title: A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway.
abstract: Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop.
authors.affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (H.L., Z.B., H.Q., L.M., G.W.);Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom (A.J.K.); andGraduate School of the Chinese Academy of Sciences, Beijing 100049, China (Z.B., L.M.).
authors.forename: Haoxun
authors.initials: H
authors.lastname: Li
authors.affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (H.L., Z.B., H.Q., L.M., G.W.);Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom (A.J.K.); andGraduate School of the Chinese Academy of Sciences, Beijing 100049, China (Z.B., L.M.).
authors.forename: Zhaonan
authors.initials: Z
authors.lastname: Ban
authors.affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (H.L., Z.B., H.Q., L.M., G.W.);Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom (A.J.K.); andGraduate School of the Chinese Academy of Sciences, Beijing 100049, China (Z.B., L.M.).
authors.forename: Hao
authors.initials: H
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (H.L., Z.B., H.Q., L.M., G.W.);Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom (A.J.K.); andGraduate School of the Chinese Academy of Sciences, Beijing 100049, China (Z.B., L.M.).
authors.forename: Liya
authors.initials: L
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (H.L., Z.B., H.Q., L.M., G.W.);Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom (A.J.K.); andGraduate School of the Chinese Academy of Sciences, Beijing 100049, China (Z.B., L.M.).
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: King
authors.affiliation: State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (H.L., Z.B., H.Q., L.M., G.W.);Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5DD, United Kingdom (A.J.K.); andGraduate School of the Chinese Academy of Sciences, Beijing 100049, China (Z.B., L.M.) gdwang@genetics.ac.cn.
authors.forename: Guodong
authors.identifier: http://orcid.org/0000-0001-9917-0656
authors.initials: G
authors.lastname: Wang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002462:Cell Membrane / Q000201:enzymology*; D003062:Codon / Q000235:genetics; D053138:Cyclohexenes / Q000737:chemistry / Q000378:metabolism*; D004122:Dimethylallyltranstransferase / Q000737:chemistry / Q000378:metabolism*; D044950:Flavanones / Q000378:metabolism; D027582:Humulus / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D054876:Prenylation; D011485:Protein Binding; D055503:Protein Multimerization; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism*; D064201:Trichomes / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism
chemical_list: D003062:Codon; D053138:Cyclohexenes; D044950:Flavanones; D013729:Terpenes; C008299:lupulon; D004122:Dimethylallyltranstransferase; C005273:naringenin; C007966:humulon