Isolation, sequence and bacterial expression of a cDNA for chalcone synthase from the cultured cells of Pueraria lobata.

Nakajima, Osamu; Akiyama, Takumi; Hakamatsuka, Takashi; Shibuya, Masabumi; Noguchi, Hiroshi; Ebizuka, Yutaka; Sankawa, Ushio

doi:10.1248/cpb.39.1911

Cited by 21 publications

(11 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A similar phenomenon was observed in the expression of intact HmS (expressed with pET‐3d). In our earlier experiments, CHS from Pueraria lobata was expressed in a soluble form even at 37 °C using pT7‐7 [14] or pET‐3d (T. Akiyama, unpublished result). The thioredoxin part, which is expected to increase the solubility of the expressed proteins, did not work in the present experiments.…”

Section: Resultsmentioning

confidence: 99%

p‐Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii

Akiyama

Shibuya

Liu

et al. 1999

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Chalcone synthase and stilbene synthase are plant-specific polyketide synthases. They catalyze three common consecutive decarboxylative condensations and specific cyclization reactions. They are highly homologous to each other, and are likely to fall into a family of polyketide synthases along with acridone synthase and bibenzyl synthase. Two cDNA clones (named HmC and HmS), both of which show high homology to the known chalcone synthases, were obtained from leaves of Hydrangea macrophylla var. thunbergii. They were expressed in Escherichia coli in order to determine their enzyme functions. Detection of chalcone formation clearly indicated that HmC encoded chalcone synthase, while HmS protein catalyzed the formation of neither chalcone nor stilbene. However, a novel pyrone, a lactonization product of a linear tetraketide was detected in reaction products of HmS protein. This proves that HmS encodes a novel polyketide synthase that catalyzes only chain elongation without cyclization.

show abstract

Section: Resultsmentioning

confidence: 99%

p‐Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii

Akiyama

Shibuya

Liu

et al. 1999

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The cloned and characterized CHS-like enzymes that form tetraketide products all have a glycine at position 256 ( − ). In comparison, the G256L mutation occurs naturally in Gerbera hybrida 2-PS, CHS-A, and CHS-B from various Ipomoea (morning glory) species, and Petunia hybrida CHS-B ( − ).…”

Section: Discussionmentioning

confidence: 99%

“…The position of Gly256 in the cavity allows for the introduction of a sterically increasing set of side chains projecting into the initiation/ elongation/cyclization cavity. Although functionally characterized tetraketide-forming type III PKS, including other CHS (16)(17)(18)(19)(20), stilbene synthase (20)(21)(22), coumaroyltriacetic acid lactone synthase (23), bibenzyl synthase (24), homoeriodictyol/eriodictyol synthase (25), and acridone synthase (26), possess a glycine at position 256, natural variation at this position occurs in Gerbera hybrida 2-PS, Petunia hybrida CHS-B and CHS-G, and Ipomoea (morning glory) CHS-A and -B (Figure 2D) (27)(28)(29). The substrate and product specificities of these CHS-like proteins with the exception of 2-PS are currently unknown.…”

mentioning

confidence: 99%

Structure-Guided Programming of Polyketide Chain-Length Determination in Chalcone Synthase

2001

View full text Add to dashboard Cite

Chalcone synthase (CHS) belongs to the family of type III polyketide synthases (PKS) that catalyze formation of structurally diverse polyketides. CHS synthesizes a tetraketide by sequential condensation of three acetyl anions derived from malonyl-CoA decarboxylation to a p-coumaroyl moiety attached to an active site cysteine. Gly256 resides on the surface of the CHS active site that is in direct contact with the polyketide chain derived from malonyl-CoA. Thus, position 256 serves as an ideal target to probe the link between cavity volume and polyketide chain-length determination in type III PKS. Functional examination of CHS G256A, G256V, G256L, and G256F mutants reveals altered product profiles from that of wild-type CHS. With p-coumaroyl-CoA as a starter molecule, the G256A and G256V mutants produce notably more tetraketide lactone. Further restrictions in cavity volume such as that seen in the G256L and G256F mutants yield increasing levels of the styrylpyrone bis-noryangonin from a triketide intermediate. X-ray crystallographic structures of the CHS G256A, G256V, G256L, and G256F mutants establish that these substitutions reduce the size of the active site cavity without significant alterations in the conformations of the polypeptide backbones. The side chain volume of position 256 influences both the number of condensation reactions during polyketide chain extension and the conformation of the triketide and tetraketide intermediates during the cyclization reaction. These results viewed in conjunction with the natural sequence variation of residue 256 suggest that rapid diversification of product specificity without concomitant loss of substantial catalytic activity in related CHS-like enzymes can occur by site-specific evolution of side chain volume at position 256.

show abstract

“…Sources for CHS sequences: Medicago sativa (Junghans et al 1993); Pisum sativum (Ichinose et al 1992); Pueraria lobata (Nakajima et al 1991); Phaseolus vulgaris (Ryder et al 1987); Glycine max (Akada et al 1990); Pinus sylvestris (Fliegmann et al 1992); Lycopersicon esculentum (O'Neill et al 1990); Petunia hybrida (Reif et al 1985;Koes et al 1989); Antirrhinum majus (Sommer and Saedler 1986); Magnolia liliiflora, Ranunculus acer, and Zea mays (NiesbachK16sgen et al 1987;Franken et al 1991); Hordeum vulgare (Rohde et al 1991); Sinapis alba (Ehmann and Schiller 1988); Arabidopsis thaliana (Feinbaum and Ausubel 1988); Matthiola incana (Epping et al 1990); Petroselinum hortense (Reimold et al 1983). Sources for STS sequences: Arachis hypogaea (Schr6der et al 1988;Lanz et al 1990); Pinus sylvestris (Fliegmann et al 1992), and unpublished; Vitis vat.…”

Section: Methodsmentioning

confidence: 99%

Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution

et al. 1994

View full text Add to dashboard Cite

Chalcone (CHS) and stilbene (STS) synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to stimulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20-25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same Km for the substrate 4-coumaroyl-CoA but a lower Vmax than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity.

show abstract

Isolation, sequence and bacterial expression of a cDNA for chalcone synthase from the cultured cells of Pueraria lobata.

Cited by 21 publications

References 0 publications

p‐Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii

p‐Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii

Structure-Guided Programming of Polyketide Chain-Length Determination in Chalcone Synthase

Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution

Contact Info

Product

Resources

About