Site-directed Mutagenesis of Benzalacetone Synthase

THE ROLE OF PHE215 IN PLANT TYPE III POLYKETIDE SYNTHASES*

Ikuro Abe {ddagger}, Yukie Sano, Yusuke Takahashi and Hiroshi Noguchi

From the School of Pharmaceutical Sciences and the 21st Century COE Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Received for publication, March 31, 2003 , and in revised form, April 30, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs) that share ~70% amino acid sequence identity. BAS catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone, whereas CHS performs sequential condensations with three malonyl-CoA to generate a tetraketide chalcone. A homology model suggested that BAS has the same overall fold as CHS with cavity volume almost as large as that of CHS. One of the most characteristic features is that Rheum palmatum BAS lacks active site Phe-215; the residues 214LF conserved in type III PKSs are uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant exhibited chalcone-forming activity in a pH-dependent manner supported a hypothesis that the absence of Phe-215 in BAS accounts for the interruption of the polyketide chain elongation at the diketide stage. On the other hand, Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) afforded increased levels of truncated products; however, none of them generated benzalacetone. These results confirmed the critical role of Phe-215 in the polyketide formation reactions and provided structural basis for understanding the structure-function relationship of the plant type III PKSs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The chalcone synthase (CHS)1 superfamily of type III polyketide synthases (PKSs) are pivotal enzymes in the biosynthesis of flavonoids as well as a wide range of structurally diverse, biologically important natural products (1, 2). Benzalacetone synthase (BAS) (EC 2.3.1.-) catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA (1) with malonyl-CoA (2) to produce a diketide benzalacetone (4) (Fig. 1), whereas CHS (EC 2.3.1.74 [EC] ) performs sequential condensations of 4-coumaroyl-CoA with three acetate units from malonyl-CoA followed by a Claisen-type cyclization reaction, leading to formation of a tetraketide naringenin chalcone (6). Further, in the CHS enzyme reaction in vitro, a triketide and a tetraketide pyrone, bisnoryangonin (BNY) (3, 4) and 4-coumaroyltriacetic acid lactone (CTAL) (4, 5) are also obtained as early released derailment by-products when the reaction mixtures are acidified before extraction (Fig. 1). BAS is thought to play a crucial role for construction of the C6-C4 moiety of a variety of biologically active phenylbutanoids, including anti-inflammatory glucoside lindleyin (7) in rhubarb (5), 6-gingerol (8), and curcumin (9) in ginger plants (6) as well as raspberry ketone (10), the characteristic aroma of raspberry fruits (7). In a previous paper (5), we reported cloning and heterologous expression of a cDNA encoding BAS from rhubarb (Rheum palmatum), a medicinal plant that produces the pharmaceutically important lindleyin. The cDNA encoded a 42-kDa protein sharing 60–75% amino acid sequence identity with other members of the CHS superfamily enzymes including stilbene synthase (STS) from groundnut (Arachis hypogaea) (8), 2-pyrone synthase (2-PS) from daisy (Gerbera hybrida) (9), and acridone synthase (ACS) from common rue (Ruta graveolens) (10).



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 1.
Conversion of 4-coumaroyl-CoA (1) and malonyl-CoA (2) to benzalacetone (3) by BAS, and to naringenin chalcone (6) by CHS. Polyketide pyrones, BNY (4), and CTAL (5) are derailment by-products of the CHS reactions in vitro when the reaction mixtures are acidified before extraction. Structures of phenylbutanoids (7–10) that are thought to be derived from benzalacetone are also included. Here the C6-C4 moieties are illustrated with thick lines.

 

Recent crystallographic and protein engineering studies on alfalfa (Medicago sativa) CHS, a homodimeric 42-kDa protein, revealed the active site machinery of the chalcone-forming reaction that proceeds through starter molecule loading at Cys-164, malonyl-CoA decarboxylation, and polyketide chain elongation, followed by cyclization and aromatization of the enzyme-bound tetraketide intermediate (Fig. 2) (1117). The catalytic center of CHS is composed of four amino acid residues: the catalytic triad of Cys-164, His-303, and Asn-336 and the "gatekeeper" Phe-215, absolutely conserved in all of the known type III PKSs. Interestingly, BAS is the only exception that lacks the active site Phe-215; the conserved residues 214LF are uniquely replaced by IL (numbering in M. sativa CHS) (Fig. 3) (5). In CHS, Phe-215, located at the junction between the active site cavity and the CoA binding tunnel, has been proposed to facilitate decarboxylation of malonyl-CoA and help orient substrates and intermediates during the sequential condensation reactions (Fig. 2) (12, 18). We proposed a hypothesis that the absence of Phe-215 in BAS may be the reason why the polyketide chain elongation is terminated at the diketide stage and followed by a second decarboxylation reaction to generate benzalacetone. In order to test the hypothesis, a mutant R. palmatum BAS in which the residues 214IL were substituted by LF was constructed. Furthermore, to investigate the structure-function relationship of the type III PKS enzymes, we also carried out functional analysis of a series of Phe-215 mutants of CHS from Scutellaria baicalensis (19).



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 2.
Proposed mechanism for the formation of chalcone by CHS (2). A, loading of 4-coumaroyl-CoA; B, decarboxylation of malonyl-CoA; C, formation of diketide intermediate; D, formation of triketide intermediate; E, formation of tetraketide intermediate; F, cyclization and aromatization to chalcone.

 


View larger version (61K):
[in this window]
[in a new window]
 
FIG. 3.
Comparison of primary sequences of BAS and other CHS superfamily enzymes. M.s CHS, M. sativa CHS; S.b CHS, S. baicalensis CHS; A.h STS, A. hypogaea stilbene synthase; G.h 2PS, G. hybrida 2-pyrone synthase; R.g ACS, R. gravenolens acridone synthase; R.p BAS, R. palmatum benzalacetone synthase. The active site residues conserved in the CHS superfamily enzymes (Cys-164, Phe-215, His-303, and Asn-336; numbering in CHS) are marked with number symbol, and residues for the CoA binding are labeled with a plus sign. Amino acid residues conserved in the CHS superfamily enzymes but absent in R. palmatum BAS are marked with an asterisk.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—4-Coumaroyl-CoA was chemically synthesized using the method of Stöckigt and Zenk (20). Malonyl-CoA, benzoyl-CoA, and hexanoyl-CoA were purchased from Sigma. [2-14C]Malonyl-CoA (48 mCi/mmol) was purchased from Moravek Biochemicals.

Site-directed Mutagenesis—R. palmatum BAS mutant (I214L/L215F) and S. baicalensis CHS mutants (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) and a pair of primers as follows (mutated codons are underlined): BAS I214L/L215F (5'-CATCTGGACTCCATGATAGGCCAAGCATTATTTGGCGATGGG-3' and 5'-AGCCCCATCGCCAAATAATGCTTGGCCTATCATGGAGTCCA-3'), CHS L214I/F215L (5'-TTGACAGCCTGGTTGGGCAGGCGATTTTAGGCGATGGCG-3' and 5'-CGCCATCGCCTAAAATCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215W (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTGGGGCGATGGCG-3' and 5'-CGCCATCGCCCCACAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215Y (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTATGGCGATGGCG-3' and 5'-CGCCATCGCCATACAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215S (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTCAGGCGATGGCG-3' and 5'-CGCCATCGCCTGACAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215A (5'-TTGACAGCCTGGTTGGGCAGGCGCTGGCAGGCGATGGCG-3' and 5'-CGCCATCGCCTGCCAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215H (5'-TTGACAGCCTGGTTGGGCAGGCGCTGCATGGCGATGGCG-3' and 5'-CGCCATCGCCATGCAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215C (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTGTGGCGATGGCG-3' and 5'-CGCCATCGCCACACAGCGCCTGCCCAACCAGGCTGTCAA-3').

Enzyme Expression and Purification—After confirmation of the sequence, the plasmid was transformed into E. coli BL21(DE3)pLysS. The cells harboring the plasmid were cultured to an A600 of 0.6 in LB medium containing 100 µg/ml ampicillin at 30 °C. Then 0.4 mM isopropyl-1-thio-{beta}-D-galactopyranoside was added to induce protein expression, and the culture was incubated further at 30 °C for 16 h. The E. coli cells were harvested by centrifugation and resuspended in 50 mM potassium phosphate buffer, pH 8.0, containing 0.1 M NaCl. Cell lysis was carried out by the freeze-thaw method and centrifuged at 15,000 g for 60 min. The supernatant was passed through a column of Pro-BondTM resin (Invitrogen), which contained Ni2+ as an affinity ligand. After washing with 50 mM potassium phosphate buffer, pH 7.9, containing 0.5 M NaCl and 40 mM imidazole, the recombinant BAS was finally eluted with 15 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 500 mM imidazole. Finally, the enzyme preparation was desalted by Bio-Gel P6DG desalting gel. Protein concentration was determined by the Bradford method (Protein Assay; Bio-Rad) with bovine serum albumin as a standard.

Enzyme Reaction—The standard reaction mixture contained 27 µM 4-coumaroyl-CoA, 54 µM malonyl-CoA, and 20 µg of the purified enzyme in a final volume of 500 µl of 100 mM potassium phosphate buffer, pH 8.0, containing 1 mM EDTA. Incubations were carried out at 30 °C for 20 min, and stopped by the addition of 50 µl of 20% HCl. By the acid treatment, naringenin chalcone is thus converted to racemic naringenin through a nonstereospecific ring-C closure. Furthermore, early released triketide and tetraketide intermediate are also converted to lactone derailment products, BNY and CTAL. The products were then extracted with 600 µl of ethyl acetate and concentrated by N2 flow. The residue was dissolved in an aliquot of MeOH containing 0.1% trifluoroacetic acid and separated by reverse-phase HPLC (JASCO 880) on a TSK-gel ODS-80Ts column (4.6 x 150 mm; TOSOH) with a flow rate of 0.8 ml/min. Gradient elution was performed with H2O and MeOH, both containing 0.1% trifluoroacetic acid: 0–5 min, 30% MeOH; 5–17 min, linear gradient from 30 to 60% MeOH; 17–25 min, 60% MeOH; 25–27 min, linear gradient from 60 to 70% MeOH. Elutions were monitored by a multichannel UV detector (MULTI 340, JASCO) at 290, 330, and 360 nm; UV spectra (198–400 nm) were recorded every 0.4 s.

On-line LC-ESIMS spectra were measured with a Hewlett-Packard HPLC 1100 series (Wilmington, DE) coupled to a Finnigan MAT LCQ ion trap mass spectrometer (San Jose, CA) fitted with an electrospray ionization source. HPLC separations were carried out under the same conditions as described above. The electrospray ionization capillary temperature and capillary voltage were 275 °C and 3.0 V, respectively. The tube lens offset was set at 20.0 V. All spectra were obtained in both negative and positive mode over a mass range of m/z 120–350, at a range of one scan every 2 s. The collision gas was helium, and the relative collision energy scale was set at 30.0% (1.5 eV). Enzyme reactions with nonphysiological substrates and analysis of their product were carried out as described before (19, 21, 22).

Enzyme Kinetics—Steady state kinetic parameters were determined by using [2-14C]malonyl-CoA (1.8 mCi/mmol) as a substrate. The experiments were carried out in triplicate using four concentrations of 4-coumaroyl-CoA (11.0, 5.5, 2.8, and 1.4 µM) in the assay mixture, containing 28 µM malonyl-CoA, 4.4 µg of purified enzyme, 1 mM EDTA, in a final volume of 500 µl of 100 mM potassium phosphate buffer. Incubations were carried out at 30 °C for 20 min. The reaction products were extracted and separated by HPLC, and the radioactivity in each fraction was determined by a liquid scintillation counter (Aloka LSC-3100). The kinetic parameters were calculated for formation of major product of the enzyme reactions at optimum pH, except for those of BAS I214L/L215F mutant, which was measured for formation of naringenin chalcone. Lineweaver-Burk plots of data were employed to derive the apparent Km and kcat values (average of triplicates ± S.D.) using EnzFitter software (BIOSOFT).

Three-dimensional Homology Modeling—The model of R. palmatum BAS was produced by the SWISS-MODEL package (available on the World Wide Web at expasy.ch/spdbv/) provided by the Swiss-PDB-Viewer program (23). A standard homology modeling procedure was applied based on the sequence homology of residues 5–384 of R. palmatum BAS and the x-ray crystal structures of CHS superfamily enzymes (63–68% identity) including M. sativa CHS (1BI5A.pdb, 1BQ6A.pdb, 1CGKA.pdb, 1CGZA.pdb, 1CHWA.pdb, 1CHWB.pdb, 1CMLA.pdb), M. sativa CHS C164A mutant (1D6FA.pdb), M. sativa CHS N336A mutant (1D6HA.pdb), M. sativa CHS H303Q mutant (1D6IA.pdb, 1D6IB.pdb), M. sativa CHS G256A mutant (1I86A.pdb), M. sativa CHS G256V mutant (1I88A.pdb, 1I88B.pdb), M. sativa CHS G256L mutant (1I89A.pdb, 1I89B.pdb), M. sativa CHS G256F mutant (1I8BA.pdb, 1I8BB.pdb), M. sativa CHS F215F mutant (1JWXA.pdb), and G. hybrida 2-PS (1EE0A.pdb, 1EE0B.pdb, 1QLVA.pdb, and 1QLVB.pdb). The corresponding Ramachandran plot was also created with Swiss PDB-Viewer software to confirm that the majority of residues grouped in the energetically allowed regions. Calculation of cavity volumes (Connolly's surface volumes) was then performed with the CASTP program (available on the World Wide Web at cast.engr.uic.edu/cast/) (24). The cavity volume of BAS was estimated to be 881 Å3, whereas that of M. sativa CHS (1CGKA.pdb) and G. hybrida 2-PS (1EE0A.pdb) were 1019 and 298 Å3, respectively.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sequence Analysis and Homology Modeling—The primary sequence of R. palmatum BAS exhibits 60–75% identity to those of CHS superfamily enzymes from other plant species including M. sativa CHS (11) and G. hybrida 2-PS (25) (Fig. 3). R. palmatum BAS maintains an almost identical CoA binding site and the catalytic triad of Cys-164, His-303, and Asn-336 (numbering in M. sativa CHS) (Fig. 3). In addition, the active site residues, including Met-137, Gly-211, Gly-216, Ile-254, Gly-256, Ser-338, and Pro-375, along with Phe-265, the second "gatekeeper" phenylalanine (2) of CHS, are well conserved in BAS. One of the most characteristic features is that R. palmatum BAS lacks active site Phe-215 absolutely conserved in all type III PKSs of plant and bacterial origin; the conserved residues 214LF are uniquely replaced by IL (5). In CHS, the hydrophobic Phe-215 located at the interface of active site cavity and the CoA-binding tunnel has been proposed to facilitate malonyl-CoA decarboxylation and help orient substrates and intermediates during the sequential condensation reactions (12, 18).

In the absence of a crystal structure of BAS, we constructed a homology model based on the sequence identity of residues 5–384 of R. palmatum BAS with those of M. sativa CHS (11) and G. hybrida 2-PS (25), whose x-ray crystal structures were recently reported by Noel and co-workers. In the Ramachandran plot calculated for the model, most amino acid residues group in the energetically allowed regions with only few exceptions, primarily glycine residues that can adopt {Phi}/{Psi} angles in all four quadrants. The homology model predicted that R. palmatum BAS has the same overall fold as M. sativa CHS (Fig. 4), with cavity volume estimated to be 881 Å3, which is almost as large as that of M. sativa CHS (1019 Å3) but much larger than that of G. hybrida 2-PS (298 Å3) (25). This suggested that the active site cavity of BAS is easily large enough to accommodate the tetraketide chalcone product.



View larger version (54K):
[in this window]
[in a new window]
 
FIG. 4.
Ribbon display (top) along with an enlargement of the active site (bottom) of three-dimensional structure of R. palmatum BAS predicted by Swiss-PDB-Viewer software (A) and crystal structure of M. sativa CHS (1CGKA.pdb) (B). Active site residues Cys-164, Leu-214 (Ile-214), Phe-215 (Leu-215), His-303, and Asn-336 (numbering in CHS) are represented with ball and stick models.

 

Enzyme Activity of BAS I214L/L215F Mutant—In order to test the hypothesis that the substitution of the conserved 214LF residues by IL in R. palmatum BAS would account for the interruption of the chain extension at the diketide stage, a mutant enzyme in which the residues 214IL were replaced by LF was constructed. The BAS I214L/L215F mutant was heterologously overexpressed in E. coli as a recombinant protein with a His tag at the C terminus as in the case of wild-type BAS, and purified to homogeneity by a nickel-chelate column. As we had expected, BAS I214L/L215F mutant exhibited chalcone-forming activity and performed sequential condensations with three acetate units from malonyl-CoA to produce a tetraketide naringenin chalcone (6), although the activity was rather weak, and a triketide pyrone, BNY (4) was obtained as a major product (Fig. 5B). It was thus confirmed that the residues 214IL are indeed involved in the formation of a diketide benzalacetone and that the polyketide chain elongation was interrupted because of the absence of the active site Phe-215 in BAS.



View larger version (11K):
[in this window]
[in a new window]
 
FIG. 5.
HPLC elution profiles of enzyme reaction products of wild-type BAS (at pH 8.0) (A), BAS I214L/L215F mutant (at pH 6.5) (B), wild-type CHS (at pH 8.0) (C), and CHS L214I/F215L mutant (at pH 7.0) (D). Note that by acid treatment, naringenin chalcone is converted to racemic naringenin through a nonstereospecific ring-C closure. HPLC separation conditions were as described under "Experimental Procedures."

 

Interestingly, the chalcone-forming activity of the mutant enzyme was dependent on pH, which was maximum at pH 6.5, whereas at pH 8.0, most of the reactions were terminated at the triketide stage, leading to formation of BNY as a major product along with a trace amount of benzalacetone and CTAL (Fig. 6B). Enzyme kinetics analysis revealed that BAS I214L/L215F mutant showed Km = 33.5 µM and kcat = 0.169 min1 for 4-coumaroyl-CoA and Km = 46.6 µM and kcat = 0.181 min1 for malonyl-CoA (chalcone-forming activity at pH 6.5) (Table I), whereas wild-type BAS showed Km = 10.0 µM and kcat = 1.79 min1 for 4-coumaroyl-CoA and Km = 23.3 µM and kcat = 1.78 min1 for malonyl-CoA (benzalacetone-forming activity at pH 8.0). The mutant enzyme thus exhibited 36-fold decreases in kcat/Km for 4-coumaroyl-CoA and 20-fold decreases in kcat/Km for malonyl-CoA compared with wild-type BAS.



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 6.
The pH dependence of enzyme activities of wild-type BAS (A), BAS I214L/L215F mutant (B), wild-type CHS (C), and CHS L214I/F215L mutant (D) in 100 mM potassium phosphate buffer (KPB) or in 100 mM Tris-HCl buffer (Tris). BA, benzalacetone; NAR, naringenin chalcone. Conditions were as described under "Experimental Procedures."

 

View this table:
[in this window]
[in a new window]
 
TABLE I
Steady state kinetic parameters for enzyme reactions

Steady state kinetic parameters were calculated for formation of major product of the enzyme reaction at optimum pH except for those of the BAS I214L/L215L mutant, which was for formation of naringenin chalcone. Lineweaver-Burk plots of data were employed to derive the apparent Km and kcat values (average of triplicates ± S.D.) using EnzFitter software (BIOSOFT). Note that by acid treatment, naringenin chalcone was converted to racemic naringenin through a nonstereospecific ring-C closure. WT, wild type.

 

The observed pH dependence of the activity of the mutant enzyme prompted us to reinvestigate the pH profile of the enzyme activity of wild-type BAS. Consistent with our previous report (5), it was reconfirmed that the formation of benzalacetone was maximum within a range of pH 8.0–8.5; however, interestingly, there was a dramatic change in the product profile under acidic pH. Instead of a diketide benzalacetone, formation of a triketide pyrone was dominant, and BNY was obtained almost as a single product at pH 6.0 (Fig. 6A). This indicated that both wild-type and I214L/L215F mutant BAS performed increased numbers of condensation reactions under acidic conditions.

Enzyme Activity of CHS L214I/F215L Mutant—In order to further investigate the structure-function relationship between BAS and CHS enzyme, the conserved active site residues 214LF of S. baicalensis CHS were in turn substituted by IL as in the case of BAS. As a result, the chalcone-forming activity of CHS L214I/F215L mutant was significantly reduced, and BNY was obtained as a major product (Fig. 5D), indicating that most of the chain elongation reactions were terminated at the triketide stage. The BNY forming activity was maximum at pH 7.0, whereas production of tetraketides, chalcone and CTAL, was prominent under acidic conditions within a range of pH 6.0–6.5 (Fig. 6D). In contrast, wild-type S. baicalensis CHS shows maximum chalcone-forming activity at pH 8.0 (Fig. 6C). Interestingly, both wild-type and L214I/F215L mutant CHS did not produce benzalacetone derived from the diketide intermediate, which was confirmed by liquid chromatography-mass spectrometry analysis.

CHS L214I/F215L mutant showed Km = 23.5 µM and kcat = 0.056 min1 for 4-coumaroyl-CoA and showed Km = 125 µM and kcat = 0.054 min1 for malonyl-CoA (BNY-forming activity at pH 7.0) (Table I), whereas wild-type S. baicalensis CHS showed Km = 5.8 µM and kcat = 0.824 min1 for 4-coumaroyl-CoA and showed Km = 25.5 µM and kcat = 0.862 min1 for malonyl-CoA (chalcone-forming activity at pH 8.0). The mutant enzyme thus exhibited 60-fold decreases in kcat/Km for 4-coumaroyl-CoA and 77-fold decreases in kcat/Km for malonyl-CoA.

Substrate Specificities of BAS I214L/L215F and CHS L214I/F215L Mutant—In previous studies, we have demonstrated that S. baicalensis CHS has remarkably broad substrate specificities toward starter and extension substrate of the polyketide formation reaction (19, 21, 22, 26, 27). Thus, instead of 4-coumaroyl-CoA, the enzyme accepted a variety of starter molecules, both aromatic and aliphatic CoA esters of different chain length including benzoyl-CoA (7) and hexanoyl-CoA (8), and efficiently converted to a series of chemically and structurally different unnatural polyketides (Fig. 7C). In contrast, R. palmatum BAS showed rather narrow substrate specificities and did not accept aliphatic CoA esters such as hexanoyl-CoA and acetyl-CoA, whereas benzoyl-CoA was converted to a triketide pyrone (9) (Fig. 7A) (5).



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 7.
Conversion of 4-coumaroyl-CoA (1) and its analogs, benzoyl-CoA (7) and hexanoyl-CoA (8), by wild-type BAS (A), BAS I214L/L215F mutant (B), wild-type CHS (C), and CHS L214I/F215L mutant (D).

 

To further investigate the effect of the mutations, the enzyme activities of BAS I214L/L215F and CHS L214I/F215L mutant were tested with the less bulky starter analogs. Benzoyl-CoA (7) as a substrate, BAS I214L/L215F, afforded a triketide and a tetraketide pyrone (9, 10), whereas hexanoyl-CoA, now accepted as a substrate, was converted to a triketide pyrone (12) (Fig. 7B). The mutation thus resulted in increased number of condensations compared with wild-type BAS. On the other hand, the CHS L214I/F215L mutant, which also afforded BNY as a major product, exhibited a similar product profile, which resulted from the decreased number of chain elongations compared with wild-type CHS (Fig. 7D). Interestingly, in either case, formation of a benzalacetone-type diketide was not detected in the assay mixture.

Enzyme Activity of CHS Phe-215 Point Mutants—In addition to CHS L214I/F215L mutant, six more Phe-215 mutants of S. baicalensis CHS (F215W, F215Y, F215S, F215A, F215H, and F215C) were constructed and tested for the enzyme activities. Previous studies by Noel and co-workers (12) showed that M. sativa CHS F215W, F215Y, and F215S mutations resulted in formation of increased levels of truncated products and that the F215S mutant preferentially accepted nonphysiological N-methylanthraniloyl-CoA as a starter to produce an unnatural tetraketide pyrone (18).

In S. baicalensis CHS, the point mutations significantly reduced chalcone-forming activities and altered functional behavior of the enzyme to various degrees (Fig. 8). Each of the Phe-215 mutants exhibited 9–209-fold decreases in kcat/Km for 4-coumaroyl-CoA and 7–2815-fold decreases in kcat/Km for malonyl-CoA compared with wild-type CHS (Table I). Consistent with the previous report, most of the S. baicalensis CHS mutants afforded increased levels of BNY derived from a triketide intermediate as in the case of the above mentioned L215I/F215L mutant (Fig. 8). Notably, F215H, F215S, and F215H mutant still retained good catalytic activities and yielded BNY as a major product. On the other hand, it was remarkable that the F215Y mutation, only a small modification of the active site by an additional hydroxyl group, resulted in almost complete loss of the enzyme activity. In contrast, M. sativa CHS F215Y mutant still retained chalcone-forming activity, suggesting subtle species difference of the active site structure between S. baicalensis and M. sativa (12). Most interestingly, none of the mutants produced benzalacetone, which indicated that additional subtle structural differences exist between the active site of BAS and CHS besides the residues 214LF.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 8.
Profile of enzyme reaction products of R. palmatum BAS, S. baicalensis CHS, and their mutants. Enzyme reactions were carried out at their optimal pH (see Table I).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
As we had expected, the BAS I214L/L215F mutant exhibited chalcone-forming activity and catalyzed sequential condensations of 4-coumaroyl-CoA with three acetate units from malonyl-CoA, then followed by cyclization and aromatization of the enzyme-bound tetraketide intermediate. Formation of products with an increased number of elongation steps was also observed with benzoyl-CoA and hexanoyl-CoA as an alternative substrate. It was thus confirmed that the residues 214IL in R. palmatum BAS are indeed involved in the formation of benzalacetone and that the polyketide chain extension was interrupted at the diketide stage because of the absence of the conserved 214LF. Only a slight modification of the active site resulted in the functional conversion of BAS into CHS, although the activity of the mutant was rather weak, and a triketide pyrone BNY was obtained as a major product. Recently, functional conversions of other plant type III PKSs (2-PS to CHS (25), acridone synthase to CHS (28), and STS to CHS (2)) by multiple amino acid substitutions have also been reported.

The CHS-based homology model of R. palmatum BAS predicted that BAS has the same overall fold as M. sativa CHS with cavity volume (881 Å3) almost as large as that of M. sativa CHS (1019 Å3) but much larger than that of G. hybrida 2-PS (298 Å3). This suggested that the active site of BAS itself is easily large enough to perform the sequential condensations and to accommodate the tetraketide chalcone product. It has been reported that the shape and volume of the active site cavity greatly influence the substrate specificity and control the final length of the polyketide (25).

In M. sativa CHS, the conformationally flexible "gatekeeper" Phe-215, located at the active site entrance, is thought to facilitate decarboxylation of malonyl-CoA and help orient substrates and intermediates during the sequential condensation reactions, thus controlling the number of the elongation steps (12, 18). Indeed, our site-directed mutagenesis of S. baicalensis CHS (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) significantly reduced chalcone-forming activities and generally afforded increased levels of BNY derived from a triketide intermediate (Fig. 8), which was agreed well with the earlier report on M. sativa CHS mutants (F215S, F215Y, and F215W) by Noel and co-workers (12).

Interestingly, none of the S. baicalensis CHS Phe-215 mutants produced benzalacetone, indicating that there are additional subtle structural differences between the active site of BAS and CHS besides the residues 214LF. Further modification of the active site was thus required for functional conversion of CHS to BAS. Point mutations such as G149D, V210I, T264S, and S332K (numbering in M. sativa CHS) may be good candidates for the termination of the elongation reaction and decarboxylation of the corresponding diketide intermediate, since these residues that are well conserved in plant CHS superfamily enzymes and located at or close to the active site are uniquely absent in R. palmatum BAS (Fig. 3). Furthermore, the T197C mutation would be also interesting because of the reasons described below (2).

The primary driving force for decarboxylation reaction is thought to be the formation of carbondioxide (an entropic effect) and a stabilized carbanion. Decarboxylation is thus facilitated by initial protonation of the {beta}-carbonyl group in {beta}-keto acid and by stabilization of the resulting anion, as proposed in the malonyl-CoA decarboxylation (Fig. 2). Alternatively, enzymatic decarboxylation may also involve a Schiff base formation between the {beta}-carbonyl and active site basic residue, which permits facile protonation of the imine (29). However, there appear to be no appropriate basic residues uniquely present in the active site of BAS for the decarboxylation of a diketide intermediate to generate benzalacetone. On the other hand, Austin and Noel (2) proposed a possibility that BAS utilizes a second active site cysteine for the decarboxylation reaction based on their observation that Thr-197 of CHS sterically altered in a number of divergent type III PKSs is replaced by a reactive cysteine in BAS. As mentioned above, T197C mutation would be thus very interesting; however, in the absence of the crystal structure of BAS, the precise geometry of the active site of the enzyme still remains to be unknown.

Alternatively, the decarboxylation of a diketide intermediate may be also catalyzed by His-303 and Asn-336, the same active site residues involved in the malonyl-CoA decarboxylation by forming an "oxyanion hole" that accommodates the negatively charged transition state (Fig. 2D). In the absence of the aromatic Phe-215, polarizing the {beta}-carbonyl group of 4-coumaroyl acetic acid (or its equivalent) derived from a diketide intermediate would similarly facilitate a second decarboxylation reaction, thus leading to formation of benzalacetone. This hypothesis may be supported by our observation that S. baicalensis CHS (as well as A. hypogaea STS) also catalyzed an unusual decarboxylation reaction of a diketide intermediate to generate a benzalacetone-type compound as a major product when incubated with 4-coumaroyl-CoA and a nonphysiological extender methylmalonyl-CoA (26). Interestingly, 4-coumaroylacetic acid, the diketide without decarboxylation process, has never been detected in the assay mixture.

The pH dependence of the polyketide chain elongations was remarkable. Except for wild-type CHS that showed maximum chalcone-forming activity at pH 8.0, most of the BAS and CHS Phe-215 mutants efficiently catalyzed an increased number of chain extensions under acidic conditions. Most notably, wild-type R. palmatum BAS exhibited a dramatic change from benzalacetone to BNY production at acidic pH. The pH change thus modulated between formation of a decarboxylated diketide product and a triketide pyrone, but interestingly products derived from decarboxylation of a triketide intermediate have never been detected. Presumably, the pH change significantly affected charge distribution in the enzyme-substrate and enzyme-intermediate complex, including the most important charged interactions of the catalytic triad of Cys-164, His-303, and Asn-336, thus greatly influenced the chain elongation reactions. The pH dependence of malonyl-CoA decarboxylation reaction of wild-type M. sativa CHS has been reported to be optimum under acidic condition (13); however, rate-limiting step of the polyketide formation reaction in each enzyme remains to be elucidated.

In summary, we have demonstrated mechanistic consequences of substitution of the crucial active site residue involved in the polyketide chain elongation reaction and provided a structural basis for understanding the structure-function relationship between BAS and CHS enzyme. Further comparative analyses of functionally divergent type III PKS enzymes promise to reveal intimate structural details of the enzyme-catalyzed processes and suggest strategies for manipulating substrate and product specificities of the polyketide formation reactions.


    FOOTNOTES
 
* This work was supported by the 21st Century COE Program and Grants-in-Aid for Scientific Research 13480188, 14580613, and 1531053 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a grant-in-aid from NOVARTIS Foundation (Japan) for the Promotion of Science and from the Tokyo Biochemical Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: University of Shizuoka, School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka 422-8526, Japan. Tel./Fax: 81-54-264-5662; E-mail: abei{at}ys7.u-shizuoka-ken.ac.jp.

1 The abbreviations used are: CHS, chalcone synthase (EC 2.3.1.74 [EC] ); BAS, benzalacetone synthase (EC 2.3.1.-); PKS, polyketide synthase; STS, stilbene synthase; 2-PS, 2-pyrone synthase; CoA, coenzyme A; BNY, bisnoryangonin; CTAL, 4-coumaroyltriacetic acid lactone; HPLC, high pressure liquid chromatography. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schröder, J. (1999) in Comprehensive Natural Products Chemistry (Sankawa, U., ed) Vol. 2, pp. 749–771, Elsevier, Oxford
  2. Austin, M. B., and Noel, J. P. (2003) Nat. Prod. Rep. 20, 79–110[CrossRef][Medline] [Order article via Infotrieve]
  3. Kreuzaler, F., and Hahlbrock, K. (1975) Arch. Biochem. Biophys. 169, 84–90[Medline] [Order article via Infotrieve]
  4. Akiyama, T., Shibuya, M., Liu, H. M., and Ebizuka, Y. (1999) Eur. J. Biochem. 263, 834–839[Abstract/Free Full Text]
  5. Abe, I., Takahashi, Y., Morita, H., and Noguchi, H. (2001) Eur. J. Biochem. 268, 3354–3359[Abstract/Free Full Text]
  6. Schröder, J. (1997) Trends Plant Sci. 2, 373–378[CrossRef]
  7. Borejsza-Wysocki, W., and Hrazdina, G. (1996) Plant Physiol. 110, 791–799[Abstract/Free Full Text]
  8. Schöppner, A., and Kindl, H. (1984) J. Biol. Chem. 259, 6806–6811[Abstract/Free Full Text]
  9. Eckermann, S., Schröder, G., Schmidt, J., Strack, D., Edrada, R. A., Helariutta, Y., Elomaa, P., Kotilainen, M., Kilpeläinen, I., Proksch, P., Teeri, T. H., and Schröder, J. (1998) Nature 396, 387–390[CrossRef]
  10. Lukacin, R., Springob, K., Urbanke, C., Ernwein, C., Schröder, G., Schröder, J., and Matern, U. (1999) FEBS Lett. 448, 135–140[CrossRef][Medline] [Order article via Infotrieve]
  11. Ferrer, J. L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775–784[CrossRef][Medline] [Order article via Infotrieve]
  12. Jez, J. M., Ferrer, J. L., Bowman, M. E., Dixon, R. A., and Noel, J. P. (2000) Biochemistry 39, 890–902[CrossRef][Medline] [Order article via Infotrieve]
  13. Jez, J. M., and Noel, J. P. (2000) J. Biol. Chem. 275, 39640–39646[Abstract/Free Full Text]
  14. Jez, J. M., Bowman, M. E., and Noel, J. P. (2001) Biochemistry 40, 14829–14838[CrossRef][Medline] [Order article via Infotrieve]
  15. Tropf, S., Kärcher, B., Schröder, G., and Schröder, J. (1995) J. Biol. Chem. 270, 7922–7928[Abstract/Free Full Text]
  16. Suh, D. Y., Fukuma, K., Kagami, J., Yamazaki, Y., Shibuya, M., Ebizuka, Y., and Sankawa, U. (2000) Biochem. J. 350, 229–235[CrossRef][Medline] [Order article via Infotrieve]
  17. Suh, D. Y., Kagami, J., Fukuma, K., and Sankawa, U. (2000) Biochem. Biophys. Res. Commun. 275, 725–730[CrossRef][Medline] [Order article via Infotrieve]
  18. Jez, J. M., Bowman, M. E., and Noel, J. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5319–5324[Abstract/Free Full Text]
  19. Abe, I., Morita, H., Nomura, A., and Noguchi, H. (2000) J. Am. Chem. Soc. 122, 11242–11243[CrossRef]
  20. Stöckigt, J., and Zenk, M. H. (1975) Z. Naturforsch. 30, 352–358
  21. Morita, H., Takahashi, Y., Noguchi, H., and Abe, I. (2000) Biochem. Biophys. Res. Commun. 279, 190–195[CrossRef][Medline] [Order article via Infotrieve]
  22. Morita, H., Noguchi, H., Schröder, J., and Abe, I. (2001) Eur. J. Biochem. 268, 3759–3766[Abstract/Free Full Text]
  23. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714–2723[Medline] [Order article via Infotrieve]
  24. Liang, J., Edelsbrunner, H., and Woodward, C. (1998) Protein Sci. 7, 1884–1897[Abstract/Free Full Text]
  25. Jez, J. M., Austin, M. B., Ferrer, J., Bowman, M. E., Schröder, J., and Noel, J. P. (2000) Chem. Biol. 7, 919–930[CrossRef][Medline] [Order article via Infotrieve]
  26. Abe, I., Takahashi, Y., and Noguchi, H. (2002) Org. Lett. 4, 3623–3626[CrossRef][Medline] [Order article via Infotrieve]
  27. Abe, I., Takahashi, Y., Lou, W., and Noguchi, H. (2003) Org. Lett. 5, 1277–1280[CrossRef][Medline] [Order article via Infotrieve]
  28. Lukacin, R., Schreiner, S., and Matern, U. (2001) FEBS Lett. 508, 413–417[CrossRef][Medline] [Order article via Infotrieve]
  29. Silverman, R. B. (2000) in The Organic Chemistry of Enzyme-catalyzed Reactions, pp. 321–357, Academic Press, Inc., San Diego