O-Methylation of benzaldehyde derivatives by “lignin specific” caffeic acid 3- O-methyltransferase

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O-Methylation of benzaldehyde derivatives by “lignin specific” caffeic acid 3- O-methyltransferase
  O -Methylation of benzaldehyde derivatives by ‘‘lignin specific’’caffeic acid 3- O -methyltransferase § Parvathi Kota a,1 , Dianjing Guo a,2 , Chloe Zubieta b,3 , Joe Noel b , Richard A. Dixon a, * a Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA b Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA Received 28 July 2003; received in revised form 10 December 2003 Abstract Although  S  -adenosyl- l -methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5- O -methyltransferase (COMT) is oneof the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivativesmore efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were thebest in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over ligninpathway intermediates for 3- O -methylation by recombinant alfalfa COMT expressed in  Escherichia coli.  OMT activity with ben-zaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMTdown-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehydederivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for ligninprecursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenyl-propanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineeringof both lignin and benzaldehyde-derived flavors and fragrances. # 2004 Elsevier Ltd. All rights reserved. Keywords: Medicago sativa ; Leguminosae; Lignification;  O -Methyltransferase; Site-directed mutants; Benzaldehydes 1. Introduction In spite of the collective complexity of plant naturalproducts, their biosynthesis is in large part broughtabout by the activities of a limited number of enzymeclasses. Much of the diversity in many natural productgroups arises from differential substitution of basic bio-synthetic skeletons. Within phenylpropanoidbiosynthesis, O -methylation and  O -glycosylation are two of the mostcommon substitution reactions, and many  O -methyl-transferases (OMTs) active against hydroxycinnamicacids, flavonoids and isoflavonoids have been reported(Gauthier et al., 1998; Frick and Kutchan, 1999; Mauryet al., 1999; Chiron et al., 2000; Wein et al., 2002). It isbecoming increasingly clear that plant small moleculeOMTs can either show exquisite specificity (Zubieta etal., 2001), or have relatively promiscuous substrate pre-ferences (Dixon, 2001; Parvathi et al., 2001; Wein et al.,2002; Zubieta et al., 2002). The latter observation hasimportant implications for genomic annotation and theevolution of plant specialized (secondary) metabolism.Caffeic acid 3- O -methyltransferase (COMT, EC.2.1.1.68) was the first plant small molecule OMT to bedescribed (Neish, 1968), its significance associated withits presumed role in the biosynthesis of lignin, the secondmost abundant polymer on earth. Lignin consists of hydroxylated and methoxylated phenylpropanoid unitscalled monolignols; in dicots, these are primarily mono-methylated guaiacyl (G) units derived from coniferylalcohol, and dimethylated syringyl (S) units derivedfrom sinapyl alcohol. The S and G units in lignin are joinedthroughdifferent types ofetherand carbon–carbon 0031-9422/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.phytochem.2004.01.017Phytochemistry 65 (2004) 837–846www.elsevier.com/locate/phytochem § This work was supported by the Samuel Roberts Noble Founda-tion.* Corresponding author. Tel.: +1-580-224-6601; fax: +1-580-224-6692. E-mail address:  radixon@noble.org (R.A. Dixon). 1 Presentaddress:DepartmentofBiology,MassachusettsInstituteof Technology, 77 Massachusetts Avenue, Cambridge, MA 02142, USA. 2 Present address: Virginia Bioinformatics Institute, 1880 PrattDrive, Blacksburg VA 24061, USA. 3 Present address: European Molecular Biology Laboratory, 38042Grenoble Cedex 9, France.  linkages (Davin and Lewis, 1992). Until recently, mostmodels of lignin biosynthesis presented the pathway as ametabolic grid, through which the side-chain reductionand successive ring hydroxylation/ O -methylation reactionsoccur at several different levels (Whetten and Sederoff,1995; Dixon et al., 2001) (Fig. 1). COMT was initially thought to be a bifunctional enzyme that used caffeic ( 1 )and 5-hydroxyferulic ( 2 ) acids as substrates duringmonolignol biosynthesis. However, recent studies haveshown that recombinant COMTs exhibit a higherpreference for 5-hydroxyconiferaldehyde ( 3 ) than forcaffeic acid ( 1 ) (Li et al., 2000; Parvathi et al., 2001). Thepreferred substrate for the enzyme srcinally known asferulate 5-hydroxylase (F5H) is coniferaldehyde ( 4 )rather than ferulic acid ( 5 ) (Humphreys et al., 1999;Osakabe et al., 1999), consistent with a pathway formonolignolformationinwhichCOMTfunctionsprimarilyto methylate 5-hydroxyconiferaldehyde ( 3 ) in the bio-synthesis of S lignin (Li et al., 2000; Parvathi et al.,2001). Consistent with this model, COMT down-regulatedtransgenic plants show a stronger reduction in S ligninthan in G lignin (Atanassova et al., 1995; Van Door-sselaere et al., 1995; Guo et al., 2000; Piquemal et al.,2002).The substrate preferences of COMT enzymes might beeven broader than initially realized. Within the context of monolignol biosynthesis, recombinant alfalfa COMTexhibits high catalytic efficiency not only with 5-hydroxy-coniferaldehyde ( 3 ) but also with caffeyl aldehyde ( 6 ),caffeyl alcohol ( 7 ), and 5-hydroxyconiferyl alcohol ( 8 )(Parvathi et al., 2001), suggesting the involvement of COMT in both 3- and 5-methylation reactions of Slignin biosynthesis at either the aldehyde or alcohol levels.These results are consistent with in vivo labeling studies Fig. 1. Hypothetical metabolic grid for the biosynthesis of the monolignols coniferyl alcohol ( 19 ) and sinapyl alcohol ( 22 ) from 4-coumaric acid( 19 ). This scheme has undergone significant revision in recent years (Humphries and Chapple, 2002). The enzymes are: C3H, ‘‘coumarate hydro-xylase’’, now known to function at the level of the corresponding shikimate ester (Schoch et al., 2001); 4CL, 4-coumarate: CoA ligase; HST,hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase; COMT, caffeic acid 3- O -methyltransferase (more accurately termed 5-hydroxy-coniferaldehyde 3- O -methyltransferase); F5H, ferulate 5-hydroxylase (more accurately termed coniferaldehyde 5-hydroxylase); CCoAOMT, caffeoylCoA 3- O -methyltransferase; CCR, cinnamyl CoA reductase; CAD, coniferyl alcohol dehydrogenase.838  P. Kota et al./Phytochemistry 65 (2004) 837–846  in  Magnolia kobus  in which S lignin can be derived fromconiferyl alcohol ( 9 ) (Matsui et al., 1994; Chen et al.,1999).The recently determined three-dimensional crystalstructure of alfalfa COMT reveals an unusually spaciouscatalytic site (Zubieta et al., 2002) and provides anexplanation for the broad specificity of the enzyme for arange of hydroxycinnamic acid, aldehyde and alcoholderivatives. Furthermore, enzymes with COMT activityfrom some species may accept substrates other thanmonolignol precursors. For example, tobacco COMT Iand COMT II have also been reported to be activeagainst protocatechuic aldehyde ( 10 ) (Maury et al.,1999), OMT from  Chrysosplenium americanum  catalyzesmethylation of both caffeic acid ( 1 ) and flavonoids(Gauthier et al., 1998), OMT from  Thalictrum tuberosum shows activity toward both alkaloids and phenylpropanoidsubstrates (Frick and Kutchan, 1999), and an OMTfrom strawberry fruit exhibits broad substrate specificityagainst monolignol precursors and furanone aromacompounds (Wein et al., 2002).We here report that benzaldehyde derivatives are themost efficient in vitro substrates for alfalfa COMTdetermined to date. We describe the pattern of OMTactivity with benzaldehydes in extracts from alfalfa steminternodes harvested at various stages of development,and the kinetic properties of recombinant alfalfa COMTfor benzaldehydes. Structurally-targeted site directedmutagenesis of alfalfa COMT can alter the relativesubstrate preference of the enzyme for monolignolprecursors and benzaldehyde derivatives. We evaluatethe significance of the broad in vitro substrate preferenceof COMT for natural product biosynthesis in vivo. 2. Results and discussion 2.1. Developmental changes in O-methyltransferaseactivities against benzaldehyde derivatives OMTs from alfalfa stem extracts have broad substratepreference against monolignol precursors (Parvathi etal., 2001). Lignin levels and OMT activities against allpotential monolignol precursors increase with thedevelopment of the stem in alfalfa, preceding theincrease in lignin methoxyl content (S/G ratio) withadvanced maturity (Inoue et al., 1998, 2000). Followinga serendipitous observation that crude extracts fromalfalfa stems are also able to methylate protocatechuicaldehyde ( 10 ), as has also been reported for tobaccoCOMT (Maury et al., 1999), crude protein extracts fromindividual internodes (first to tenth) were assayed fortheir ability to catalyze methylation of a range of benzaldehyde derivatives.Typical developmental profiles of OMT activities areshown in Fig. 2. The highest activities for all substrateswere recorded in the sixth to eighth internodes.Surprisingly, the highest activities were obtained withprotocatechuic aldehyde ( 10 ) and 3,4-dihydroxy,5-methoxybenzaldehyde ( 11 ). The extracts catalyzedmethylation of these compounds to a greater extentthan the preferred monolignol precursor 5-hydroxy-coniferaldehyde ( 3 ) (Fig. 2). 2.2. O-Methyltransferase activities in stem extracts of transgenic alfalfa down-regulated in COMT  COMT activity and protein level are strongly down-regulated in alfalfa by expression of homologous senseor antisense COMT sequences driven by the vasculartissue-specific bean  PAL2  promoter (Guo et al., 2000;Parvathi et al., 2001). Control alfalfa cv Regen SYplants transformed with an empty vector that exhibitwild-type COMT expression have also been produced(Guo et al., 2000). We took advantage of the transgenicalfalfa lines to demonstrate that the above activitieswith benzaldehyde derivatives in crude extracts wereindeed due to the activity of COMT rather than a secondOMT specific for benzaldehyde derivatives. It is veryunlikely that the antisense strategy used targets otherOMT enzymes based on the known sequences of COMT-like enzymes in the model legume  Medicagotruncatula  (information available at the TIGR website,http://www.tigr.org/tdb/tgi.shtml), genes from whichshare very high sequence identity to their orthologs inalfalfa.OMT activities against benzaldehyde derivatives andmonolignol precursors in stem extracts from the sixth toeighth internodes of COMT down-regulated and emptyvector control lines are shown in Table 1. The stemsamples were collected from plants at the same develop-mental stage grown together under the same environ-mental conditions. Down-regulation of COMT had a Fig. 2. Developmental changes in  O -methyltransferase activities inalfalfa stem internodes (cultivar Apollo). Enzyme activities with theindicated substrates (50  m M) and  14 C-SAM were determined in crudestem protein extracts from internodes 1–10. Duplicate assays wereperformed (maximum analytical variation less than   5%). P. Kota et al./Phytochemistry 65 (2004) 837–846  839  larger impact on the methylation of protocatechuicaldehyde( 10 )and3,4-dihydroxy,5-methoxybenzaldehyde( 11 ) than on the monolignol precursors. However, thelow level of activity against protocatechuic acid ( 12 ) wasmuch less affected. These results indicate that the majoractivity catalyzing methylation of benzaldehydes inalfalfa stems is the enzyme known as COMT. 2.3. Kinetic properties of recombinant alfalfa COMT with benzaldehyde derivatives The alfalfa COMT cDNA was cloned in pET15b andexpressed in  E. coli   (Gowri et al., 1991; Inoue et al.,1998) as an N-terminally hexahistidine tagged protein.COMT expression was induced by IPTG and the enzymepurifiedtohomogeneityfromthe solubleproteinfractionsby nickel columnaffinity chromatography(Parvathi etal.,2001; Zubieta et al., 2002). This recombinant ‘‘wild-type’’enzyme was used to study the catalytic behavior of COMT towards benzaldehyde derivatives. Kineticanalyses were performed by measuring the initial velocityagainst a range of substrate concentrations at a fixedconcentration (60 m M)of  14 C-labeled SAM. The substratepreferences and kinetic constants are presented inTables 2 and 3. The smallest  K  m  values obtained werefor protocatechuic aldehyde ( 10 ) and 5-hydroxy-coniferaldehyde ( 3 ), the latter of which is the favoredsubstrate (expressed as Km) for COMT in the mono-lignol biosynthetic pathway. The highest  V  max / K  m values observed were for protocatechuic aldehyde ( 10 )and 3,4-dihydroxy, 5-methoxybenzaldehyde ( 11) ,whereas protocatechuic acid ( 12 ) was a much poorersubstrate in terms of catalytic efficiency expressed as V  max / K  m  (Table 3). The difference in catalytic efficiencybetween aldehydes and their corresponding acids wasmuch greater for the benzaldehyde derivatives than forthe phenylpropanoid derived monolignol precursors.To confirm the nature of the products formed by theCOMT-mediated methylation of protocatechuic alde-hyde ( 10 ) and 3,4-dihydroxy, 5-methoxybenzaldehyde( 11 ) with  14 C-labeled SAM, HPLC/diode-array analysiswith parallel radiodetection was performed. HPLC/UVabsorption traces of the reaction products from reactionmixtures of recombinant COMT with protocatechuicaldehyde( 10 )and3,4-dihydroxy,5-methoxybenzaldehyde( 11 ) are presented in Fig. 3. The terminated reaction was spiked with the potential methylated products,vanillin ( 13 ) and syringaldehyde ( 14 ) respectively. InFig. 3A, 3,4-dimethoxybenzaldehyde ( 15 ) was alsoadded to the reactions after termination, to check whetherCOMT could methylate both hydroxyls on protocatechuic Table 1OMT activities (pkat mg  1 protein) in stem material from wild-type (empty vector control) and COMT down-regulated (line 310) transgenic alfalfaSubstrate Wild type COMTdown-regulatedReduction in COMTactivity (-fold)Protocatechuic aldehyde ( 10 ) 7.1  0.04 0.5  0.01 14.23,4-Dihydroxy, 5-methoxybenzaldehyde ( 11 ) 14.6  0.9 1.0  0.06 14.6Protocatechuic acid ( 12 ) 0.18  0.01 0.1  0.04 1.8Caffeic acid ( 1 ) 3.8  0.06 0.7  0.14 5.75-Hydroxy coniferaldehyde ( 3 ) 4.5  0.30 1.7  0.003 2.6Values are from duplicate assays with pooled stem material from multiple plants, expressed as average  spread of values.Table 2Activities of recombinant alfalfa COMT against benzaldehyde deriva-tives and protocatechuic acid (100  m M)Substrate (100  m M) COMT activitypkat mg  1 3-Hydroxy benzaldehyde ( 16 ) 04-Hydroxy benzaldehyde ( 17 ) 0Protocatechuic aldehyde ( 10 ) 949Isovanillin ( 18 ) 10Vanillin ( 13 ) 03,4-Dihydroxy, 5-methoxybenzaldehyde ( 11 ) 1277Syringaldehyde ( 14 ) 0Protocatechuic acid ( 12 ) 20A. 16  3-Hydroxybenzaldehyde: R1=OH, R2=H, R3=H 17  4-Hydroxybenzaldehyde: R1=H, R2=OH, R3=H 10  Protocatechuic aldehyde: R1=OH, R2=OH, R3=H 18  Isovanillin: R1=OH, R2=OCH3, R3=H 13  Vanillin: R1=OCH3, R2=OH, R3=H 11  3,4-Dihydroxy 5-methoxybenzaldehyde:R1=OH, R2=OH, R3=OCH3 14  Syringaldehyde: R1=OCH3, R2=OH, R3=OCH3 15  3,4-Dimethoxybenzaldehyde: R1=OCH3, R2=OCH3, R3=HB. 12  Protocatechuic acid840  P. Kota et al./Phytochemistry 65 (2004) 837–846  aldehyde ( 10 ). The elution profiles of incorporation of radioactivity in the products from  14 C-labeled SAM aresuperimposed on the UV traces. The collective resultsindicate that the only methylation product of proto-catechuic aldehyde ( 10 ) was, vanillin ( 13 ), and that syring-aldehyde ( 14 ) was the product formed from 3,4-dihydroxy, 5-methoxybenzaldehyde ( 11 ). Because of thepotential volatility of benzaldehydes, total radioactivitywas determined before and after enzymatic incubation.No loss was observed.Table 2 shows the extent of methylation of differentbenzaldehyde derivatives by recombinant alfalfa COMT.3-Hydroxybenzaldehyde ( 16 ) and 4-hydroxybenzldehyde( 17 ) were not methylated. Similarly, vanillin ( 13 ) andsyringaldehyde ( 14 ) could not be methylated by COMT.In contrast, protocatechuic acid ( 12 ) and isovanillin ( 18 )were methylated, although to a lesser degree than proto-catechuic aldehyde ( 10 ) and 3,4-dihydroxy, 5-methoxy-benzaldehyde ( 11 ). Together with the results presentedin Fig. 3, it is clear that COMT can effectively catalyzethe SAM-dependent methylation of 3,4-dihydroxy-substituted benzaldehydes at the 3-OH ( meta ) position,but not at the 4-OH (  para ) position. 2.4. Kinetic discrimination of COMT by site-directed mutagenesis The crystal structure of COMT explains the broadsubstrate specificity of the enzyme (Zubieta et al., 2002).The active site of COMT is more spacious than that of other small molecule OMTs (Zubieta et al., 2001, 2002)and accommodates both 3- and 5-substituted C6–C3substrates (phenylpropanoids), with either acid or alde-hyde groups at the end of the 3-carbon propanoid tail.Whether C6–C3 or C6–C1, the aldehydes would ingeneral undergo more facile deprotonation of the tar-geted hydroxyl moiety in preparation for  O -methylationthan the corresponding acids. Indeed, the preferencewould be for the  meta -position deprotonation/methyl-ation (3- and 5-hydroxyl) rather than  para  directeddeprotonation/methylation (4-hydroxyl). Owing to theversatile structure of the binding site, aromatic compoundssmaller than phenylpropanoids will fit albeit with apossibly loose arrangement in the active site cavity nearthe putative histidine general base (His 269) and thereactive methyl group of a firmly bound SAM molecule.However, their binding might lack the constraints con-tributed by residues surrounding the propanoid tail. Onthe other hand, aldehydes lack a negative charge thatwould clash sterically and electronically with residuesincluding I316, M130, I319, and M180 that surroundthe propanoid tail (Zubieta et al., 2002). In total, thedifference in the  K  m  values between aldehydes andacids can be explained by effects due to the p K  a  shift of the –OHs and/or loss of repulsive interactions betweenthe acid tail and M180, I316, M130, and I319.Based on the crystal structure of alfalfa COMT, aseries of mutations was designed to alter residues thatcontact the aromatic ring and side chain of naturalCOMT substrates. They were then constructed by site-directed mutagenesis to facilitate the functional deter-mination of the catalytic and substrate recognition rolesof the residues lining the active site surface. These studiesultimately will help resolve the relative importance of key active site residues in the kinetic discriminationbetween monolignol precursors and various benzalde-hydes. The mutant enzymes were expressed in  E. coli  and purified to homogeneity. Table 3 shows the kinetic Table 3Kinetic properties of purified recombinant alfalfa COMT (WT) and a series of site-directed mutantsCOMT mutants  V  max / K  m Caffeicacid ( 1 )5-OH Coniferaldehyde( 3 )Protocatechuicaldehyde ( 10 )3,4-Dihydroxy, 5-methoxybenzaldehyde ( 11 )Protocatechuicacid ( 12 )WT 833/43 (19) 500/5 (100) 476/5 (102) 2000/16 (122) 128/515 (0.3)L136Y 303/10 (29) 278/13 (21) 909/15 (60) 1250/31 (31) 196/52 (52)A162T 476/12 (39) 455/11 (44) 556/10 (57) 1667/39 (43) NDM130L ND 909/28 (32) 43/19 (2) 250/12 (21) NDF172Y ND 333/10 (33) 526/153 (3) 1429/27 (52) NDN131L 250/335 (0.8) 500/13 (40) 179/10 (18) 1000/13 (79) NDN131K 556/9 (60) 417/12 (34) 3333/55 (61) 2500/57 (44) 250/352 (1)N131D 278/121 (2) 5000/120 (42) ND ND NDN131E 9/214 (0.04) 68/1 (60) ND ND NDN324Y 83/133 (0.6) 714/17 (42) 57/6 (10) 370/5 (71) NDN324HM130L 250/32 (8) 1000/41 (24) 222/6 (37) 3333/87 (39) 667/357 (2)H183K 167/140 (1) 455/8 (56) ND ND NDThe mutations are given as single-letter amino acid codes. For each mutant, values are given for  V  max  and  K  m  and numbers in parentheses show the V  max / K  m  ratio.  V  max  values are given as pkat/mg COMT, and  K  m  values are expressed in  m M. ND, no activity determined. Assays were performed induplicate or triplicate (maximum analytical variation less than   5%). P. Kota et al./Phytochemistry 65 (2004) 837–846  841
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