Biochemical Characterization of Sinapoylglucose:Choline Sinapoyltransferase, a Serine Carboxypeptidase-like Protein That Functions as an Acyltransferase in Plant Secondary Metabolism*

Amber M. Shirley {ddagger} and Clint Chapple §

From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Received for publication, March 6, 2003
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, serine carboxypeptidase-like (SCPL) proteins that catalyze transacylation reactions in plant secondary metabolism have been identified from wild tomato and Arabidopsis. These include sinapoylglucose: choline sinapoyltransferase (SCT), an enzyme that functions in Arabidopsis sinapate ester synthesis. SCT and the other known SCPL acyltransferases all share the conserved serine, aspartic acid, and histidine residues employed for catalysis by classical serine carboxypeptidases, although the importance of these residues and the mechanism by which this class of SCPL proteins catalyze acyltransferase reactions is unknown. To characterize further SCT and its catalytic mechanism, we have employed the Saccharomyces cerevisiae vacuolar protein localization 1 mutant, which secretes the serine carboxypeptidase, carboxypeptidase Y, and other proteins normally targeted to the vacuole. When expressed in this strain, SCT is similarly secreted. SCT has been purified from the yeast medium and used for kinetic characterization of the protein. Immunological analysis of SCT has revealed that the expected 50-kDa mature protein is proteolytically processed in yeast and in planta, most likely resulting in the production of a heterodimer derived from a 30- and 17-kDa polypeptide.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Arabidopsis and some other members of the Brassicaceae, the end products of the phenylpropanoid pathway include the sinapate esters, sinapoylmalate and sinapoylcholine. Sinapoylglucose:malate sinapoyltransferase (SMT)1 catalyzes the final reaction in the formation of sinapoylmalate, the predominant sinapate ester and a major UV protectant in Arabidopsis leaves (1, 2). The most abundant sinapate ester in seeds is sinapoylcholine, also known as sinapine, the formation of which is catalyzed by sinapoylglucose:choline sinapoyltransferase (SCT) (Fig. 1). Radiotracer feeding experiments with [14C]choline and [14C]ethanolamine indicated that sinapoylcholine may serve as an important seed storage form of choline for the subsequent synthesis of phosphatidylcholine in developing seedlings (3). In contrast, the isolation of the fah1 and sng2 mutants, each of which accumulates free choline instead of sinapoylcholine (4, 5), suggests that the function of sinapoylcholine is still unclear. Nevertheless, the accumulation of sinapoylcholine in seeds is of agronomic importance because it has a negative impact on the value of canola (Brassica sp.) meal (6).



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FIG. 1.
The reaction catalyzed by sinapoylglucose:choline sinapoyltransferase (SCT).

 

Both SMT and SCT belong to a newly described class of proteins from plant secondary metabolism that are homologous to serine carboxypeptidases and yet function as acyltransferases (5, 7, 8) or lyases (9). Until recently, serine carboxypeptidase-like (SCPL) proteins from plants had been identified and characterized based primarily upon their ability to degrade model peptide substrates (10, 11, 12, 13). Although the in vivo targets of most of these enzymes remain unknown, serine carboxypeptidases have traditionally been thought to function in protein turnover and processing because they catalyze the hydrolytic cleavage of C-terminal peptide bonds.

Serine carboxypeptidases belong to the larger {alpha}/{beta} hydrolase superfamily of proteins and contain conserved serine, aspartic acid, and histidine residues that make up a catalytic triad (14, 15, 16, 17, 18, 19). The proposed mechanism of catalysis is similar to serine endopeptidases, which involves the formation of an acyl enzyme intermediate followed by its subsequent hydrolysis.

Considering their apparent evolutionary relationship with hydrolases, the mechanism by which SCPL proteins act as acyltransferases is of significant interest. Kinetic analysis of SCT has been reported previously following full or partial purification of the enzyme from seeds of Raphanus sativus, Sinapis alba, and Brassica napus (20, 21); however, these studies have provided conflicting reports concerning the catalytic mechanism of the enzyme. In light of the recent cloning of SCT from Arabidopsis and the identification of SCT as an SCPL protein (5), in this paper we describe a detailed analysis of activity, substrate specificity, and processing of SCT. These results were obtained using a novel heterologous Saccharomyces cerevisiae expression system that lacks the carboxypeptidase Y (CPY) gene and is defective in vacuolar targeting, resulting in secretion of heterologously expressed SCT.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains—The construction of the S. cerevisiae strain W2579 (MATa {Delta}prc1 vpl1-1 leu2-3 leu2-112 ura3-52) has been described (22). The W2579 strain was obtained from Carlsberg Research Center, Copenhagen, Denmark.

Constructs for Expression of SCT in S. cerevisiae—Standard techniques were used for DNA manipulations (23). For construction of the SCT yeast expression vector, two oligonucleotides were designed to amplify the SCT cDNA. The 5'-oligonucleotide (5'-GAATTCAAAATGGGAAACCTTAGC-3') incorporated an EcoRI restriction site before the SCT start codon, and the 3'-oligonucleotide (5'-GCATGCGTCTTAAACTGTAACAAC-3') incorporated an SphI restriction site after the stop codon. The SCT gene was amplified by PCR, subcloned into EcoRV-digested pBS KS-, and sequenced using thermosequenase fluorescent-labeled primer cycle sequencing kit (Amersham Biosciences) with the IRD-800 M13 forward and IRD-700 M13 reverse primers (LICOR, Lincoln, NE). The reaction products were analyzed with the LongReadIR DNA 4200 automated DNA sequencer (LICOR, Lincoln, NE). The coding sequence was then excised by EcoRI/SphI digestion and cloned into EcoRI/SphI-digested pYES2 vector (Invitrogen) to yield pYES2-SCT. For analysis of SCT expression and activity, the S. cerevisiae host W2579 (22) was transformed with the empty pYES2 vector and pYES2-SCT as described (24).

Yeast Growth Conditions and Preparation of Yeast Extracts—W2579 cells carrying pYES2 and pYES2-SCT were grown in media supplemented with 100 mg liter-1 of L-leucine to complement the leucine-deficient W2579 strain. SCT production was induced by the addition of galactose to stationary phase cultures (25). After 24 h, cultures were centrifuged at 3,000 x g for 10 min. To assay SCT retained within cells, cell pellets were resuspended in two volumes of 50 mM MOPS (pH 7.0). One cell volume of 0.5 mm of zirconia/silica beads (Biospec Products Inc., Bartlesville, OH) was added, and the cell suspension was vortexed at high speed for 1 min and then placed on ice for 1 min. This process was repeated four additional times. The cell lysate was then centrifuged at 3,000 x g for 10 min and assayed for SCT activity as described below. To assay SCT secreted from cells, protein was precipitated from the yeast medium by adding solid ammonium sulfate to obtain 50% saturation. After stirring for 30 min, the precipitate was removed by centrifugation at 12,000 x g for 20 min and discarded. The supernatant was then raised to 70% saturation with ammonium sulfate, and after stirring for 30 min, the precipitated protein was collected by centrifugation at 12,000 x g for 20 min. The ammonium sulfate precipitate was dissolved in 50 mM Tris-HCl (pH 7.5), containing 1 mM 1,4-dithio-DL-threitol, and was dialyzed for 18 h against two changes of 4 liters of the same buffer. The dialysate was then incubated at 60 °C for 10 min, cooled to 4 °C, and centrifuged at 12,000 x g for 20 min. The supernatant was then passed through a Q-Sepharose fast flow (Sigma) column (30-ml bed volume) that had been equilibrated in 50 mM Tris-HCl (pH 7.5). The column flow-through was collected and made to 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2. The sample was then applied to a concanavalin A-Sepharose 4B (Sigma) column (3-ml bed volume) at a flow rate of 0.5 ml min-1. The column was washed with 3 times with 4 ml of 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and then the bound protein was eluted with 3 times with 4 ml of wash buffer containing 0.6 M methyl-{alpha}-D-mannopyranoside. The bound protein fraction was dialyzed for 18 h against two changes of 4 liters of 50 mM MOPS (pH 7.0) containing 20% (v/v) glycerol. The protein was desalted using PD-10 Sephadex® G-25 M columns (Supelco; Belefonte, PA) into 50 mM MOPS (pH 7.0) immediately prior to enzyme assays. Protein content was determined using the Bio-Rad Protein Assay (Bio-Rad) (26).

Enzyme Assays—SCT assays were initiated by the addition of 5 µl of protein to tubes containing substrate in 20 µl of 50 mM MOPS (pH 7.0). The assays were incubated for 60 min at 30 °C, terminated by the addition of 37.5 µl of methanol, and centrifuged for 10 min at 5,000 x g. In each case, 20 µl of sample was analyzed directly by HPLC on a PuresilTM C18 column (Waters) (120-Å pore size, 5-µm particle size) using a 12-min gradient from 1.5% acetic acid to 22.5% acetonitrile at a flow rate of 1 ml min-1 using UV detection at 335 nm, except for caffeic acid and caffeoylcholine, which were detected at 315 nm. Hydroxycinnamate esters were quantified using the extinction coefficient of the corresponding hydroxycinnamic acids. Sinapoylglucose for use in enzyme assays was purified from the sng1 mutant of Arabidopsis (27).

Expression of CPY in S. cerevisiae—The p72UG vector (22) was obtained from Carlsberg Research Center, Copenhagen, Denmark, and was transformed into the host W2579 as described (24). Transformed cells were grown in media for 18 h and then induced with galactose (25). After 24 h, the cultures were centrifuged at 2,000 x g for 10 min to pellet the cells, and the supernatant was then assayed directly for CPY activity by monitoring the decrease in absorbance at 339 nm of 0.2 mM N-(3-[2-furyl]acryloyl)-Phe-Phe (Sigma) in 50 mM MES and 1 mM EDTA (pH 6.5) over a 30-min period, as described previously (28). Assays to test SCT for carboxypeptidase activity were performed similarly.

Plant Material and Preparation of Protein Extracts—Arabidopsis thaliana L. Heynh. ecotype Landsberg erecta (Ler) was cultivated at a light intensity of 100 microeinsteins m-2 s-1 at 23 °C under a photoperiod of 16 h light/8 h dark in Redi-Earth potting mix (Scotts-Sierra Horticultural Products; Marysville, OH). For preparation of protein extracts for gel blot analysis, 10 g of light yellow to fully mature siliques from Ler or sng2 were frozen in liquid nitrogen and ground to a fine powder after which desalted protein extracts were prepared as described previously (5).

Generation of Polyclonal SCT Antibodies—To generate anti-SCT polyclonal antibodies, the predicted mature SCT protein was expressed in Escherichia coli and isolated from the insoluble fraction as described previously (5). The insoluble fraction containing SCT was electrophoretically separated by SDS-PAGE on a 10% acrylamide gel (29). The gel was copper-stained (30), and the SCT band was extracted from the gel by electroelution into 20 mM Tris and 150 mM glycine buffer using the ElutrapTM Electroelution System (Schleicher & Schuell). The purified protein was used as antigen for the generation of polyclonal antibodies in rabbit using Cocalico Biologicals, Inc., antibody production services (Reamstown, PA).

Immunological Analysis of SCT—Protein gel blotting was performed using a recently developed technique for direct quantification of Cy3 fluorescence.2 Briefly, following SDS-PAGE, proteins were transferred to Westran® polyvinylidene difluoride membrane (Schleicher & Schuell) in 20 mM Tris, 150 mM glycine, and 20% (v/v) methanol buffer at 50 V for 1 h. The membrane was then washed for 1 h in PBS buffer (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl) containing 5% (w/v) powdered milk. The membrane was washed with PBS buffer containing 0.1% (v/v) Tween 20 for 15 min and then incubated for 1 h in 2% powdered milk in PBS buffer containing a 1:1000 dilution of the polyclonal SCT antibody. The membrane was washed again with PBS buffer containing 0.1% (v/v) Tween 20 for 15 min and then incubated in 2% (w/v) powdered milk in PBS buffer containing a 1:1000 dilution of anti-rabbit IgG Cy3TM conjugated secondary antibody (Sigma) for 1 h. The membrane was finally washed with PBS buffer containing 0.1% (v/v) Tween 20 for 15 min. Cy3 fluorescence was visualized using a Typhoon 8600 Variable Mode Imager (Amersham Biosciences) in fluorescence mode using the green 532 nm laser with Tamra 580 BP 30 filter at 400 PMT sensitivity.

Deglycosylation of SCT—SCT protein purified from yeast media was concentrated using a Microcon YM-30 (Millipore, Bedford, MA). Concentrated protein (150 µg) was deglycosylated using Recombinant N-Glycanase FTM (Glyko, Novato, CA) according to the manufacturer's instructions.

Matrix-assisted Laser Desorption Ionization/Mass Spectrometry— Matrix-assisted laser desorption ionization-mass spectrometry was performed using a PerSeptive Biosystems (Framingham, MA) Voyager mass spectrometer, equipped with a nitrogen laser (337 nm UV laser) for ionization and a time-of-flight mass analyzer. The sample and matrix ({alpha}-cyano-4-hydroxycinnamic acid) were mixed in a ratio of 1 to 1 µl on the sample plate and allowed to air-dry prior to analysis.

LC/MS—LC-MS analyses were performed on a Micromass Q-TOF hybrid time-of-flight/quadrupole LC/MS system. Samples were separated on a Hewlett-Packard HP-1100 binary liquid chromatography (LC) system with UV diode-array detection. LC separations were performed using a PuresilTM C18 column (Waters) (120-Å pore size, 5-µm particle size) with a 23-min gradient from 1.5% acetic acid to 70% acetonitrile at a flow rate of 1 ml min-1 using UV detection at 335 nm. The post-column effluent stream was split ~1:4 (ESI:Waste), and the ESI portion was combined with a flow of internal standard using an LC Tee. An internal standard mixture containing ~10 µg ml-1 spinosad and ~20 µg ml-1 caffeine was supplied via a Hamilton syringe pump at flows from 1 to 10 µl min-1. The resulting sample stream was introduced into the Q-TOF using the Z-Spray electrospray source. The Q-TOF was operated in both positive electrospray (+ESI) and negative electrospray (-ESI) modes using data-dependent triggering between MS and MS/MS modes. Theoretical values for the [M + H]+ internal standards ions in +ESI were m/z 195.0882 (caffeine) and m/z 732.4687 (spinosyn A). Accurate mass measurements were made using spinosyn A as the reference lock mass. Prior to analysis, the Q-TOF was calibrated to ±0.005 Da (+ESI) using a solution of sodium trifluoroacetate.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of SCT in Yeast and Purification of SCT from Yeast Medium—The Carlsberg yeast strain W2579 was generated to synthesize large amounts of CPY and secrete the protein to the medium (22). To determine whether this yeast strain could be used to express and secrete SCT, cultures of cells harboring pYES2 and pYES2-SCT were grown to stationary phase and then induced to produce protein by the addition of galactose. Cell extracts and the medium supernatant were then assayed by HPLC for SCT activity. No SCT activity was detected in either the cells or supernatant fractions of cultures harboring pYES2. In cultures carrying pYES2-SCT, SCT activity was readily detected in the medium (Fig. 2), and only low levels (~10% of the total) of SCT activity were recovered in the yeast cell lysate. In contrast, when pYES2-SCT was transferred into a yeast strain not defective in vacuolar targeting, SCT activity could only be found in yeast cell extracts (data not shown). These data demonstrate that like CPY, SCT is secreted in prc1/vpl1 yeast.



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FIG. 2.
Assays of SCT activity from yeast medium. SCT assays from yeast harboring pYES2-empty vector (top) and pYES2-SCT (bottom) were analyzed by HPLC with UV detection at 335 nm. SC, sinapoylcholine.

 

In order to conduct a detailed characterization of SCT, the protein was partially purified from the medium with an overall yield of 3.8% and a 12-fold increase in specific activity (Table I). After precipitation with ammonium sulfate, the protein was dialyzed and then heat-treated to denature temperature-sensitive proteins. SCT has been reported previously to be a heat-stable enzyme (20), and the yeast-expressed protein was similarly stable at 60 °C for at least 1 h. Having found that SCT does not bind to ion exchange media at neutral pH (data not shown), the heat-treated preparation was passed through a Q-Sepharose fast flow column to remove all anionic protein species. Finally, because preliminary data indicated that SCT is a glycoprotein, the protein was further purified using concanavalin A-Sepharose 4B. In assays of the final enzyme fraction, SCT activity was linear for at least 60 min over a wide range of protein concentrations and was stable at -20 °Cinthe presence of 20% glycerol for several months without loss of activity (data not shown).


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TABLE I
Purification of SCT from S. cerevisiae vpl1 mutant medium

 

Kinetic Analysis of SCT Activity—Unlike other methods previously used for the measurement of SCT activity (21), the HPLC method used in this study enabled the simultaneous detection of sinapoylcholine and any other sinapoylglucose-derived products, including sinapic acid, a potential spontaneous or enzyme-catalyzed hydrolytic product. Indeed, initial experiments demonstrated the enzyme-catalyzed formation of a compound that co-chromatographed with sinapic acid in SCT assays conducted at non-saturating choline concentrations, suggesting that SCT can function as an esterase. Surprisingly, LC-MS analysis of the product (accurate mass of [M-H]-= 297.1038 ± 0.010) indicated that the compound was sinapoylglycerol, not sinapic acid, suggesting that SCT can use the glycerol found in the SCT assay buffer as a sinapate acceptor. To test this hypothesis, SCT was desalted into MOPS, with and without glycerol, and was used in conventional SCT assays. In these experiments, the production of the putative sinapoylglycerol was dependent upon the presence of glycerol in the buffer, providing conclusive evidence that SCT can use glycerol as a sinapate acceptor (data not shown). In order to avoid this side reaction in future assays, a new protein preparation was made and exchanged into MOPS buffer without glycerol as the final step in purification. Unfortunately, in the absence of glycerol, SCT was completely inactivated during the overnight dialysis step; therefore, all further protein preparations were stored in buffer containing 20% glycerol and desalted into buffer without glycerol just prior to kinetic analysis. Under these conditions, no spontaneous or enzyme-catalyzed sinapoylglucose esterase or sinapoylcholine esterase activity was detectable under standard assay conditions (data not shown).

In addition to the unexpected sinapoylglycerol product, it was noted that a novel SCT-dependent peak with a retention time 2 min less than sinapoylcholine was observed in SCT assays conducted during purification of the protein using Tris buffer. In contrast, when protein was desalted into phosphate buffer prior to assay, this novel peak was not present (data not shown). LC-MS analysis of the product (accurate mass of [M + H]+ 328.1396 ± 0.005) confirmed that it was sinapoyl-2-amino-2-hydroxymethyl-1,3-propanediol. Based upon these results, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) was analyzed as a sinapate acceptor and was found to demonstrate Michaelis-Menten kinetics with regard to transesterification (Table II). Following the identification of this activity, SCT was routinely exchanged into MOPS buffer as a final step in purification to avoid background activity against Tris.


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TABLE II
Kinetic constants for SCT assayed with various sinapate donors and acceptors

 

To examine the in vitro kinetics of SCT with regard to its natural in vivo substrates, the conversion of sinapoylglucose to sinapoylcholine in the presence of choline was assayed by HPLC. As reported previously (5), the reaction catalyzed by SCT was observed to proceed spontaneously at a readily measurable rate in the absence of protein; therefore, all assay results were corrected for spontaneously produced sinapoylcholine. When assayed in the presence of 100 mM choline, semi-purified SCT appeared to demonstrate Michaelis-Menten kinetics with regard to sinapoylcholine transesterification with an apparent Km of 160 µM for sinapoylglucose and an apparent Vmax of 2.8 x 103 picokatals mg-1 (Fig. 3A). SCT also demonstrated Michaelis-Menten kinetics with regard to sinapoylcholine transesterification in the presence of 1 mM sinapoylglucose with an apparent Km of 3.2 mM for choline and an apparent Vmax of 2.5 x 103 picokatals mg-1 (Fig. 3B).



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FIG. 3.
Kinetic analysis of SCT-catalyzed substrate transacylation. Analysis with sinapoylglucose at 100 mM choline (A) and analysis with choline at 1 mM sinapoylglucose (B). For each data point, spontaneous activity was subtracted. Error bars represent 1 ± S.D. for triplicate assays.

 

In order to investigate the determinants for substrate binding, the substrate specificity of SCT was analyzed with a variety of hydroxycinnamoylglucose esters and potential sinapate acceptors. In assays containing each of the hydroxycinnamoylglucose esters, a novel peak was identified by HPLC, and the identity of each product was confirmed by matrix-assisted laser desorption ionization mass spectroscopy (feruloylcholine M = 280.054; caffeoylcholine M = 266.017; p-coumaroylcholine M = 250.17). All of the hydroxycinnamoylglucose esters assayed demonstrated similar kinetics with regard to transesterification (Table II). Although the apparent Km value for caffeoylglucose was lower than the apparent Km value for sinapoylglucose, the apparent Vmax value for caffeoylglucose was ~3-fold less than the apparent Vmax value for sinapoylglucose. Conversely, the apparent Km value for p-coumaroylglucose was 10-fold higher than the apparent Km value for sinapoylglucose, but the apparent Vmax value for p-coumaroylglucose was greater than the apparent Vmax value for sinapoylglucose. As a result, the Vmax/Km values for each substrate were similar. In contrast, analysis of the substrate specificity of SCT for the sinapate acceptor indicated a clear specificity for choline (Table II). In assays containing each of the sinapate acceptors analyzed, a novel peak was identified by HPLC, and the identity of each product was confirmed by LC-MS (sinapoyl-N,N-dimethylethanolamine, accurate mass of [M + H]+ 296.1463 ± 0.005; sinapoyl-2-methylaminoethanol, accurate mass of [M + H]+ 282.1378 ± 0.005). All of the sinapate acceptors assayed, except for ethanolamine, demonstrated Michaelis-Menten kinetics with regard to transesterification (Table II). For ethanolamine, a novel peak dependent upon the presence of ethanolamine was present in the HPLC chromatograms; however, the rate of product formation in the reactions was insufficient for determination of an apparent Km or Vmax. Analysis of the putative sinapoylethanolamine peak by LC-MS gave a mass consistent with sinapoylethanolamine, but its concentration was too low for an accurate mass determination. Choline analogs with successively fewer N-methyl substitutions displayed progressively higher apparent Km and lower apparent Vmax values for transacylation activity (Table II).

In addition to the ethanolamine derivatives, neopentyl alcohol (3,3-dimethylpropan-1-ol) was analyzed as a sinapate acceptor. Neopentyl alcohol has a structure similar to that of choline, but it does not contain a nitrogen atom and its accompanying positive charge. A novel peak dependent upon the addition of neopentyl alcohol was detected by HPLC, but neopentylsinapate was not amenable to ionization by LC-MS. Although this characteristic precluded positive identification of the product, neopentyl alcohol appeared to demonstrate Michaelis-Menten kinetics with regard to transesterification (Table II).

Analysis of SCT Serine Carboxypeptidase Activity—Even though SCT-catalyzed hydrolysis of sinapoylglucose and sinapoylcholine was not detectable; SCT was analyzed for its ability to catalyze an authentic serine carboxypeptidase reaction. Because SCT presumably evolved from enzymes capable of binding peptide substrates, it was of interest to determine whether SCT retains any residual hydrolytic activity toward a model peptide substrate. Although CPY readily degraded N-(3-[2-furyl]acryloyl)-Phe-Phe, SCT could not catalyze the hydrolysis of the substrate under the conditions assayed (Fig. 4).



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FIG. 4.
Analysis of carboxypeptidase activity of CPY and SCT. The hydrolysis of 0.2 mM N-(3-[2-furyl]acryloyl)-Phe-Phe was followed spectrophotometrically by a decrease in absorption at 339 nm. •, CPY; {blacktriangledown} and {circ}, independent SCT samples.

 

Analysis of SCT Transesterification Mechanism—To investigate the order of the SCT transacylation reaction, SCT assays were conducted at three fixed concentrations of choline and four fixed concentrations of sinapoylglucose. Unexpectedly, these data were not clearly consistent with plots expected for enzymes with a double displacement or bi-bi mechanism (Fig. 5, A and B). Lineweaver-Burk plots of SCT activity at 10 and 1 mM choline with variable sinapoylglucose concentrations generated a pair of parallel lines, consistent with a double displacement mechanism (Fig. 5A) (31). In contrast, at the lowest concentration of sinapoylglucose, assay velocities measured in the presence of 100 mM choline were lower than when the enzyme was assayed in the presence of 10 mM choline. Because these data cause this line of the plot to cross the other two, it is difficult to conclude which type of mechanism SCT employs. Furthermore, these findings are indicative of substrate inhibition by choline (Fig. 5A). Similarly, at 0.07 mM sinapoylglucose, the lowest fixed concentration assayed, SCT activity decreased in the presence of high concentrations of choline, again indicative of substrate inhibition (Fig. 5B). Because Lineweaver-Burk plots led to inconclusive results with regard to mechanism, the data from these experiments were analyzed using the method of Hanes (32). Assays conducted with variable concentrations of sinapoylglucose at three fixed concentrations of choline generated lines with no common intersection point and with all pairwise intersects at positive values of sinapoylglucose concentration (Fig. 5C). Furthermore, comparable analysis of assays conducted with choline at four fixed concentrations of sinapoylglucose led to parabolic lines with a common intersection at the y axis (Fig. 5D). The results of both of these plots are indicative of substrate inhibition and, most importantly, provide strong evidence of a double displacement mechanism for SCT transacylation (33).



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FIG. 5.
Kinetic analysis of the SCT reaction. A, Lineweaver-Burk plots of SCT activity at three fixed concentrations of choline: •, 100 mM; {circ}, 10 mM; {blacktriangledown}, 1 mM. B, Lineweaver-Burk plots of SCT activity at four fixed concentrations of sinapoylglucose: •, 1 mM; {circ}, 0.375 mM; {blacktriangledown}, 0.15 mM; {triangledown}, 0.07 mM. C, Hanes plots of SCT activity at three fixed concentrations of choline: •, 100 mM; {circ},10mM; {blacktriangledown},1mM. D, Hanes plots of SCT activity at four fixed concentrations of sinapoylglucose: •, 1 mM; {circ}, 0.375 mM; {blacktriangledown}, 0.15 mM; {triangledown}, 0.07 mM. For each data point, spontaneous activity was subtracted. Error bars represent 1 ± S.D. for triplicate assays.

 

Immunological Analysis of SCT—The first 19 amino acids of SMT is a propeptide that is cleaved in planta to generate the mature protein (7), and SCT is predicted to carry a similar propeptide (5). To determine whether SCT was N-terminally processed in the prc1/vpl1 yeast expression system, SCT expressed in E. coli (Fig. 6A) was used to generate rabbit polyclonal anti-SCT antibodies that were then used to probe a protein gel blot. Unexpectedly, instead of a single band at the predicted size of the unprocessed protein (52 kDa) or the N-terminally processed protein (50 kDa), the SCT antibody recognized bands at ~35, 31, 29, and 27 kDa (Fig. 6C) that were not present in yeast containing the pYES2 empty vector (Fig. 6B). In order to determine whether the immunoreactive bands were degradation products of SCT or if the protein was processed in a unique and artifactual way in the yeast expression system, a gel blot of Arabidopsis silique extracts was similarly probed with the anti-SCT antibody. Like the data obtained when SCT was expressed in yeast, the SCT antibody recognized several bands in Ler silique extracts at ~31, 28, 26, and 22 kDa (Fig. 6E), although the band at 22 kDa was present in the preimmune serum blot (Fig. 6D). Extracts of the sng2 mutant were included as a control because the mutant is known to accumulate low levels of SCT mRNA (5), and thus might accumulate low levels of SCT protein. As predicted, the bands present in the Ler extracts were dramatically reduced in extracts of sng2 siliques, indicating that these bands were all attributable to SCT (Fig. 6E). Considering that all of the immunoreactive peptides are smaller than the predicted intact polypeptide, and that in sum they exceed its mass, these data suggest that both in planta and in yeast, SCT is proteolytically processed and possibly post-translationally modified.



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FIG. 6.
Protein gel blot analysis of SCT. A, SDS-PAGE analysis of SCT expressed in E. coli. Lane 1, total E. coli insoluble fraction containing SCT; lane 2, SCT protein after electroelution. B and C, protein gel blot analysis of SCT expressed in yeast and probed with preimmune serum (B), and polyclonal SCT antibodies (C). Lane 1, SCT purified from E. coli; lane 2, ammonium sulfate precipitate of medium from yeast harboring pYES2; lane 3, ammonium sulfate precipitate of medium from yeast harboring pYES2-SCT. D and E, protein gel blot analysis of SCT expressed in planta and probed with preimmune serum (D), and polyclonal SCT antibodies (E). Lane 1, crude extract from Arabidopsis Landsberg erecta maturing siliques; lane 2, crude extract from Arabidopsis sng2 maturing siliques.

 

In order to investigate the possibility that the multiple bands present on the protein gel blot represented differential glycosylation, SCT purified from yeast medium was deglycosylated using N-glycanase to remove asparagine-linked glycosyl chains. When the deglycosylated protein was subjected to protein gel blot analysis, two bands at ~30 and 17 kDa were present (Fig. 7). In this context, it is important to note that wheat serine carboxypeptidase II (CPDW-II) is proteolytically processed. The CPDW-II protein is synthesized as a single polypeptide chain and post-translationally processed to remove a 27-amino acid polypeptide in order to form a heterodimer of the A and B chain (17, 34). To determine whether SCT might be processed at a similar site, SCT was aligned with CPY, SMT, and the entire CPDW-II primary translation product using the ClustalW algorithm. SCT contains a 26-amino acid sequence, not found in CPY and SMT, that corresponds with the site of processing in CPDW-II (Fig. 8). Removal of this sequence in SCT would result in two polypeptides, 17 and 30 kDa in size. These masses are consistent with the two bands on the protein gel blot after deglycosylation, suggesting that SCT is proteolytically processed, probably generating a heterodimeric protein derived from the primary translation product (Fig. 8).



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FIG. 7.
Protein gel blot analysis of SCT deglycosylation. SCT purified from yeast medium before (lane 1) and after (lane 2) deglycosylation with N-Glycanase FTM.

 


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FIG. 8.
Alignment of SCT with SCPL proteins. Alignment of SCT with the yeast CPY (GenBankTM accession number 1YSC [PDB] ), serine CPDW-II from wheat (GenBankTM accession number P08819 [GenBank] ) including the 27-amino acid sequence from residues 260 to 286 which is removed post-translationally, SMT (GenBankTM accession number AF275313 [GenBank] ), and SCT (GenBankTM accession number AY033947 [GenBank] ) prepared by using ClustalW algorithm. Amino acids that are identical in two or more proteins are shaded in black, and conservative amino acid substitutions are shaded in gray. Dashes denote gaps introduced to optimize the amino acid alignment. Putative active site residues in SCT, based upon their alignment with the CPY catalytic triad, are designated with asterisks. The underlined region of CPDW-II bordered by inverted triangles corresponds to the residues proteolytically removed from the mature protein. The double underlined regions of SCT correspond to the consensus sequences for N-glycosylation.

 

The SCT protein was scanned for potential N-glycosylation sites with the consensus sequence of Asn-Xaa-(Ser/Thr) (35) in order to determine whether differential glycosylation of the 30- and 17-kDa polypeptides could explain the multiple bands present in the yeast protein fractions before de-glycosylation. Four potential N-glycosylation sites were identified in the SCT protein: one in the proposed 30-kDa polypeptide, two in the proposed 17-kDa polypeptide, and one in the 26-amino acid sequence presumably removed by processing (Fig. 8). Glycosylation at the potential N-glycosylation site within the 30-kDa SCT polypeptide could account for the shift in the largest immunoreactive SCT band on the protein gel blot after deglycosylation. Variable glycosylation at the two potential sites within the 17-kDa polypeptide could account for three immunoreactive bands below 30 kDa before deglycosylation (Fig. 7).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CPY is a yeast vacuolar protein that, when overexpressed in the vpl1 mutant, accumulates in the yeast medium to concentrations of over 30 mg liter-1 (22). Although the intracellular location of SCT is unknown, it is most likely targeted to the plant vacuole where SMT, another enzyme of sinapate ester biosynthesis, is found (36, 37). Because of the homology of SCT to serine carboxypeptidases, the probable similarity in intracellular location, and the inability to express active SCT protein in a previously described E. coli system, the vpl1 mutant was employed to express SCT. Although active SCT protein is secreted to the medium, the same high level of protein production obtained with CPY was not achieved with SCT. A possible explanation for this observation is that, unlike the vector used for CPY expression, the pYES2 vector used for SCT expression does not take advantage of the LEU2d marker, which maintains high plasmid copy number (22). Alternatively, the low level of secreted protein could also result from inability of the yeast to properly recognize the plant signal sequences of SCT. This could lead to degradation of the protein or to secretion of the unprocessed protein; however, SCT is not secreted in a VPL1 host, indicating that SCT is not shuttled through a default secretory pathway. Even though the level of secreted SCT protein is much less than that of CPY, the vpl1 mutant expression system provides an alternative for expression of other vacuolar plant proteins in a eukaryotic system. If SCT is representative of other vacuolar proteins, this expression system can be used to produce protein preparations that are not contaminated with cytosolic proteins and that can be used directly for assays of enzymatic activity. For example, with the completion of the Arabidopsis Genome Initiative, 51 SCPL genes have been identified in Arabidopsis; however, SMT and SCT are the only members of this group with known function. The prc1/vpl1 yeast expression system may be valuable for the expression and rapid identification of activity for the remaining proteins.

The immunological analysis of SCT from the yeast expression system revealed that the protein is proteolytically processed and differentially glycosylated. These results are consistent with a previous purification of SCT from B. napus which reported the presence of multiple polypeptides even in the most highly purified fractions. SCT was attributed to a 28-kDa band, whereas the other bands were thought to represent contaminating proteins (21). Expression of SCT in yeast and immunoblot analysis of the sng2 mutant has demonstrated that all of these species can be attributed to SCT. Similarly, the native molecular mass of B. napus SCT was estimated to be 65 kDa, an observation that led to the hypothesis that the enzyme is a dimer of two 28-kDa subunits (21). Instead, immunoblot analysis of SCT expressed in yeast indicates that the protein is likely to be a heterodimer of 30- and 17-kDa subunits, each of which contains some but not all of the proposed catalytic serine-, aspartic acid-, and histidine-active site residues. Furthermore, the discrepancy between the 65-kDa native molecular mass observed for B. napus SCT and the Arabidopsis protein is probably explained by glycosylation. The multiple bands reported for B. napus SCT are almost certainly the result of differential glycosylation that is seen in the yeast expression system as well as in Arabidopsis. This degree of glycosylation is consistent with that observed for CPDW-II and CPY that contain 17–20% N-linked carbohydrate (34, 38).

The proposed heterodimeric nature of SCT is consistent with that of the 1-O-isobutyryl-{beta}-glucose SCPL acyltransferase isolated from the wild tomato species Lycopersicon pennellii, for which highly purified fractions contained both a 34- and 24-kDa band (39). In fact, of the 51 SCPL genes that have been annotated in the Arabidopsis genome, approximately half of the deduced amino acid sequences of these proteins contain sequences that coincide with the insertion in SCT and the polypeptide sequence in CPDW-II that is removed during maturation of the protein. Thus, internal proteolytic processing may be a widespread phenomenon in plant SCPL proteins. Interestingly, a mechanism for proteolytic processing of SCT exists in both plants and yeast. Determining the mechanism by which this insertion is removed and whether or not SCT can be expressed without this insertion may provide insight into the possible role or requirement for these insertions in SCPL proteins. For example, when the inferred mature coding sequence of SCT was expressed using an E. coli expression system, it resulted in the production of insoluble protein of which only a very small percentage could be re-folded (5). Based upon this observation, it is tempting to speculate that the removal of the 26-amino acid polypeptide sequence in SCT may be required for proper folding of the protein and its enzymatic activity.

SCT did not demonstrate a distinct substrate preference for the hydroxycinnamoylglucose ester donor compounds analyzed. Although SCT purified from B. napus has been reported to exhibit a distinct preference for sinapoylglucose as the acyl donor (21), the relative apparent Km and Vmax values obtained for Arabidopsis SCT expressed in yeast compare well with those reported for SCT partially purified from R. sativus and S. alba (20). This broad substrate specificity may explain the accumulation of the various hydroxycinnamoylcholine esters in other members of the Brassicaceae, such as feruloylcholine from Cleome pungens (40) and isoferuloylcholine from Sibara virginica (L.) (41), and indicates that a separate acyltransferase activity is not necessarily required for their formation.

Interestingly, neopentyl alcohol was capable of functioning as a sinapate acceptor, indicating that SCT does not have an absolute requirement for binding a positively charged substrate. Similarly, the identification of Tris as a substrate for SCT and the unexpected finding that glycerol can also function as a sinapate acceptor may indicate that at sufficiently high concentrations, SCT can use a diverse array of alcohols as substrates. Tris has been suggested previously (42) to function as a sinapate acceptor for SCT, an observation that is confirmed by the LC-MS accurate mass obtained for sinapoyl-2-amino-2-hydroxymethyl-1,3-propanediol. The production of a novel product in the presence of Tris has also been reported for the 1-O-isobutyryl-{beta}-glucose SCPL acyltransferase purified from L. pennellii (39). Although this observation is probably not relevant in vivo, Tris buffer was used in the early purification steps of SCT from the yeast medium, and the presence of this alternate sinapate acceptor may explain the decrease in both total activity and specific activity when Tris was introduced as a buffer. Finally, although kinetic analysis of SCT required that the glycerol be removed from the protein buffer, the glycerol was required for stability of the protein. This finding is in agreement with previous reports (21) describing SCT purified from B. napus, which required the addition of 1 mM choline chloride in highly purified fractions to prevent rapid loss of activity. Stabilization by choline and glycerol may indicate that any SCT sinapate acceptor at sufficient concentrations can stabilize SCT, and as a result Tris may also have contributed to the stabilization of the protein during the purification process.

Because traditional serine carboxypeptidases catalyze hydrolytic reactions, it was of interest to determine whether SCT could function in an analogous manner as an esterase to convert sinapoylglucose to sinapic acid. Previously, purified extracts of a serine carboxypeptidase-like 1-O-isobutyryl-{beta}-glucose acyltransferase from wild tomato species L. pennellii have been reported to have esterase activity (39, 43, 44). SCPL acyltransferases have most likely been co-opted through evolutionary time from traditional serine carboxypeptidases and have evolved novel acyltransferase activity to function in plant secondary metabolism. The failure to detect any hydrolytic activity of SCT suggests that at least certain SCPL acyltransferases have become sufficiently refined to function as non-hydrolytic enzymes in plant secondary metabolism.

Defining the order of the SCT reaction is important for understanding the differences in the mechanism of catalysis used by SCPL acyltransferases and traditional serine carboxypeptidases. Traditional serine carboxypeptidases employ a double displacement mechanism of catalysis. Such a mechanism would require SCT to exclude water from its active site in order to prevent rapid hydrolysis of the proposed sinapoylated enzyme intermediate. In order to prevent this rapid hydrolysis, it has been suggested that SCT may employ a general base mechanism (5) such as that proposed for MhpC, a C-C hydrolase from E. coli (45). In this model, the catalytic serine of SCT would act as a general base to deprotonate the hydroxyl of choline with resulting direct nucleophilic attack on the ester of sinapoylglucose, a mechanism consistent with a random or sequential bi-bi mechanism. Two previous publications have addressed the order of the SCT reaction with conflicting results. Although kinetic analysis of SCT partially purified from R. sativus indicated a random or sequential bi-bi mechanism (20), SCT purified from B. napus exhibited a double displacement mechanism. The anomalous results observed at high choline and low sinapoylglucose concentrations with both the enzyme from B. napus (21) and with Arabidopsis SCT purified from the yeast expression system strongly suggest that SCT employs a double displacement mechanism of catalysis.

Although a double displacement reaction mechanism is in agreement with that employed by traditional serine carboxypeptidases, it leaves unknown the manner in which SCT excludes water from its active site, thereby preventing a hydrolytic esterase reaction. With a large family of genes annotated as encoding SCPL proteins in Arabidopsis and the continually increasing number of SCPL cDNAs from other plant genome sequencing projects, assigning a function to all of these proteins will become important. Understanding the determining factors for altering a serine carboxypeptidase so that it may function as an acyltransferase will hopefully provide the insight required to classify SCPL proteins as either carboxypeptidases or acyltransferases. Based upon the analysis of SCT, it appears that the transacylation activity of these enzymes is not engendered by an alteration of catalytic mechanism.


    FOOTNOTES
 
* This work was supported by a grant from the National Science Foundation (to C. C.) and graduate fellowships from the United States Department of Agriculture and Purdue University (to A. M. S.). This is Journal Paper Number 17084 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address, BASF Plant Science LLC, 26 Davis Dr., Research Triangle Park, NC 27709. Back

§ To whom correspondence should be addressed. Tel.: 765-494-0494; Fax: 765-496-7213; E-mail: chapple{at}purdue.edu.

1 The abbreviations used are: SMT, sinapoylglucose:malate sinapoyltransferase; SCT, sinapoylglucose:choline sinapoyltransferase; MOPS, 4-morpholinepropanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; PBS, phosphate-buffered saline; SCPL, serine carboxypeptidase-like; CPY, carboxypeptidase Y; LC, liquid chromatography, HPLC, high pressure liquid chromatography; MS, mass spectrometry, ESI, electrospray ionization; Q-TOF, quadrupole-time-of-flight; CPDW-II, carboxypeptidase II. Back

2 J. M. Humphreys and C. Chapple, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank the Carlsberg Research Center for the generous donation of the yeast vpl1 strain and the CPY p72UG plasmid. We thank Joelene Smith and Dr. Jeff Gilbert for the LC-MS analysis conducted at Dow Agrosciences, Indianapolis, IN. We thank Professor Dieter Strack for providing sinapoylcholine and Dr. Knut Meyer for providing feruloylglucose, caffeoylglucose, and p-coumaroylglucose.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Landry, L. G., Chapple, C. C. S., and Last, R. (1995) Plant Physiol. 109, 1159–1166[Abstract/Free Full Text]
  2. Booij-James, I. S., Dube, S. K., Jansen, M. A. K., Edelman, M., and Mattoo, A. K. (2000) Plant Physiol. 124, 1275–1283[Abstract/Free Full Text]
  3. Strack, D. (1981) Z. Naturforsch. 36, 215–221
  4. Chapple, C. C. S., Vogt, T., Ellis, B. E., and Somerville, C. R. (1992) Plant Cell 16, 735–743[CrossRef]
  5. Shirley, A. M., McMichael, C. M., and Chapple, C. (2001) Plant J. 28, 83–94[CrossRef][Medline] [Order article via Infotrieve]
  6. Hobson-Frohock, A., Fenwick, G. R., Heaney, R. K., Land, D. G., and Curtis, R. F. (1977) Br. Poult. Sci. 18, 539–541
  7. Lehfeldt, C., Shirley, A. M., Meyer, K., Ruegger, M. O., Cusumano, J. C., Viitanen, P. V., Strack, D., and Chapple, C. (2000) Plant Cell 12, 1295–1306[Abstract/Free Full Text]
  8. Li, A. X., and Steffens, J. C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6902–6907[Abstract/Free Full Text]
  9. Wajant, H., Mundry, K., and Pfizenmaier, K. (1994) Plant Mol. Biol. 26, 735–746[Medline] [Order article via Infotrieve]
  10. Bamforth, C. W., Martin, H. L., and Wainwright, T. (1979) J. Inst. Brew. 85, 334–338
  11. Umetsu, H., and Ichishima, E. (1983) Phytochemistry 22, 591–592[CrossRef]
  12. Hammerton, R. W., and Ho, T. D. (1986) Plant Physiol. 80, 692–697
  13. Mikola, L. (1986) Plant Physiol. 81, 823–829
  14. Bech, L. M., and Breddam, K. (1989) J. Biol. Chem. 262, 13726–13735
  15. Hayashi, R., Moore, S., and Stein, W. H. (1973) J. Biol. Chem. 248, 8366–8369[Abstract/Free Full Text]
  16. Hayashi, R., Bai, Y., and Hata, T. (1975) J. Biol. Chem. 250, 5221–5226[Abstract]
  17. Liao, D., and Remington, S. J. (1990) J. Biol. Chem. 265, 6528–6531[Abstract/Free Full Text]
  18. Liao, D., Breddam, K., Sweet, R. M., Bullock, T., and Remington, S. J. (1992) Biochemistry 31, 9796–9812[Medline] [Order article via Infotrieve]
  19. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., Schrag, J., Sussman, J. L., Verschueren, K. H. G., and Goldman, A. (1992) Protein Eng. 5, 197–211[Abstract]
  20. Gräwe, W., and Strack, D. (1986) Z. Naturforsch. 41, 28–33
  21. Vogt, T., Aebershold, R., and Ellis, B (1993) Arch. Biochem. Biophys. 300, 622–628[CrossRef][Medline] [Order article via Infotrieve]
  22. Nielsen, T. L., Holmberg, S., and Petersen, J. G. L. (1990) Appl. Microbiol. Biotechnol. 33, 307–312[Medline] [Order article via Infotrieve]
  23. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  24. Johnston, J. R. (1994) in Molecular Genetics of Yeast: A Practical Approach, (Johnston, J. R., ed) pp. 121–134, IRL Press at Oxford University Press, Oxford
  25. Urban, P., Werck-Reichhart, D., Teutsch, H., Durst, F., Regnier, S., Kazmaier, M., and Pompon, D. (1994) Eur. J. Biochem. 222, 843–850[Abstract]
  26. Bradford, M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
  27. Lorenzen, M., Racicot, V., Strack, D., and Chapple, C. (1996) Plant Physiol. 112, 1625–1630[Abstract/Free Full Text]
  28. Peterson, L. M., Holmquist, B., and Bethune, J. L. (1982) Anal. Biochem. 125, 420–426[Medline] [Order article via Infotrieve]
  29. Laemmli, U. K. (1970) Nature 227, 680–685[Medline] [Order article via Infotrieve]
  30. Lee, C., Levin, A., and Branton, D. (1987) Anal. Biochem. 166, 308–312[Medline] [Order article via Infotrieve]
  31. Lineweaver, H., and Burk, D. (1934) J. Am. Chem. Soc. 56, 658–666
  32. Hanes, C. S. (1932) Biochem. J. 26, 1406–1421
  33. Cornish-Bowden, A. (1995) Fundamentals of Enzyme Kinetics, pp. 148–149, Portland Press Ltd., London
  34. Breddam, K., Sørensen, S. B., and Svendsen, I. B. (1987) Carlsberg Res. Commun. 52, 297–311
  35. Marshall, R. D. (1972) Annu. Rev. Biochem. 41, 673–702[CrossRef][Medline] [Order article via Infotrieve]
  36. Sharma, V., and Strack, D. (1985) Planta 163, 563–568
  37. Hause, B., Meyer, K., Viitanen, P. V., Chapple, C., and Strack, D. (2002) Planta 215, 26–32[CrossRef][Medline] [Order article via Infotrieve]
  38. Endrizzi, J. A., Breddam, K., and Remington, S. J. (1994) Biochemistry 33, 11106–11120[Medline] [Order article via Infotrieve]
  39. Li, A. X., Eannetta, N., Ghangas, G. S., and Steffens, J. C. (1999) Plant Physiol. 121, 453–460[Abstract/Free Full Text]
  40. Clausen, S., Olsen, O., and Sørensen, H. (1982) Phytochemistry 21, 917–922[CrossRef]
  41. Gmelin, R., and Kjaer, A. (1970) Phytochemistry 9, 667–669[CrossRef]
  42. Strack, D., Knogge, W., and Dahlbender, B. (1983) Z. Naturforsch 38, 21–27
  43. Ghangas, G. S., and Steffens, J. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9911–9915[Abstract]
  44. Ghangas, G. S., and Steffens, J. C. (1995) Arch. Biochem. Biophys. 316, 370–377[CrossRef][Medline] [Order article via Infotrieve]
  45. Fleming, S. M., Robertson, T. A., Langley, G. J., and Bugg, T. D. H. (2000) Biochemistry 39, 1522–1531[CrossRef][Medline] [Order article via Infotrieve]