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...

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Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro-organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOC1) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide-linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two protomers, 'hemi-myeloperoxidase'.We studied the initial activities of human dimeric MPO and hemi-MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds I1 and 111, both inactive forms concerning HOCl generation, and the rate constants of their formation were the same for dimeric MPO and hemi-MPO, but hemi-MPO required a slightly larger excess of HzOz for complete conversion. Hemi-MPO was less stable at a high temperature (SO'C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi-MPO against its oxidative product hypochlorous acid was somewhat lower (ICso = 32 pM HOC1) compared to dimeric MPO (ICs0 = 50 pM HOCl). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.The enzyme myeloperoxidase (donor: H 2 0 2 oxidoreductase; MPO) is a major azurophil granule component of normal human ncutrophils and makes up 2 -5% of the total dry mass of these cells [l -41. MPO is involved in the defence mechanism against invading micro-organisms. In the presence of H202, which is generated from 0; produced by NADPH oxidase [5, 61, MPO so-called 'hemi-MPO' [18]. From kinetic studies of the chlorinating and peroxidase (in the absence of CI -) activities in the pH range of 4.4-6.2 of hemi-MPO (generated from normal dimeric MPO [20]), and of monomeric MPO (isolated from HL-60 cells [21]) no significant differences were found between dimeric enzyme, and hemi-or monomeric enzyme. At neutral pH, however, the peroxidase activities of monomeric MPO (79 kDa) [23], both isolated from human leukemia cells, were slightly lower as compared to those of the dimeric or 'large' MPO.Based on the assumption that dimcrization might confer higher catalytic activity or increased stability to the enzyme, we investigated and compared some properties of hemi-MPO with those of dimeric MPO. If the dimer was more stable or more active, this could explain its exclusive occurrence in normal human neutrophils. Besides chlorinating activity at a neutral physiological pH, we also examined compound I1 and compound 111 formation, since both compounds are inactive forms of MPO resulting from side-reactions in the catalytic process [24, 251. Furthermore, we investigated the heat stability and the resistance of both enzyme preparations towards their own product HOCl and the secondary oxid...

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