Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis

Abstract

Isoflavonoids are ecophysiologically active secondary metabolites of the Leguminosae and known for health-promoting phytoestrogenic functions. Isoflavones are synthesized by 1,2-elimination of water from 2-hydroxyisoflavanones, the first intermediate with the isoflavonoid skeleton, but details of this dehydration have been unclear. We screened the extracts of repeatedly fractionated Escherichia coli expressing a Glycyrrhiza echinata cDNA library for the activity to convert a radiolabeled precursor into formononetin (7-hydroxy-4'-methoxyisoflavone), and a clone of 2-hydroxyisoflavanone dehydratase (HID) was isolated. Another HID cDNA was cloned from soybean (Glycine max), based on the sequence information in its expressed sequence tag library. Kinetic studies revealed that G. echinata HID is specific to 2,7-dihydroxy-4'-methoxyisoflavanone, while soybean HID has broader specificity to both 4'-hydroxylated and 4'-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, HID proteins were members of a large carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions with no intrinsic catalytic activities identified. Site-directed mutagenesis with soybean HID protein suggested that the characteristic oxyanion hole and catalytic triad are essential for the dehydratase as well as the faint esterase activities. The findings, to our knowledge, represent a new example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.

Figure 1.

Isoflavonoid biosynthesis in leguminous plants. 4′-Hydroxylated isoflavones are biosynthesized from (2S)-flavanones by the reaction catalyzed by IFS and subsequent intramolecular 1,2-dehydration of 2-hydroxyisoflavanones. 4′-Methoxylated isoflavones are produced from the IFS reaction products in 2 steps: 4′-O-methylation catalyzed by HI4′OMT and subsequent dehydration. In this study, HIDs of distinct substrate specificity toward 4′-hydroxylated and 4′-methoxylated 2-hydroxyisoflavanones were characterized.

Figure 2.

Amino acid sequence of G. echinata HIDM aligned with soybean HIDH (A) and the phylogenetic relationship of G. echinata HIDM and homologous proteins found in L. japonicus, M. truncatula, and soybean EST databases (B). A, The amino acid residues of which two sequences are identical are in reverse type. Gaps (-) are inserted to optimize alignment. Putative residues that constitute the catalytic triad (Thr, Asp, and His) are indicated with asterisks (*), and the oxyanion hole (His-Gly-Gly) is boxed. B, A total of seven amino acid sequences were analyzed using the ClustalW program (Thompson et al., 1994), and the tree was displayed by TreeView software (Page, 1996). Numbers indicate bootstrap value from 1,000 replicates. Nicotiana tabacum hsr203j was defined as the out group.

Figure 3.

The phylogenetic relationship of G. echinata HIDM and the carboxylesterase family in various organisms. The complete phylogenetic tree comprising all the proteins examined (102 sequences) is shown in Supplemental Figure 1. The nomenclature of proteins in the figure is either after the names used in the original reports or abbreviated names of the species producing the proteins, followed by the numbers of distinct sequences in parentheses: AtCXE, Arabidopsis; AN, Aspergillus nidulans; EC, Escherichia coli; GM, Glycine max; HS, Homo sapiens; ML, Mesorhizobium loti; MT, Medicago truncatula; OS, Oryza sativa; PS, Pseudomonas syringae; SC, Streptomyces coelicolor; SP, Schizosaccharomyces pombe; ST, Solanum tuberosum; BIG8.1, Vitis vinifera BIG8.1; E86, Pisum sativum E86; hsr203j, Nicotiana tabacum hsr203j; PepEST, Capsicum annuum PepEST; PrMC3, Pinus radiata PrMC3. Plant clades are enclosed by a dotted line.

Figure 4.

Putative reaction mechanism for HID. The amino acid residues of the catalytic triad and the oxyanion hole cooperatively eliminate water from a 2-hydroxyisoflavanone to produce an isoflavone. His, instead of Thr, of the catalytic triad could perform the initial proton abstraction (see “Discussion”).