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- Materials and methods
- Results and discussion
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.
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 . 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 .
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 , 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 ).
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 . 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.
Results and discussion
- Top of page
- Materials and methods
- Results and discussion
The highest specific activity for the formation of phlorisovalerophenone and phlorisobutyrophenone was reported for the young cones . 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 .
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).
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 .
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, 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 sequence||Identity (%)||Protein||Source|
|SLIEAFTPIGINDWNNIFWIAHPGGPAILDEIEAK|| ||VPS-peptide 1||Humulus lupulus|
|SLIEAFTPIGINDWNNIFWIAHPGGPAILDEIEAK||100||CHS-like protein||Humulus lupulus|
|SLIEAFKPIGINDWNSIFWIAHPGGPAILDQVEHK|| 85||CHS||Chrysosplenium americanum|
|SLVEAFTPIGISDWNSLFWIAHPGGPAILDQVELK|| 80||CHS||Vitis vinifera|
|SLIEAFQPLGISDWNSIFWIAHPGGPAILDQVELK|| 80||CHS||Solanum tuberosum|
|SLIEAFQPLGISDWNSIFWIAHPGGPAILDQVELK|| 80||CHS||Lycopersicon esculentum|
|SLVEAFKPIGISDWNSLFWIAHPGGPAILDQVELK|| 77||CHS||Vitis vinifera|
|SLVEAFQPLGISDWNSIFWIAHPGGPAILDQVELK|| 77||CHS||Petunia hybrida|
|STTGDGLEWGALFGFGPGLTVETVVLHSVP|| ||VPS-peptide 2||Humulus lupulus|
|STTGDGLEWGALFGFGPGLTVETVVLHSVP||100||CHS-like protein||Humulus lupulus|
|ATTGDGLEWGVLFGFGPGLTVETVVLHSVP|| 93||CHS||Chrysosplenium americanum|
|ATTGDGLDWGVLFGFGPGLTVETVVLHSVP|| 90||CHS2||Daucus carota|
|ATTGEGLEWGVLFGFGPGLTVETVVLHSVP|| 90||CHS3||Sinapis alba|
|ATTGEGLEWGVLFGFGPGLTVETVVLHSVP|| 90||CHSA1||Brassica napus|
|STTGEGLDWGVLFGFGPGLTVETVVLHSVP|| 90||CHS WHP1||Zea mays|
|ATTGEGLEWGVLFGFGPGLTVETVVLHSVP|| 90||CHS B2||Brassica napus|
|ATTGEGLEWGVLFGFGPGLTVETVVLHSVP|| 90||CHS||Raphanus sativus|
|ATTGEGLEWGVLFGFGPGLTVETVVLHSVP|| 90||CHS B1||Brassica napus|
|ATTGEGLEWGVLFGFGPGLTVETVVLHSVP|| 90||CHS||Arabidopsis thaliana|
|ATTGEGLEWGVLFGFGPGLTVETVVLHSVP|| 90||CHS 1||Sinapis alba|
|STTGEGLDWGVLFGFGPGLTVETVVLHSVP|| 90||CHS||Digitalis lanata|
|STTGEGLDWGVLFGFGPGLTVETVVLHSVP|| 90||CHS||Antirrhinum 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 ). 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 . 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.