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Keywords:

  • Humulus lupulus;
  • chalcone synthase;
  • polyketide synthase;
  • hop bitter acids;
  • phlorisovalerophenone synthase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Phlorisovalerophenone synthase (VPS), a novel aromatic polyketide synthase, was purified to homogeneity from 4.2 mg protein extract obtained from hop (Humulus lupulus L.) cone glandular hairs. The enzyme uses isovaleryl-CoA or isobutyryl-CoA and three molecules of malonyl-CoA to form phlorisovalerophenone or phlorisobutyrophenone, intermediates in the biosynthesis of the hop bitter acids (α- and β-acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The pI of the enzyme was 6.1. Km values of 4 µm for isovaleryl-CoA, 10 µm for isobutyryl-CoA and 33 µm for malonyl-CoA, were found. The amino-acid sequences of two peptides, obtained by digestion of VPS, showed that the enzyme is highly homologous to plant chalcone synthases. The functional and structural relationship between VPS and other aromatic polyketide synthases is discussed.

Abbreviations
VPS

phlorisovalerophenone synthase.

The cones of the hop plant (Humulus lupulus L.) have been used for centuries in the beer-brewing process. Their major contribution to beer is the characteristic bitterness that results from the isomerization of the hop α-acids into a more soluble and stable form during the brewing process; isomerized α-acids are the main bitter substances in beer. In the plant, hop bitter acids consist of both α-acids, mainly humulone, cohumulone and adhumulone, and β-acids, mainly lupulone, colupulone and adlupulone [1]. These compounds are synthesized during the development of the H. lupulus female inflorescences into cones and are accumulated in the yellow glands covering the basal part of the bracteoles of the cones [2,3]. In general, glands are a rich source of secondary metabolites. Recently, strategies for bioengineering the development and metabolism in glandular tissues have been reviewed [4].

In previous papers [5,6] a novel biosynthetic pathway leading to the bitter acids in H. lupulus was proposed. The suggested intermediates phlorisovalerophenone and phlorisobutyrophenone were also detected in hop cones. Furthermore, protein extracts from the cones were able to synthesize these compounds from malonyl-CoA plus either isovaleryl-CoA or isobutyryl-CoA. Apparently, the catalytic mechanism involved in this biosynthesis is similar to that observed in other plant condensing enzymes, like chalcone synthase and stilbene synthase (Fig. 1). These enzymes catalyze a reaction which proceeds by a sequential condensation of three acetate units to a starter residue to form the tetraketide intermediate that is folded to form a ring [7,8]. This type of reaction, which was first described by Birch and Donovan [9], classifies chalcone synthase and stilbene synthase as polyketide synthases. Chalcone synthase is a key enzyme in the biosynthesis of flavonoids. It catalyzes the formation of naringenin from three molecules of malonyl-CoA and coumaroyl-CoA. Stilbene synthase, an enzyme in the biosynthesis of stilbene phytoalexins, is structurally and functionally related to chalcone synthase. Using the same substrates and the same catalytic mechanism as chalcone synthase, stilbene synthase folds the intermediate polyketide in a different way, yielding resveratrol. At present, several chalcone synthase and stilbene synthase-related proteins have been described. All of these enzymes perform a chalcone synthase/stilbene synthase-type reaction, but with substrates different from coumaroyl-CoA (reviewed in [8]).

image

Figure 1. Reactions catalyzed by the plant polyketide synthases. Chalcone (naringenin) synthase (CHS), stilbene (resveratrol) synthase (STS) and phlorisovalerophenone synthase (VPS).

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Chalcone synthase protein and its enzymatic activity were also detected in protein extracts from hop [5]. However, the profile of phlorisovalerophenone and phlorisobutyrophenone formation during cone development was different from that of naringenin formation. The present results confirm the presence of a new polyketide synthase in H. lupulus catalyzing the formation of the bitter acid precursors. This new enzyme, named phlorisovalerophenone synthase (VPS), catalyzes the formation of phlorisovalerophenone and phlorisobutyrophenone accepting only isovaleryl-CoA or isobutyryl-CoA as starter molecules (Fig. 1). Here we report on the purification, characterization and partial amino-acid sequence of VPS from the glands of hop inflorescences and cones.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Plant material

Different cultivars of Humulus lupulus L. were grown in the garden of the Division of Pharmacognosy, Leiden. From August to September 1995 hop female inflorescences and cones were collected at different developmental stages, frozen in liquid N2 and stored at −80 °C.

Gland isolation

The glands from the basal part of the hop bracteoles were isolated according to a modified method for the isolation of secretory cells [10] using a cell disrupter (Bead Beater, Biospec Products, Bartlesville, OK, USA). The Bead Beater 300 mL polycarbonate chamber was filled with 10 g of frozen young cones, 150 g of glass beads (0.5-mm diameter) and cold distilled water plus 0.01% Chaps. Plant material was abraded for 1 min, the rotor speed was controlled with a rheostat set between 90 and 100 V. After abrasion the glands were separated from the leaves by Buchner filtration. The glands were collected by passing the filtrate through a nylon filter and washed twice with cold distilled water. The glands were used immediately for protein extraction.

Protein extraction

Crude extracts were obtained using the extraction method described earlier [5]. After centrifugation, the supernatant was desalted by use of a PD10 column (Sephadex G-25 M, Pharmacia), equilibrated with buffer A (10 mm KPi, pH 7.5, 1 mm 2−mercaptoethanol, 1 mm EDTA, 1% trehalose, 5 mm cysteine), according to the manufacturer’s directions for use. The eluted fraction was immediately used for protein purification.

Enzyme assays

Chalcone synthase and VPS activities were determined by HPLC as described before [5,11].

Protein purification

All the following operations were performed at 4 °C. Anion exchange chromatography and chromatofocusing were carried out with an FPLC system (Pharmacia). The desalted protein solution was applied to a Mono Q HR 5/5 column (Pharmacia), which had been equilibrated with buffer A. Unretained protein was removed by eluting with two bed volumes of buffer A. Retained proteins were eluted by applying a linear gradient of seven column volumes from 0 to 200 mm NaCl in buffer A. The flow rate was 1 mL·min−1. Fractions of 2 mL were collected. Centricon-10 centrifugal concentrators (Amicon) were used to concentrate the combined active fractions and to change from buffer A to B (25 mm imidazol-HCl pH 7, 1 mm 2-mercaptoethanol, 1 mm EDTA, 1% trehalose, 5 mm cysteine). The resulting protein solution (4 mL) was applied to a Mono P HR 5/20 column (Pharmacia) which had been equilibrated with buffer B. After sample application the column was eluted with 5 mL of buffer B and then with 50 mL of buffer C (10% polybuffer 74 pH 4.5 with HCl, 1 mm 2-mercaptoethanol, 1 mm EDTA, 1% trehalose, 5 mm cysteine) giving a pH gradient from 7.5 to 4.5. The flow rate was 1 mL·min−1 and 2 mL fractions were collected. Fractions containing VPS activity were combined and concentrated by ultrafiltration (Centricon-10, Amicon). Subsequently, buffer C was changed to buffer A and the enzyme solution (100 µL) was applied in two separate runs to HPLC analysis, using a Shodex KW 803 size-exclusion column (Waters) equilibrated with buffer A. The chromatography was carried out using a Waters 626 pump, a Waters 486 tunable absorbance detector, a Waters 6005 Controller and millennium data acquisition software. The flow rate was 0.6 mL·min−1, fractions were collected during 60 min, detection was at 280 nm.

Analytical methods

Protein concentration was measured in duplicate by the method of Peterson [12]. SDS and native PAGE were carried out using precast gels (10–15% gradient and 12.5 homogeneous) and a PhastSystem (Pharmacia). The proteins were visualized by silver staining [13]. For comparison of molecular mass, HMW-SDS and HMW marker sets (Pharmacia) were used. For amino-acid sequence analysis, 50 µg of purified protein were electrophoresed on SDS/PAGE, stained with Coomasie Brilliant Blue, and destained with 30% methanol at room temperature. The sequencing was performed by Eurosequence (Groningen, the Netherlands).

Enzyme characterization

The molecular mass was determined by SDS/PAGE and PAGE of the native enzyme as described above. The pH optimum of the enzyme was determined using KPi in the range of pH 5.5–7.5 using the assay condition described earlier by Zuurbier et al. [5,11]. The pI of the enzyme was determined during chromatofocusing as described above. For kinetic studies, a VPS preparation obtained after chromatofocusing was used. The reaction mixtures contained 35 µg protein dissolved in 61 µL assay buffer, with varying the malonyl-CoA concentration (between 10 and 50 µm) in the presence of 10 µm of isovaleryl-CoA, or with varying isovaleryl-CoA or isobutyryl-CoA concentration (between 0.25 and 20 µm), in the presence of 50 µm malonyl CoA, in a total volume of 100 µL. Reaction mixtures were incubated at 30 °C for 10 min. Enzyme activity was measured as described above.

Immunoblotting of proteins after PhastGel electrophoresis

Electrophoresed native proteins were electroblotted onto nitrocellulose sheets according to Harlow and Lane [14]. Rainbow molecular markers (Pharmacia) were used to estimate the transfer efficiency. Polyclonal rabbit antiserum against chalcone synthase from Pinus sylvestris was used for immunodetection. Immunostaining was performed using goat anti-(rabbit IgG) conjugates to alkaline phosphatase (Promega) as secondary antibody and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as reagents in the colour reaction [14].

Results and discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

The highest specific activity for the formation of phlorisovalerophenone and phlorisobutyrophenone was reported for the young cones [5]. In preliminary experiments complete (young) cones were used, allowing, however, only partial purification of the enzyme. The enzyme showed higher affinity to isovaleryl-CoA; the enzyme was therefore called phlorisovalerophenone synthase (VPS). After protein precipitation, size-exclusion and anion-exchange chromatography, SDS/PAGE showed, besides some other polypeptides, a protein band at about 45 kDa, which seemed to be related to VPS activity (data not shown). Chalcone synthase and chalcone synthase-related proteins are known to be homodimers, with subunits of 40–45 kDa [8].

The bitter acids are synthesized and accumulated in glands of the female cones. The glands could be separated by mechanical abrasion using glass beads in the presence of Chaps, a zwitterionic detergent, which prevented the glands from sticking to the vessel wall or to the plant material. This method provided an efficient separation, and the final amount of glands collected was ≈ 0.10–0.15% of the initial fresh weight. The gland extract contained a considerably lower amount of contaminating proteins than the extract of the complete cone; both chalcone synthase (formation of naringenin) and VPS activity were detected. The glands were now used as a source for VPS. VPS was purified to apparent homogeneity, starting with only 4.2 mg of protein extract. Due to the low stability of this enzyme, gland extraction and all the purification steps had to be carried out in one run (13–14 h). The first steps of the purification procedure were centrifugation and desalting of the supernatant using a PD10 column for the removal of resinous material, bitter acids, phenolics and essential oils. The desalting removed most of the compounds that could interfere with the following purification steps and allowed the buffer to be changed to one suitable for the next chromatography step. The buffers used throughout the procedure shared a basic formulation, which included protective compounds such as trehalose, cysteine, 2-mercaptoethanol and EDTA. From the anion-exchange chromatography, the VPS activity eluted between 45 and 79 mm NaCl (Fig. 2A). The active fractions were combined and subjected to chromatofocusing. VPS activity eluted from Mono P at pH 6.1 (Fig. 2B). The last impurities were separated from VPS by HPLC size-exclusion column (Fig. 2C). Chalcone synthase activity co-eluted with VPS activity until chromatofocussing, afterwards this activity was lost (data not shown).

image

Figure 2. Purification of VPS from hop glandular hairs. (A) Anion-exchange chromatography profile (Mono Q HR 5/10, Pharmacia). (B) chromatofocusing profile (Mono P HR 5/20, Pharmacia). (C) Size-exclusion chromatography profile (Shodex KW 803, Waters). Absorbances (….) were measured at 280 nm, specific phlorisovalerophenone formation (–•–) in pkat·mg−1 protein and NaCl gradient (––) in mm. PIVP, phlorisovalerophenone.

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The purification of VPS was monitored by SDS and native PAGE (Fig. 3). After SDS/PAGE and silver staining of the purified enzyme only one polypeptide was detected at ≈ 45 kDa. Native PAGE indicated a molecular mass for VPS of 110 ± 0.5 kDa. The pH optimum of the enzyme was about 7. The pI, as shown by chromatofocussing, was 6.1. In kinetic studies carried out using partially purified protein, the enzyme showed a Km of 4 µm for isovaleryl-CoA. The apparent Km value for malonyl-CoA was 33 µm and for isobutyryl CoA 10 µm. These kinetic characteristics closely resemble those described in literature for chalcone synthase, stilbene synthase and other related polyketide synthases for their corresponding substrates [15–18]. Moreover, VPS cross-reacted with antibodies raised against P. sylvestris chalcone synthase (data not shown). When enzymatic assays were conducted with partially and purified proteins under favorable conditions for naringenin formation, no chalcone synthase activity was found in those fractions. On the contrary, it was shown that chalcone synthase can perform the function of VPS, but not perfectly, because the majority of the reactions terminated after two condensation reactions [19].

image

Figure 3. Purification of VPS from 1.2 g of hop glandular hairs and electrophoretic analysis of the different fractions. (A) SDS/PAGE of the different purification steps of VPS: lane 1, crude extract; lane 2, desalted crude extract; lane 3, fraction obtained by anion-exchange chromatography (AEC; Mono Q); lane 4, fraction obtained by chromatofocussing (CF; Mono P); lane 5, fraction obtained by size-exclusion chromatography (SEC; Shodex KW 803). The purification process is also described numerically, below. (B) Native PAGE of purified VPS. Molecular markers in kDa.

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For peptide sequencing, Edman-degradation analysis failed, indicating that the VPS N-terminal was blocked. After digestion, two peptides were selected yielding internal sequences of 35 and 30 amino acids (Table 1). Both sequences showed 100% homology with a recently cloned gene coding for a chalcone synthase-like protein, isolated from H. lupulus[20], which suggests that this gene encodes hop VPS. Table 2 shows the homology of the peptides with some other polyketide synthases, indicating the close relationship between VPS and chalcone synthase.

Table 1. Amino-acid sequence of two peptic fragments of VPS and homology of these peptides with other polyketide synthases (presented are identities to peptide 1 of at least 75% and at least 90% to peptide 2). CHS, chalcone synthase.
Amino acid sequenceIdentity (%)ProteinSource
SLIEAFTPIGINDWNNIFWIAHPGGPAILDEIEAK VPS-peptide 1Humulus lupulus
SLIEAFTPIGINDWNNIFWIAHPGGPAILDEIEAK100CHS-like proteinHumulus lupulus
SLIEAFKPIGINDWNSIFWIAHPGGPAILDQVEHK 85CHSChrysosplenium americanum
SLVEAFTPIGISDWNSLFWIAHPGGPAILDQVELK 80CHSVitis vinifera
SLIEAFQPLGISDWNSIFWIAHPGGPAILDQVELK 80CHSSolanum tuberosum
SLIEAFQPLGISDWNSIFWIAHPGGPAILDQVELK 80CHSLycopersicon esculentum
SLVEAFKPIGISDWNSLFWIAHPGGPAILDQVELK 77CHSVitis vinifera
SLVEAFQPLGISDWNSIFWIAHPGGPAILDQVELK 77CHSPetunia hybrida
STTGDGLEWGALFGFGPGLTVETVVLHSVP VPS-peptide 2Humulus lupulus
STTGDGLEWGALFGFGPGLTVETVVLHSVP100CHS-like proteinHumulus lupulus
ATTGDGLEWGVLFGFGPGLTVETVVLHSVP 93CHSChrysosplenium americanum
ATTGDGLDWGVLFGFGPGLTVETVVLHSVP 90CHS2Daucus carota
ATTGEGLEWGVLFGFGPGLTVETVVLHSVP 90CHS3Sinapis alba
ATTGEGLEWGVLFGFGPGLTVETVVLHSVP 90CHSA1Brassica napus
STTGEGLDWGVLFGFGPGLTVETVVLHSVP 90CHS WHP1Zea mays
ATTGEGLEWGVLFGFGPGLTVETVVLHSVP 90CHS B2Brassica napus
ATTGEGLEWGVLFGFGPGLTVETVVLHSVP 90CHSRaphanus sativus
ATTGEGLEWGVLFGFGPGLTVETVVLHSVP 90CHS B1Brassica napus
ATTGEGLEWGVLFGFGPGLTVETVVLHSVP 90CHSArabidopsis thaliana
ATTGEGLEWGVLFGFGPGLTVETVVLHSVP 90CHS 1Sinapis alba
STTGEGLDWGVLFGFGPGLTVETVVLHSVP 90CHSDigitalis lanata
STTGEGLDWGVLFGFGPGLTVETVVLHSVP 90CHSAntirrhinum majus

Summarizing, a novel enzyme, involved in the biosynthesis of bitter acids, was purified to apparent homogeneity from hop glands. According to its molecular characteristics and cross reactivity with chalcone synthase antibodies, VPS is considered as a new product of the plant polyketide gene superfamily. VPS preferentially uses isovaleryl CoA as starter molecule, but it is also active with isobutyryl CoA. Evidence for the existence of a polyketide gene superfamily has been accumulated from the comparative study of chalcone synthase and stilbene synthase (reviewed in [8]). It is known that slight changes in the primary structure of chalcone synthase or stilbene synthase are sufficient to change their substrate preferences. Stilbene synthase has probably evolved from chalcone synthase on several independent occassions during seed-plant evolution It was suggested that the conversion of chalcone synthase into VPS would require less dramatic changes [19]. Characterization of the VPS cDNA clone will be the next step in the process of elucidation of the regulatory mechanisms involved in the biosynthesis of hop bitter acids.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

N.P. wishes to express her gratitude to the Consejo Nacional de Investigaciones Cientificas y Tecnicas (Argentina) for their support. This study was partly financed by Technology Foundation from the Netherlands. We thank Mr W. Snoeijer for growing the hop plants, Dr H. Bouwmeester (Wageningen) for his helpful suggestion about gland isolation techniques and Mr A. Geerlings for his help on sample preparation for internal sequence analysis. We thank Prof. J. Schröder (University Freiburg, Germany) for his generous gifts of antibodies raised against P. sylvestris chalcone synthase.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References
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    Zuurbier, K.W.M., Fung, S.-Y., Scheffer, J.J.C. & Verpoorte, R. ( 1995) Formation of aromatic intermediates in the biosynthesis of bitter acids in Humulus lupulus. Phytochem. 38, 7782.
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Footnotes
  1. Enzymes: chalcone (naringenin) synthase (EC2.3.1.74); stilbene (resveratrol) synthase (EC2.3.1.95).

  2. *Present address: Institute of Evolutionary and Ecological Sciences, Leiden University, PO Box 9516, 2300 RA Leiden.