyl-DPs were more than 99% pure. PDP was kindlyprovided by Dr. Stephanie Sen (Purdue University, India-napolis), and GGDP and SDP were purchased from Amer-ican Radiolabeled Chemicals (St. Louis). (U-14C)-HPP (0.6–1.5 �m) was prepared from (U-14C)-Tyr (specific activity464 mCi mmol�1; Amersham, Arlington Heights, IL) asdescribed by Schulz et al. (1993). Tocol was a gift from EisaiCompany (Tokyo). A mixture of various cis- and trans-methyl-phytyl-1,4-benzoquinone isomers was synthesizedby Dr. Daniel Liebler (University of Arizona, Tucson). 2�-trans-2-Me-6-Ph-1,4-BQ was purified from the mixture by acombination of TLC and HPLC (Henry et al., 1987). PQ-9was extracted from Iris hollandica bulbs and purified byTLC (Pennock, 1985) and HPLC (Johnson et al., 2000).Wild-type Synechocystis sp. PCC 6803 was grown on BG-11plates or liquid media (Williams, 1988) either photohetero-trophically (with 15 mm Glc) or photoautotrophically(without Glc) at 20 to 30 �E m�2 s�1 and 30°C unlessotherwise stated. Synechocystis sp. PCC 6803 cells weresubcultured at least three times in liquid media prior togrowth experiments. Escherichia coli strains DH5� (Strat-agene, La Jolla, CA) and BL-21 (DE3; Novagen, Milwaukee,WI) were used for conventional subcloning and proteinexpression, respectively.

Plasmids and Mutants

Primers 5�-TATTCATATGGCAACTATCCAAGCTTTT-TG-3� (SLR1736F) and 5�-GGATCCTAATTGAAGAAGA-TACTAAATAGTTC-3� (SLR1736R) containing engineeredNdeI and BamHI sites (underlined) and Vent DNA poly-merase (New England Biolabs, Beverly, MA) were used toamplify the SLR1736 ORF (GenBank accession no.BBA17774) from Synechocystis sp. PCC 6803 genomic DNA.The amplified fragment was subcloned into the EcoRV site





of pBluescript II KS (�) to generate pKS1736. pKS1736 wasdigested with MfeI and ligated with the EcoRI-digestedkanamycin resistance cassette from pUC4K (Taylor andRose, 1988). Two constructs with opposite orientation ofthe kanamycin resistance cassette relative to the SLR1736ORF were used to transform wild-type Synechocystis sp.PCC 6803 and generate disruption mutants by homologousrecombination (Williams, 1988). Transformants were sub-cultured on kanamycin containing media for several plat-ing cycles and the absence of wild-type SLR1736 genecopies was confirmed by PCR using SLR1736F and R prim-ers followed by Southern-blot analysis (Fig. 3, A and B).Because the two orientation disruption mutants were phe-notypically indistinguishable (data not shown), that withthe Kmr cassette in the same orientation as the SLR1736ORF, referred to hereafter as the SLR1736::Kmr mutant(Fig. 3A), was used for further analyses. The NdeI-BamHIfragment from pKS1736 encoding the entire SLR1736 ORFwas ligated into NdeI-BamHI-digested pET30b (Novagen)to create pSynHPT, which was transformed into BL-21(DE3) cells for protein expression.

The SLR1736 protein sequence was used to search theArabidopsis database and identify a single genomic clone,F19F24, containing a homologous sequence. Primers 5�-TTGTTTTCAGGCTGTTGTTGCAGCTCTC-3� and 5�-CGT-TTCTGACCCAGAGTTACAGAGAATG-3� were used toamplify a 977-bp fragment from F19F24 for use as a probeto screen an Arabidopsis seed cDNA library (a gift of Dr.John Ohlrogge, Michigan State University, East Lansing).The longest of 15 positive clones was sequenced and shownto encode a protein similar to SLR1736 that was designatedAtHPT. For protein expression purposes the full-lengthclone encoding AtHPT was amplified using primers (5�-CCATGGAGTCTCTGCTCTC-3� and 5�-GGATCCCAAG-CAGAGACTTCTTTACC-3�) and subcloned into NcoI-BamHI-digested pET3d vector (Novagen) to generatepAtHPT.

Prenyllipid Analysis

Fifteen to 20 mg of 14-d-old plate-grown Synechocystissp. PCC 6803 cells were harvested, their lipids extracted(Bligh and Dyer, 1959), and dissolved in 100 �L of hexaneor ethyl acetate. Ten microliters of each sample was with-drawn for chlorophyll determination (Lichtenthaler, 1987),whereas 50 �L was subjected to HPLC (Hewlett-Packard1100, Wilmington, DE) on a LiChrosorb 5 Si60A 4.6- �250-mm normal phase column (Phenomenex, Torrance,CA) at 42°C as described by Syvaoja et al. (1986). Toco-pherols were detected by fluorescence using 290 nm exci-tation and 325 nm emission. For plastoquinone and phyl-loquinone analysis, separation was achieved on a reversephase column (Spherisorb 5 ODS2 4.6 � 250 mm, Waters,Marlborough, MA) as described by Johnson et al. (2000).PQ-9 and phylloquinone were detected at 250 and 275 nm,respectively.

Homogentisate Polyprenyltransferase Assay

Each 0.2-mL reaction contained freshly prepared (U-14C)-HPP (approximately 0.2 �m, specific activity 464 mCimmol�1), 50 mm HEPES [4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid], pH 7.6, 4 mm MgCl2, 50 mm potas-sium ascorbate, 100 �m KF, 0.2% (w/v) CHAPS, and 0.1 mgof total protein extracted from E. coli expressing HPPD(Norris et al., 1995). Individual reactions contained 100 �mPDP, GGDP, or SDP and the insoluble protein fraction fromE. coli expressing pSynHPT (0.03 mg of protein), pAtHPT (1mg of protein), or the empty pET vector (0.03 or 1 mg ofprotein). Reactions were incubated for 1 h at room temper-ature, extracted with two volumes of methanol:chloroform(1:1, v/v), and any newly formed prenylquinols were oxi-dized with AgO (Pennock, 1985). The organic phase wastransferred to a fresh tube, evaporated to dryness, dis-solved in ethyl acetate, and subjected to TLC on silica gel(J.T. Baker, Phillisburg, NJ) developed with 20% (v/v)ethyl ether in petroleum ether (Pennock, 1985). Labeledprenylquinones were detected by autoradiography for14 d.

For HPLC analyses, the polyprenyltransferase assayswere performed as described above except that larger vol-umes were used for the individual reactions to ensureformation of sufficient products: 0.5 mL for SynHPT withPDP and 5 mL for SynHPT with GGDP, and AtHPT withPDP or GGDP. At the end of incubation, reactions werespiked with 2�-trans-2-Me-6-Ph-1,4-BQ and extracted pre-nyllipids resolved by TLC as above. The areas correspond-ing to prenylquinones (RF 0.36–0.67) were scraped from theTLC plates, eluted with ethyl ether, dried under nitrogen,and dissolved in hexane. Samples were then subjected toHPLC on a normal phase column (LiChrosorb 5 Si60A,4.6 � 250 mm) using 0.1% (v/v) dioxane in hexane as amobile phase to separate various methyl-phytyl benzoqui-none isomers (Henry et al., 1987). For geranylgeranylatedquinone products, a mobile phase consisting of 0.15% (v/v)dioxane in iso-octane was used (Hutson and Threlfall,1980). Prenylquinones were detected at 252 nm. Eluentswere collected at 30- to 60-s intervals and the associatedradioactivity determined by liquid scintillation counting.

Growth Curves

Wild-type Synechocystis sp. PCC 6803 and theSLR1736::Kmr mutant were inoculated to a final opticaldensity at 730 nm of 0.05 in 50 mL of liquid BG-11 mediumand grown at 30°C with vigorous shaking in four possiblecombinations: with or without 15 mm Glc and at 20 to 30(low light) or 105 to 110 (high light) �E m�2 s�1. Theoptical density at 730 nm was measured every 6 to 12 h andused to calculate cell density (Williams, 1988).

Oxygen Evolution

Liquid cultures of photoautotrophically grown wild-type and mutant Synechocystis sp. PCC 6803 cells werewashed twice and resuspended in fresh BG-11 medium ata concentration of 3 mg chlorophyll mL�1. The cells were





exposed to three different high-light intensities for 5 min(0.75, 2, and 5 mE m�2 s�1). Oxygen measurements wereperformed with a Clark-type electrode at 25°C using aHansatech CB1-D3 recording unit with Minirec recordingsoftware (Hansatech Instruments, King’s Lynn, England).The oxygen evolution rate was calculated from the slopewithin the linear region of the curves.

Phylogenetic Analysis

Sequence alignment (ClustalW alignment using theBLOSUM series matrix) and subsequent phylogenetic anal-ysis were performed using MacVector software (GeneticComputer Group). The N-terminal 96-amino acid extensionof AtHPT and the corresponding N termini of the otherpolyprenyltransferases were not included in the phyloge-netic analysis. The following protein sequences were used:Synechocystis sp. PCC 6803 ChlG (accession no. BAA10281),Arabidopsis ChlG (accession no. S60222), Synechocystis sp.PCC 6803 HPT (accession no. S74813), Nostoc HPT (480–1,445 bp of contig 566), Anabaena HPT (6,672–7,625 bp ofcontig c295), rice HPT (accession no. AX046728), maizeHPT (accession no. AX046716), Arabidopsis HPT (acces-sion no. AF324344), soybean HPT (accession no.AX046734), and wheat HPT (accession no. BE471221, over-lapped BE471221 and BG604641 corresponded to theC-terminal part of AtHPT starting at Asp-160). For phylo-genetic analysis, distances between the amino acid se-quences were estimated by using the uncorrected p dis-tance method with gaps distributed proportionally. Thebest tree was constructed by the Unweighted Pair-GroupMethod with Arithmetic Mean with random tie breaking.Bootstrapping (10,000 repetitions) confirmed the confi-dence of the best tree structure.

ACKNOWLEDGMENTS

We would like to thank Dr. Heiko Lokstein for technicalassistance with oxygen evolution measurements and Dr.Zigang Cheng for purification of individual methyl-phytylbenzoquinone isomers. We are very grateful to Dr. DaveShintani and the members of the DellaPenna laboratory forreviewing the manuscript and great moral support.

Received May 7, 2001; returned for revision June 25, 2001;accepted August 1, 2001.

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Isolation and Functional Analysis of Homogentisate Phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis

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