Supporting Information

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SI Materials and Methods

Protein concentration was determined by using the Bradford method (1) with BSA as a standard. The reaction mixture (100 ml) contained 2 mM (7E,7'E)-4-coumaryl 4-coumarate, 50 mM potassium phosphate buffer (pH 6.0), and enzyme (20 ml). The reaction was carried out at 30°C for 10 min. The reaction was stopped by the addition of EtOAc (200 ml), and then 2,4-dihydroxyacetophenone (10 mg) was added to the mixture as an internal standard. The mixture was centrifuged briefly and the organic layer was collected and dried. The resultant residue was redissolved in 25 ml of MeOH, from which 10 ml was injected into a Shimadzu SCL-6A HPLC (column: Waters Nova-Pak C₁₈; elution condition: CH₃CN:H₂O = 40:60, isocratic, 1 ml/min, monitored at 254 nm). The retention times were: 2,4-dihydroxyacetophenone, 2.0 min; (7E,7'E)-4-coumaryl 4-coumarate, 5.5 min; (E)-hinokiresinol, 6.4 min; (Z)-hinokiresinol, 6.6 min. The amount of product was calculated based on the ratio of the (Z)-hinokiresinol peak area to the internal-standard peak area.

Native PAGE was conducted on 2-15% or 10-20% polyacrylamide gradient gels by using the method of Davis (2). SDS/PAGE was performed on a 15-25% polyacrylamide gradient gel following the method of Laemmli (3). After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. The subunit molecular weight of the purified enzyme was estimated by calibration with standard proteins (LMW Electrophoresis Calibration Kit, Pharmacia).

Purified HRS (30 ml) was diluted with SDS/PAGE loading dye (30 ml, Daiichi Pure Chemicals) supplemented with 40 mM DTT, warmed at 37°C for 2 h, and carboxymethylated for 30 min by adding 800 mM iodoacetamide (6 ml) under N₂. The carboxymethylated protein was separated on SDS/PAGE (0.1% sodium thioglycolate was added to the anode buffer). After staining with Coomassie Brilliant Blue R-250, the gel was washed with EtOH/AcOH/H₂O (25:8:68, vol/vol) and H₂O (twice), and two bands (21 and 23 kDa, ~10 and 15 mg, respectively) were excised with a blade. The amount of protein in each band was estimated by comparing the stain strength of the polypeptide bands with that of marker protein bands. The gels were incubated with 0.02 AU of lysylendopeptidase (Wako) in 0.1 M Tris·HCl (pH 8.8) containing 0.08% SDS at 37°C for 15 h. The reaction solution was filtered (0.45 mm) and the gel was homogenized with a small pestle in a microcentrifuge tube. Extraction buffer [0.1 M Tris·HCl (pH 8.8) containing 0.08% SDS] was added to the gel debris, and the mixture was gently vortexed for 1 h at room temperature. This mixture was filtered and combined with the overall filtrate obtained above, and subjected to reversed-phase HPLC (Shimadzu LC-10Ai) separation. The peptide mixture was applied to the column (Cosmosil 5C₁₈-AR-II, Nacalaitesque), which had been equilibrated with H₂O containing 0.05% trifluoroacetic acid (TFA), and eluted with a linear gradient of acetonitrile (from 0 to 100%) containing 0.05% TFA. The separated peptides were collected individually, blotted onto PVDF membrane, and submitted to amino acid sequencing with a Procise Sequencing System, Model 491 (Applied Biosystems) according to the manufacturer's instructions. The peptide sequences are shown in SI Table 2.

For determination of the optimum pH, assays were conducted as above, but with 50 mM potassium acetate (pH 5.0-5.5), 50 mM potassium phosphate (pH 5.5-8.0), and 50 mM Tris·HCl (pH 7.5-9.0) buffers containing a mixture of rHRSa and rHRSb(final concentration, 100 mg/liter each), 100 mM (7E,7'E)-4-[7',9',9'-²H₃]coumaryl 4-coumarate, and a reaction period of 10 min. The optimum temperature was determined under the typical assay conditions, except that measurements were conducted at different temperatures. For determination of the K_m value for (7E,7'E)-4-[7',9',9'-²H₃]coumaryl 4-coumarate, assays were conducted in 50 mM potassium phosphate buffer (pH 6.0) containing 0.1-20 mM (7E,7'E)-4-[7',9',9'-²H₃]coumaryl 4-coumarate with a reaction period of 10 min.

Standard proteins, natural HRS, rHRSa, rHRSb, and the 1:1 mixture of rHRSa and rHRSb were separately passed through a TSKgel G3000SW_XL column (Tosoh) at 0.2 ml/min (0.1 M potassium phosphate buffer, pH 6.0). The molecular weight of this purified enzyme was estimated by standard calibration. The standard proteins used were apoferritin, b-amylase, alcohol dehydrogenase, BSA, and carbonic anhydrase (Sigma). A similar experiment was also carried out by passing the proteins above through a HiLoad Superdex 200 pg (Pharmacia) column at 0.5 ml/min [0.1 M Tris·HCl (pH 7.8) containing 2 mM DTT, 1 mM EDTA-Na₂, 2.5 mM 2-mercaptoetanol, 0.5 mM PMSF, and 5% glycerol (vol/vol)].

1. Bradford MM (1976) Anal Biochem 72:248-254.

2. Davis BJ (1964) Ann N Y Acad Sci 121:404-427.

3. Laemmli UK (1979) Nature 227:680-685.