Phlorisovalerophenone synthase, a novel polyketide synthase from hop (Humulus lupulus L.) cones

Abstract

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 (a-and b-acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The pI of the enzyme was 6.1. K m values of 4 mm for isovaleryl-CoA, 10 mm for isobutyryl-CoA and 33 mm 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.Keywords: Humulus lupulus; chalcone synthase; polyketide synthase; hop bitter acids; 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 a-acids into a more soluble and stable form during the brewing process; isomerized a-acids are the main bitter substances in beer. In the plant, hop bitter acids consist of both a-acids, mainly humulone, cohumulone and adhumulone, and b-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. Usi...

Analysis of the chalcone synthase from Humulus lupulus L. and biotechnology aspects of medicinal hops

“…T197, which contributes to the coumaroyl binding site in chalcone synthases, has no homologous position in VPS. This amino acid difference is consistent with the different initial capture specificity of VPS, which utilises isovaleryl-CoA or isobutyryl-CoA as substrate components, instead of 4-coumaroyl-CoA, to which it has either no or low affinity [see Paniego et al (1999) for substrate specificities]. This assumption is strongly supported by recent work published by Jez et al (2000), who performed site-specific mutagenesis of several plant-specific polyketide synthases including position of T197 and found differences in substrate specificities of corresponding mutants.…”

“…This enzyme was proven to be involved in the first steps of biosynthesis of hop bitter acids (Paniego et al 1999, Okada andIto 2001). However, because there is either no or very low true CHS activity caused by this enzyme (Paniego et al 1999), it has to be assumed that true chalcone synthase(s), if expressed in glandular tissue, has to fulfil this function.…”

Likelihood Analysis of the Chalcone Synthase Genes Suggests the Role of Positive Selection in Morning Glories ( Ipomoea )

“…The evolution of new catalytic functions from CHS genes is also found elsewhere, for example, at least three independent shifts from CHS to stilbene synthase (STS) have arisen in seed plant evolution (Tropf et al 1994). Catalytic shifts from CHS to acridone synthase (ACS) (Lukacin et al 1999), bibenzyl synthase (BBS) (Preisig-Mu¨ller et al 1995), 2-pyrone synthase (2PS) (Eckermann et al 1998), and phlorisovalerophenone synthase (PVPS) (Paniego et al 1999) are also evident. Therefore, gene duplication, coupled with functional divergence, is a recurrent pattern in the evolution of the CHS gene family (Clegg and Durbin 2003).…”

“…Analysis of CHS multigene family suggested recurrent gene duplications and subsequent adaptive differentiation among duplicated copies Helariutta et al 1996). Some other plant-specific polyketide synthases, including STS, ACS, BBS, 2PS, and PVPS, are proposed to have evolved from CHSs by the same mechanism (Schro¨der 1997 Paniego et al 1999). Those enzymes share a common chemical mechanism with CHS but differ from CHS in their substrate specificity and/or in the stereochemistry of the polyketide cyclization reaction.…”

“…There is growing evidence that chalcone synthase is closely related to other plant-specific polyketide synthases, including stilbene synthase (STS) (Scho¨ppner and Kindl 1984;Schro¨der 1997), acridone synthase (ACS) (Lukacin et al 1999), bibenzyl synthase (BBS) (Preisig-Mu¨ller et al 1995), 2-pyrone synthase (2PS) (Eckermann et al 1998), and phlorisovalerophenone synthase (PVPS) (Paniego et al 1999). It is also observed that very few amino acid changes in those proteins may result in shifts in enzyme function.…”