Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae) Vera Hemleben1,*, Angela Dressel1, Bernhard Epping1, Richard Lukačin2, Stefan Martens2

and Michael B. Austin3

1Department of General Genetics, Center of Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany (*author for correspondence; e-mail vera.hemleben@ uni-tuebingen.de);2Institute for Pharmaceutical Biology, Philipps-University Marburg, Deutschhausstr. 17A, 35037 Marburg/Lahn, Germany; 3Structural Biology Laboratory – Noel group, The Salk Institute for Bio-logical Studies, 10010 North Torrey Pines Road, La Jolla, California, CA 92037, USA Received 18 February 2004; accepted in revised form 5 July 2004 Key words: anthocyanin, evolution, flavonoid biosynthesis, Medicago sativa, polyketide synthase, protein structure Abstract For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/ anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in Escherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes. Introduction Secondary compounds play important roles in plants, especially in flower pigmentation, pollen fertility, and as stress and defence substances. The biosynthetic pathways leading to flavones, flavo- nols, differentially modified anthocyanins, and isoflavonoids involved in pathogen defence is one of the most extensively studied and biochemically and genetically characterized pathways in higher plants (Seyffert, 1982; Dooner et al., 1991; Fork-mann and Heller, 1999; Springob et al., 2003). Plant anthocyanins may possess antioxidant

activity, and, therefore, are discussed for their potential health benefits (Dixon and Steele, 1999; Forkmann and Martens, 2001; Kowalczyk et al., 2003). Delivered from the phenylpropanoid bio-synthetic pathway, p-coumaroyl-CoA is iteratively condensed with malonyl-CoA, resulting in a linear tetraketide intermediate that undergoes intramo-lecular cyclization and aromatization to form chalcone. This process is catalyzed in plants by chalcone synthase (CHS), the central member of a superfamily of interesting plant (Schröder, 1997) and bacterial (Moore and Hopke, 2001) type III polyketide synthase (PKS) enzymes (Austin and

Noel, 2003). More than 100 different lower andhigher plant chs genes were studied and used asgenetic marker in phylogenetic and evolutionaryinvestigations (Niesbach-Klosgen et al., 1987;Martin, 1993; Rausher et al., 1999; Koch et al.,2001). Often chs gene families are present in theplant genome, and the individual members aredifferentially regulated and fulfil different func-tions (Chappel and Hahlbrock, 1984; Weisshaaret al., 1991; Hartmann et al., 1998; Lloyd et al.,1999; Durbin et al., 2000; Hirner and Seitz, 2000).

Structural analyses combined with re-combinant gene technology helped to elucidate thebiochemical characters of the homodimeric en-zyme (Ferrer et al., 1999; Jez and Noel, 2000; Jezet al,. 2000a, b). In each CHS monomer an inde-pendently-functioning buried active site cavity(Tropf et al., 1995), containing the absolutelyconserved catalytic Cys-His-Asn triad as well thep-coumaroyl and malonyl binding regions, isconnected to the outside by a long, narrow CoA-binding tunnel.

For the Brassicaceae M. incana (stock), previ-ously used as a suitable system to characterize theflavonoid/anthocyanin biosynthetic pathway, var-ious nearly isogenic red-flowering lines (01–16)were established which are homozygous for thegenes e, f, and g known to be involved in antho-cyanin biosynthesis of M. incana (Kappert, 1949;Seyffert, 1971, 1982). In addition, three white-

flowering mutant lines (17–19) are available each ofwhich is affected in one of the genes e, f, and g,respectively (see Figure 1). Flower pigmentationand trichome formation occur correlated in theanthocyanin producing wild type lines (Kappert,1949). The white-flowering mutant lines 17 and 19with defects in gene e and g, respectively (Helleret al., 1985) possess leaves without trichomes(glabra phenotype), indicating that factors knownto be involved in the regulation of trichome for-mation and pigment biosynthesis, TTG1 (Trans-parenta Testa Glabra 1; Larkin et al., 1994; Walkeret al., 1999; Szymanski, 2000) or a transcriptionfactor (Ramsay et al,. 2003) are affected. Line 18,the white-flowering mutant, especially investigatedhere, appears hairy (incana; see Figure 1). Geneti-cal and biochemical studies (enzyme assays andfeeding of the respective precursor, dihydroqu-ercetin, showed in line 18 a defect in CHS enzymeactivity (Spribille and Forkmann, 1981), suggestinga mutation in the chs (gene f); CHS protein is alsopresent in flower petals, although in reducedamounts, as detected by Western blot (Rall andHemleben, 1984).

Here we wanted to characterize the mutation inline 18, which obviously affects a functional regionof the CHS. Since for M. incana only the cDNAwas previously characterized (Epping et al., 1990),firstly the genomic sequence was obtained and thesite of the chs mutation in line 18 was localized.

Figure 1. Flower pigmentation, trichome formation on the leaf surface (SEM pictures), and genetic constitution of various M. incana

inbred lines. Line 04: wild type; lines 17–19: white-flowering mutant lines exhibiting different genetic defects in the respective gene loci

responsible for anthocyan biosynthesis (e, f, and g; see Seyffert 1971; 1982); gene f is coding for CHS.

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Gene expression was studied by Northern blot andRT-PCR and by an in vitro translation approach.Wild type and mutated CHS cDNAs were alsoexpressed in E. coli for comparison of their cata-lytic properties. In the light of the new structuralinformation on CHS (Ferrer et al., 1999; Jez et al.,2000a, b), structural implications of the mutationfound are discussed in comparison to the infor-mation obtained for the alfalfa (Medicago sativa)CHS structure (Ferrer et al., 1999).

Materials and methods

Plant material and SEM studies

Plants of M. incana R. Br. (Brassicaceae), wildtype line 04 and mutant lines 17–19 (Figure 1;Seyffert, 1971, 1982) were cultivated in the green-house. Trichome formation on the leaf surface wasanalyzed by scanning electron microscopy (SEM):Leaves were fixed in 100% methanol for 1 h, thenwashed 3 · with 100% ethanol, and stored in100% ethanol. Dry leaf pieces were then coatedwith gold and observed directly with a SEM.

Molecular characterization and expression of chsgene

DNA and RNA extraction from flower petals werecarried out according to the methods described byGebhardt et al. (1991), modified after Saghai-Maroof et al. (1984), and Kay et al. (1987),respectively. For southern and northern hybrid-ization standard protocols were used. PCR of thegenomic regions and RT-PCR were performedwith chs-specific primers deduced from the cDNAsequence of the clone pMI112 (Epping et al., 1990;acc. #. X17577; see Figure 1), which was also used

as 32P-labeled probe for hybridization studies. Forcharacterization of the promoter region, primerswere designed according to the respective chs se-quences of Sinapis alba (acc. no. X16437; Batsc-hauer et al., 1991) and Arabidopsis thaliana (acc.no. AF012810; Hartmann et al., 1998). For RT-PCR, total RNA was used as template followingthe protocol of ‘‘Life Technologies’’ for super-scriptTMII reverse transcriptase and using an oli-go-dT anchor-primer (5¢/3¢ RACE Kit, Roche),and subsequently PCR was carried out with theoligo-dT anchor-primer and the specific chs 5¢primer (see Figure 2). Nucleotide sequencing ofthe genomic chs and of the RT-PCR products werecarried out after ligation of the PCR product intopUC18 and cloning in E. coli using universe andreverse primers (ABI, PE, Biosystems). CHS-PCRprimers used for chs gene characterization or RT-PCR (see Figure 2): CHS 5¢, GGG GGA ATTCAA GTC TAC TAA CAA ACC CCA TTT GG;CHS 3¢, GGG GGA ATT CGA ATT CCG GACCAT CAA CAC TGC GTT CAG C; CHS-M 5¢,TCC TCG TCG TCT GCT CTG AA; CHS-M 3¢,GGA AGG TGA CGG CTG TGA TT; CHSPromoter, GGA AGC AAG AAG GAG AACCA; dT-anchor: GAC CAC GCG TAT CGATGT CGA CTT TTT TTT TTT TTT TTV(V ¼ A,C, or G).

The nucleotide sequences appeared in theEMBL Nucleotide Database for the wild typegenomic Matthiola chs under the acc. no. #AJ427536 and for the mutant allele (G487T) underthe acc. no. # AJ427537.

In vitro translation and radioimmunoprecipitation

In vitro translation was carried out in a rabbitreticulocyte lysate system with 35S-methionine(NEN) and with total RNA of M. incana lines 04

Figure 2. Scheme of the chs gene of M. incana. Locations of the primers used for PCR, for RT-PCR and nucleotide sequencing

(including internal sequencing primers) are indicated. The mutation in the second exon of the chs gene in line 18 is marked.

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and 18, respectively, according to the method de-scribed by Mettenleiter et al. (1985). CHS proteinwas detected by radioimmunoprecipitation with apolyclonal antibody against CHS (kindly providedby Prof. Klaus Hahlbrock, MPI for Plant Breed-ing Research, Cologne, Germany). The translationmix was incubated in 200 ll PBS-lysis buffer and100 ll purified Staphylococcus aureus cell suspen-sion (Kessler, 1981) for 30 min at 4 �C, and sub-sequently centrifuged for 2 min at 14,000 rpm in aBiofuge in order to bind unspecific translationproducts. To the supernatant 5 ll of the CHSantiserum was added and incubated for 90 min at4 �C; subsequently; then S. aureus cell suspensionwas added and the mixture again incubated for30 min at 4 �C. Several washing steps followedwith 600 ll PBS-lysis buffer, and the last pelletswere solved in 75 ll SDS-buffer, heated for 5 minat 95 �C, and electrophoretically separated on adiscontinous SDS-polyacrylamide gel (PAGE;Lammli, 1970). The dried gel was exposed to X-rayfilm for 6 weeks.

Construction of recombinant expression vectors

The CHS cDNAs from Matthiola incana lines 04and 18 were amplified by proofreading PCR fromthe cCHS-M-04 and cCHS-M-18 plasmids usingappropriate oligonucleotide primers and subcl-oned into the pET101/D-TOPO expression vectoraccording to the manufacturer instructions (Invi-trogen, Groningen, The Netherlands). Expressionconstructs were verified by restriction analysis andDNA sequencing, using the universal and reversesequencing primers.

Heterologous expression and activityof recombinant CHSs

E. coli strain BL 21 Star (Invitrogen) was trans-formed with the CHS-pET101/D-TOPO con-structs containing the coding sequence of the wildtype or mutant enzymes and subcultured subse-quently to a density of 0.8 in Luria-Bertanimedium as described elsewhere (Lukacin et al.,1999). Isopropyl thio-ß-D-galactoside (1 mM)was added for the induction of CHS expression,and the bacteria were harvested after another 3 hat 28 �C and stored frozen at )70 �C. Both theCHSs were partially purified from the crudebacterial extracts by a modified procedure devised

for Ruta graveolens ACS (acridone synthase) andCHS. Briefly, the bacterial pellets (approx. 1 gwet mass) were suspended in 10 ml of 50 mMHEPES buffer pH 7.0, 10 mM EDTA, and thesuspensions were sonicated for 1 min and clearedby centrifugation (30,000 g, 10 min, 4 �C). Theclear supernatants were fractionated on a Frac-togel EMD DEAE 650 (S) anionic exchanger(Merck, Darmstadt, Germany) as described pre-viously (Lukacin et al., 1999). Collected fractionscontaining the wild type or mutant CHSs wereconcentrated 20-fold by ultrafilter centrifugation(Centriprep 10 and Centricon 10, Amicon, Wit-ten, Germany). After this purification step about85% of the contaminating protein of the bacterialextracts had been removed from the CHS poly-peptides. Specific activities of the wild type andthe mutant enzyme, as well as Western blotprotein analysis, were determined in parallel atthis stage.

General assays

The protein composition of samples was exam-ined by SDS-PAGE (Laemmli, 1970). Westernblotting was carried out as described in the lit-erature (Towbin et al., 1979) and using the Rutagraveolens CHS and ACS polyclonal antiseraraised in rabbits (Lukacin et al., 2001). Theproteins bound to the nitrocellulose filters werevisualized by staining with Ponceau S solutionafter immunostaining. Protein amounts werequantified after precipitation with trichloroaceticacid and employing the Lowry procedure (San-dermann and Strominger, 1976) with bovineserum albumin as a reference. The activities ofthe CHSs were determined with 1 nmol 4-cou-maroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA andferuloyl-CoA, respectively, in the presence of1.5 nmol [2-14C]-malonyl-CoA (Hartmann Ana-lytic, Braunschweig, Germany) and protein in atotal volume of 200 ll employing 15–80 lg pro-tein (Lukacin et al., 2001). Additionally, [14C]-acetyl-CoA (Hartmann Analytic, Braunschweig,Germany) was used as single precusor in thedescribed assay. All determinations were made atleast in triplicates. The products were extractedfrom the incubation mixtures and identified byradio-thin layer chromatography on cellulose inthree different solvent systems as described pre-viously (Spribille and Forkmann, 1981).

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Structural CHS analysis and computer modelling

Primary sequence alignments were made using theprogram Clustal W (Thompson et al., 1994).Three-dimensional computer homology models ofMatthiola wild type and mutant CHS amino acidsequences were carried out with the programModeller 4 (Sali and Blundell, 1993), using theknown structure of alfalfa CHS (PDB accession1BI5) as structural template.

Results

Anthocyanin synthesis and trichome formationin Matthiola lines

For all experiments the M. incana red-floweringline 04 and the white-flowering chs mutant line 18(sometimes also lines 17 and 19 for comparison)were used. Matthiola anthocyanin-producing wildtype lines all exhibit the hairy (incana) phenotypewith trichomes on the leaf surface. In the mutantline 18 trichomes appear perfectly normal leadingto an incana phenotype (Figure 1), whereas theother white-flowering mutant lines 17 and 19 arecharacterized by the glabra phenotype (withouttrichomes). This indicates that the white-floweringmutant line 18, previously characterized by thelack of CHS activity (Spribille and Forkmann,1981) and, therefore, assumed to be affected by achs mutation in gene f is still able to form tric-homes. Defects affecting later stages of theanthocyanin biosynthesis pathway (in lines 17and 19, genes e and g) obviously prevent tri-chome formation (Figure 1; see Discussion).

Genomic organization of chs gene and sequenceanalysis in wild type and mutant lines

To characterize the chs mutation, the genomic chswas characterized for the wild type line (04) andlines 17–19, since only the chs cDNA was se-quenced for the M. incana wild type (Eppinget al., 1990). To clarify the general organizationof the genomic chs gene, Southern blots werehybridized with a 32P-labeled cDNA insert ofclone pMI112 under various stringency condi-tions, and similar hybridization patterns wereobtained for the wild type and mutant lines.These patterns (not shown) and sequencing of

several genomic and cDNA clones (see below)indicated that in M. incana CHS is encoded by asingle-copy gene.

To obtain the complete genomic sequence,PCR products amplified with gene-specificprimers starting from the first LRE (light-regu-lating element; Koch et al., 2001) were cloned,and several clones for each line were sequenced.The chs gene contains one intron with 80 bp inlength, separating the triplet coding for cysteineat amino acid (aa) position 65 in the polypeptidechain (Epping et al., 1990), as found in all higherplants investigated, and showed no differencesbetween the Matthiola wild type and the mutantlines.

One specific nucleotide difference could be de-tected between the wild type line 04 chs and themutant line 18 chs sequences in the second exon(G487T for the genomic sequence), whereas thewhite-flowering mutant lines 17 and 19 showed thewild type sequence. This nucleotide change in line18 chs results in an amino acid substitution at theprotein level from arginine (triplet AGG) intoserine (triplet AGT) at residue 72 in the polypep-tide chain (R72S; Figuers 2 and 5).

Transcription studies and in vitro translation of chsmRNA

Previously, in petals of the wild type and mutantline 18 CHS protein was detected by Western blotin reduced levels relative to the wild type lines(Rall and Hemleben, 1984). Therefore, to inves-tigate whether in flowers of both lines chs mRNAis similarly produced, Northern blots and RT-PCR were performed with total RNA extractedfrom young flower buds of lines 04 and 18,respectively, in a stage where the wild type startedto exhibit red-colored petals. Northern blotshybridized with the 32P-labeled cDNA showed asingle band with the expected size of approx.1.4 kb with both lines, only for line 18 a weakerhybridization signal was observed, indicating areduced chs gene expression in the mutant line(Figure 3a). Furthermore, RT-PCR productsfrom lines 04 and 18 were cloned and sequenced,and, actually, the mutation from G to T in thesecond exon of chs (see Figure 2) could be con-firmed for line 18.

To see, whether the mutant chs mRNA couldbe translated into CHS protein, in vitro translation

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of total RNA from petals of both lines was per-formed in an in vitro translation system fromrabbit reticulocytes, CHS protein then was radio-immunoprecipitated with a polyclonal CHS anti-body and the precipitate electrophoreticallyseparated on SDS-PAGE. With equal amounts oftotal RNA from both, wild type and mutant line, aprotein band appeared with the size of the CHSmonomer (43 kDa; Figure 3b; Rall and Hemle-ben, 1984). The strength of the protein band ap-peared to be approximately four times less for themutant line 18; that is corresponding to theamount of mRNA visualized in Northern blots(see Figure 3a), indicating a reduced amount ofmRNA in the mutant. Nevertheless, chs mRNA ofboth lines can be translated in vivo and in vitro.

Recombinant CHS expression

For further functional characterization, Matthi-ola wild type and mutant CHS cDNAs were ex-pressed in E. coli, prominent protein bands of43 kDa were observed upon SDS-PAGE andsubsequent Western blot analysis of the partiallypurified recombinant wild type (line 04) andmutant (line 18) enzymes (Figure 4a). During thepurification procedure of the CHS polypeptides adegradation product of lower molecular mass

was observed to increase as it was also reportedfor recombinant flavanone 3b-hydroxylase fromPetunia hybrida (Lukacin et al., 2000). Never-theless, the partially purified wild type CHS re-vealed specific activity with 4-coumaroyl-CoAand malonyl-CoA as substrates, whereas themutant enzyme totally lacked CHS activity(Figure 4b). The same results were observed withcaffeoyl-CoA and malonyl-CoA as substrates(data not shown). The formation of eriodictyolfrom these starter molecules was reported fromenzyme assays using crude extracts of petals fromMatthiola wild type plants (Spribille and Fork-mann, 1981). Furthermore, none of the othertested CoA esters (cinnamoyl-CoA and feruloyl-CoA) led to the formation of specific products ordetectable side products; also [14C]-acetyl-CoAused as single precusor in the described assay didnot give rise to a product (data not shown). Theresults demonstrate that this one mutationalamino acid exchange (R72S) is sufficient toabolish CHS activity. Notably, protein solubilityis maintained in the mutant enzyme, ruling outthe most likely effect of this mutation: protein

Figure 3. CHS expression studies in the wild type line 04 and

the mutant line 18, respectively, of M. incana with RNA on

Northern blots or with an in vitro translation approach.

(a) Northern blot with total RNA of young flower buds of the

wild type line 04 and the mutant line 18 (20 lg per lane, loadingcontrol confirmed similar amounts; not shown), hybridized with

the radioactively labelled insert of cDNA clone pMI112.

(b) In vitro translation of 2 lg of total RNA of the mutant line

18 and wild type line 04, respectiviely, and radioimmunopre-

cipitation of CHS with a polyclonal anti-CHS serum separated

on SDS-PAGE. The size of the CHS mRNA and of the protein

are marked on the right, respectively.

Figure 4. Recombinant CHS characterization. Western blot

analysis (a) and radioscan of separated extractions from CHS

enzyme assays (b) of partially purified wild type line 04 and

mutant line 18 CHS from M. incana. Both the recombinant

enzymes cross-reacted with a polyclonal anti-CHS serum after

nitrocellulose blotting. The molecular mass marker sizes are

indicated on the left. NAR: naringenin.

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aggregation and precipitation resulting fromgross misfolding.

Structural features of the mutated enzyme

A three-dimensional structural homology model ofthe wild type Matthiola CHS was constructedusing Modeller 4 (Sali 1993) from a primary se-quence alignment of the Matthiola (Figure 5) andalfalfa (Medicago sativa) CHS protein sequences(which are 84% identical) and the known 3-Dstructure of alfalfa CHS. The program threads thealigned sequence onto the structural template,utilizing conjugate gradients and moleculardynamics with simulated annealing techniques tooptimize the resulting model’s scoring function,while relieving any intramolecular steric clashes.The likely effects of the line 18 CHS R72S muta-tion (Figure 5) were evaluated in the context ofthis wild type Matthiola CHS homology model(see Discussion).

Discussion

The unique chs gene of Matthiola contains an in-tron of 80 bp which is not altered in the mutantline 18. This intron, separating a conserved cyste-ine as in other plants, is similar in length as theArabidopsis chs intron (86 bp; Feinbaum andAusubel, 1988) and shorter than in S. alba(522 bp; Batschauer et al., 1991). For character-ization of the promoter, only a short regionincluding the TATA box was amplified, containingthe LRE present upstream of the TATA box ofother Brassicaceae (Feinbaum and Ausubel, 1988;Batschauer et al., 1991; Hartmann et al., 1998;Koch et al., 2001; this part showed no differencebetween the wild type and mutant line.

Previous genetic and biochemical investigationson Matthiola line 18 with incana phenotypeshowed that the defect in anthocyanin formation iscaused by the inability to produce active CHS(Spribille and Forkmann, 1981), although the

Figure 5. Homology model of M. incana CHS, showing the location and conserved local environment (nearly identical in all known

plant type III PKS enzyme crystal structures, see text) of R72. Hydrogen bond N-O distances (in Angstroms) are labeled in red. The

white-flowering M. incana line 18 CHS R72S mutation is modeled in cyan. The side chains of F219 and the catalytic triad (C168, H

308, and N341) line the CHS internal active site cavity, which is occupied in this model by bound naringenin (shown in rose). The

position and orientation of this chalcone-derived product analogue is taken directly from the complexed crystal structure of alfalfa

CHS (Ferrer et al., 1999; PDB accession 1CGK). Inset: Ribbon diagram showing the physiological CHS homodimer, with N- and C-

termini labeled. Naringenin (space-filling model) is shown occupying each identical active site cavity. A red box identifies the region of

the protein depicted in greater detail.

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protein is present in reduced amounts (Rall andHemleben, 1984), whereas the other white-flower-ing lines 17 and 19 obviously have a defect in laterstages of anthocyanin biosynthesis (Seyffert, 1971,1982; Heller et al., 1985). Recent studies suggestedthat in line 17 ttg1 is affected (A.R. Walker, N.A.Ramsay and J.C. Gray, unpublished; V. Hemlebenand A. Dressel, in preparation). Line 19 could becomplemented with two basic-helix-loop-helix(bHLH) genes (MYC-146 and GL3 ) from Ara-bidopsis (Ramsay et al., 2003), and a mutation waslocated in a bHLH (TT8-like) gene (Ramsay et al.,2003; N.A. Ramsay, A.R. Walker and J.C. Gray,unpublished). In line 18, chs mRNA from petals isdetected in reduced amounts relative to the wildtype, and it is translated in vivo and in vitro intoCHS protein; however, as shown earlier, thisprotein is inactive in vivo (Spribille and Forkmann,1981). This could also be confirmed here by het-erologous recombinant expression of the cDNA inE. coli (Figure 4), independent of the starter mol-ecules tested. The chs mutation is located at posi-tion nt 253 of the cDNA (Epping et al., 1990) or atnt 487 in the genomic sequence presented here(G487T) leading to an amino acid exchange fromarginine (triplet: AGG) to serine (triplet AGT) inthe polypeptide chain (R72S). Remarkably, thisarginine residue is absolutely conserved in CHSs,as well as in all known functionally divergent plantand bacterial CHS-like enzymes (Niesbach-Klos-gen et al., 1987; Martin, 1993; Jez et al., 2001);however, other Brassicaceae like A. thaliana andS. alba realize this with a different triplet forarginine (CGT; Feinbaum and Ausubel, 1988;Batschauer et al., 1991).

Since the alfalfa CHS structure has beenextensively studied using recombinant proteinanalysis following amino acid substitution by site-directed mutagenesis (Ferrer et al., 1999; Jez et al.,2000a, b), we can conclude that the CHS R72Smutation described here (Figure 5) is not directlyaffecting the catalytic Cys-His-Asn triad. (As dif-ferent residue numbers correspond to the equiva-lent Matthiola and alfalfa CHS residues, theresidue number for alfalfa CHS is listed here inparenthesis following the numbering of the Mat-thiola sequence.) In the wild type Matthiola CHShomology model (Figure 5) one of the R72 (68)terminal nitrogens is within hydrogen-bondingdistance of the mainchain carbonyl oxygens ofF219 (215) and Q216 (212). The distances of these

interactions are 2.6 and 2.8 A, respectively. Theother terminal nitrogen of R72 (68) is withinhydrogen-bonding range of both the mainchaincarbonyl oxygen of A217 (213) and the side chainoxygen of Q37 (33), 3.4 and 3.2 A, respectively.This latter glutamine residue, like R72 (68), is alsoabsolutely conserved among plant CHS-like en-zymes (including those with differing substratepreferences and product profiles). The buriednature of these interactions with R72 (68) andtheir conservation in CHS-like proteins of varyingactivity suggest that this residue is important inmaintaining or achieving the proper fold. Indeed,identical hydrogen bonds to R72 (68) are observedin each of the four plant CHS-like enzymes whosecrystal structure has been elucidated to date(Ferrer et al., 1999; Jez et al., 2000b; Austin et al.,in press).

One curiosity in the wild type Matthiola CHSprotein sequence is the occurrence of serine at the220 (216) position (Figure 5). The main chaincarbonyl oxygen of this residue contributes to thecoumaroyl-binding pocket of the active site and isin fact located 2.6 A from the para-hydroxy of thebound coumaroyl ring, as seen in all coumaroylmoity-containing CHS and STS complexes crys-tallized to date (Ferrer et al., 1999; Austin et al., inpress). However, while residue 220 is most often aglycine, an alanine at this position has been crys-tallographically observed in a CHS-like enzymewith no drastic effects to the position of the adja-cent arginine (Austin and Noel, 2003). Further-more, a bona fide CHS with a serine at thisposition was recently discovered (Yamazaki,2001). Thus, it seems unlikely that the unusualoccurrence of serine at this position in the wildtype Matthiola CHS would alter the otherwiseconserved hydrogen bonding interactions of R72(68).

Taken together, the structural considerationsimply that R72 (68) is most likely involved in CHSfolding or stabilization. The mutation of this res-idue to serine not only abolishes these four stabi-lizing interactions, but also introduces a furtherpotentially destabilizing effect. If the mutant en-zyme is in fact able to achieve the wild type CHSfolding pattern, then the polar serine side chainoxygen will be sandwiched between two otherelectronegative oxygens: the neighboring mainchain carbonyl oxygen of residue 71 (67) and theaforementioned S220 (216) side chain oxygen. The

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modeled mutant S72 (68) side chain hydroxyl is 3.2and 2.6 A, respectively, from these two oxygens.Thus, the absolute conservation of R72 (68)among both plant and bacterial CHS-like enzymesof varying function as well as the destabilizinglocal environment of the R72S mutation couldimplicate protein misfolding as the most likelycause of CHS inactivation in Matthiola line 18.However, results obtained from our heterologousexpression of this mutant M. incana CHS proteinin E. coli are inconsistent with gross misfolding,which typically leads to protein aggregation andprecipitation. Rather, these results show that themutant enzyme maintains solubility throughoutpurification, despite its total lack of catalyticactivity.

Consequently, it seems probable that ratherthan grossly misfolded protein, what results issome slight perturbation of the nearby coumaroyl-binding pocket of the active site cavity thatdestroys substrate binding (in an otherwise native-seeming protein). Notably, the adjacent phenylal-anine F219 (215) is of demonstrated catalyticimportance to the malonyl decarboxylation stepcrucial for starter molecule elongation (discussedin Austin and Noel, 2003). Mutating this F215 inalfalfa CHS greatly diminishes activity (Jez et al.,2000b), and a similar naturally-occuring mutationin benzalacetone synthase (BAS), a related type IIIPKS, is responsible for that enzyme’s unusuallynon-iterative single polyketide extension step (Abeet al., 2003). Thus, it is conceivable that the R72(68) hydrogen bond to the F219 (215) carbonyloxygen provides some necessary positioning orstabilizing effect on the phenylalanine side chain,and that elimination of such stabilization impairsthe mechanism without causing gross misfolding.

Although the wild type R72(68) residue dis-cussed here is absolutely conserved in CHS, as wellas in most known functionally divergent plant andbacterial CHS-like enzymes (Niesbach-Klosgenet al., 1987; Martin, 1993; Jez et al., 2001; Austinand Noel, 2003), there has been no previousmutagenic or biochemical examination of the roleof this buried arginine. Our current analysis of anatural chs mutation in M. incana confirms thisposition to be crucial for normal enzymatic activ-ity. Although the removal of this buried arginine’sstabilizing hydrogen bonds does not cause grossprotein misfolding or directly effect the Cys-His-Asn catalytic triad, this mutation apparently alters

the normal topology of the nearby CHS active sitecavity, and likely perturbs a conserved and mech-anistically-important active site F219 (215).

Known CHS enzymes share more than 50% se-quence identity across their nearly 400-residuesequences. As would be expected, previous charac-terizations of induced and natural CHS knockoutmutations in A. thaliana (Saslowsky et al., 2000)and Rubus idaeus (raspberry; Zheng, et al., 2001)enzymes suggest mutation of conserved residues inany part of the enzyme has the potential to disruptcatalytic function. While most of such non-activesite mutations likely destroy activity by disruptingenzyme folding or stability, a subset of mutationspreserve the stability of the type III PKS fold, butnonetheless disrupt wild type substrate or productspecificity. The identification and characterizationof such crucial remote positions, including thearginine at CHS position 72(68) described here,contributes to our growing understanding of thestructural basis for evolutionary functional diver-gence in type III PKS enzymes.

Acknowledgements

We thank Prof. W. Seyffert, em., University ofTubingen, for providing the genetically definedlines of Matthiola incana. Previous contributionsof Dr. Hans-Michael Rupp are gratefullyacknowledged. E. Schoppmann, University ofTubingen, helped with the SEM studies.

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