Biphenyl synthase,a novel type III polyketide synthase

Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60% amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.     

Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants

Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS¹ type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.     

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

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 simulate 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 K_m for the substrate 4-coumaroyl-CoA but a lower V_max 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.Abbreviations AA amino acid - CHS chalcone synthase - STS stilbene synthase Correspondence to: J. Schröder 0592The data are discussed on the level of the presently available CHS and STS sequences although many were published after beginning the experiments several years ago. The new information changed the CHS consensus in some details but otherwise confirmed the deductions on the potential significance of amino acid differences between CHS and STS     

Covalent binding of chloroacetamide herbicides to the active site cysteine of plant type III polyketide synthases

Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides.     

Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning,functional expression,and site-directed mutagenesis of two polyketide synthases

Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.     

A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis

Chalcone synthase (CHS) related type III plant polyketide synthases (PKSs) are likely to be involved in the biosynthesis of diarylheptanoids (e.g. curcumin and polycyclic phenylphenalenones), but no such activity has been reported. Root cultures from Wachendorfia thyrsiflora (Haemodoraceae) are a suitable source to search for such enzymes because they synthesize large amounts of phenylphenalenones, but no other products that are known to require CHSs or related enzymes (e.g. flavonoids or stilbenes). A homology-based RT-PCR strategy led to the identification of cDNAs for a type III PKS sharing only approximately 60% identity with typical CHSs. It was named WtPKS1 (W. thyrsiflora polyketide synthase 1). The purified recombinant protein accepted a large variety of aromatic and aliphatic starter CoA esters, including phenylpropionyl- and side-chain unsaturated phenylpropanoid-CoAs. The simplest model for the initial reaction in diarylheptanoid biosynthesis predicts a phenylpropanoid-CoA as starter and a single condensation reaction to a diketide. Benzalacetones, the expected release products, were observed only with unsaturated phenylpropanoid-CoAs, and the best results were obtained with 4-coumaroyl-CoA (80% of the products). With all other substrates, WtPKS1 performed two condensation reactions and released pyrones. We propose that WtPKS1 catalyses the first step in diarylheptanoid biosynthesis and that the observed pyrones are derailment products in the absence of downstream processing proteins.     

Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae

Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75-50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.     

Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins

Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C₂-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7–4.2% of naringenin) and naringenin production by STS (1.4–2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Benzalacetone synthase. A novel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum.

Benzalacetone synthase (BSA) is a novel plant-specific polyketide synthase that catalyzes a one step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 skeleton of phenylbutanoids in higher plants. A cDNA encoding BAS was for the first time cloned and sequenced from rhubarb (Rheum palmatum), a medicinal plant rich in phenylbutanoids including pharmaceutically important phenylbutanone glucoside, lindleyin. The cDNA encoded a 42-kDa protein that shares 60-75% amino-acid sequence identity with other members of the CHS-superfamily enzymes. Interestingly, R. palmatum BAS lacks the active-site Phe215 residue (numbering in CHS) which has been proposed to help orient substrates and intermediates during the sequential condensation of 4-coumaroyl-CoA with malonyl-CoA in CHS. On the other hand, the catalytic cysteine-histidine dyad (Cys164-His303) in CHS is well conserved in BAS. A recombinant enzyme expressed in Escherichia coli efficiently afforded benzalacetone as a single product from 4-coumaroyl-CoA and malonyl-CoA. Further, in contrast with CHS that showed broad substrate specificity toward aliphatic CoA esters, BAS did not accept hexanoyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and acetyl-CoA as a substrate. Finally, besides the phenylbutanones in rhubarb, BAS has been proposed to play a crucial role for the construction of the C6-C4 moiety of a variety of natural products such as medicinally important gingerols in ginger plant.     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.     

Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase

Aloesone synthase (ALS) and chalcone synthase (CHS) are plant-specific type III poyketide synthases sharing 62% amino acid sequence identity. ALS selects acetyl-CoA as a starter and carries out six successive condensations with malonyl-CoA to produce a heptaketide aloesone, whereas CHS catalyses condensations of 4-coumaroyl-CoA with three malonyl-CoAs to generate chalcone. In ALS, CHS's Thr197, Gly256, and Ser338, the active site residues lining the initiation/elongation cavity, are uniquely replaced with Ala, Leu, and Thr, respectively. A homology model predicted that the active site architecture of ALS combines a 'horizontally restricting' G256L substitution with a 'downward expanding' T197A replacement relative to CHS. Moreover, ALS has an additional buried pocket that extends into the 'floor' of the active site cavity. The steric modulation thus facilitates ALS to utilize the smaller acetyl-CoA starter while providing adequate volume for the additional polyketide chain extensions. In fact, it was demonstrated that CHS-like point mutations at these positions (A197T, L256G, and T338S) completely abolished the heptaketide producing activity. Instead, A197T mutant yielded a pentaketide, 2,7-dihydroxy-5-methylchromone, while L256G and T338S just afforded a triketide, triacetic acid lactone. In contrast, L256G accepted 4-coumaroyl-CoA as starter to efficiently produce a tetraketide, 4-coumaroyltriacetic acid lactone. These results suggested that Gly256 determines starter substrate selectivity, while Thr197 located at the entrance of the buried pocket controls polyketide chain length. Finally, Ser338 in proximity of the catalytic Cys164 guides the linear polyketide intermediate to extend into the pocket, thus leading to formation of the hepataketide in Rheum palmatum ALS.     

A new family of type III polyketide synthases in Mycobacterium tuberculosis

The Mycobacterium tuberculosis genome has revealed a remarkable array of polyketide synthases (PKSs); however, no polyketide product has been isolated thus far. Most of the PKS genes have been implicated in the biosynthesis of complex lipids. We report here the characterization of two novel type III PKSs from M. tuberculosis that are involved in the biosynthesis of long-chain alpha-pyrones. Measurement of steady-state kinetic parameters demonstrated that the catalytic efficiency of PKS18 protein was severalfold higher for long-chain acyl-coenzyme A substrates as compared with the small-chain precursors. The specificity of PKS18 and PKS11 proteins toward long-chain aliphatic acyl-coenzyme A (C12 to C20) substrates is unprecedented in the chalcone synthase (CHS) family of condensing enzymes. Based on comparative modeling studies, we propose that these proteins might have evolved by fusing the catalytic machinery of CHS and beta-ketoacyl synthases, the two evolutionarily related members with conserved thiolase fold. The mechanistic and structural importance of several active site residues, as predicted by our structural model, was investigated by performing site-directed mutagenesis. The functional identification of diverse catalytic activity in mycobacterial type III PKSs provide a fascinating example of metabolite divergence in CHS-like proteins.     

Enzymatic formation of unnatural aromatic polyketides by chalcone synthase

Substrate specificity of recombinant chalcone synthase (CHS) from Scutellaria baicalensis (Labiatae) was investigated using chemically synthesized aromatic and aliphatic CoA esters. It was demonstrated for the first time that CHS converted benzoyl-CoA to phlorobenzophenone (2,4,6-trihydroxybenzophenone) along with pyrone by-products. On the other hand, phenylacetyl-CoA was enzymatically converted to an unnatural aromatic polyketide, phlorobenzylketone (2, 4,6-trihydroxyphenylbenzylketone), whose structure was finally confirmed by chemical synthesis. Furthermore, in agreement with earlier reports, S. baicalensis CHS also accepted aliphatic CoA esters, isovaleryl-CoA and isobutyryl-CoA, to produce phloroacylphenones. In contrast, hexanoyl-CoA only afforded pyrone derivatives without formation of a new aromatic ring. It was noteworthy that both aromatic and aliphatic CoA esters were accepted in the active site of the enzyme as a starter substrate for the complex condensation reaction. The low substrate specificity of CHS thus provided further insight into the structure and function of the enzyme.     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Structure and mechanism of chalcone synthase-like polyketide synthases

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure–function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 393–398. Received 14 June 2001/ Accepted in revised form 15 July 2001     

Fatty acid specificity of bile salt-dependent lipase: enzyme recognition and super-substrate effects

A putative fatty acid specificity of bile salt-dependent lipases (BSDLs) has been re-investigated. The strategy was to use two evolutionally distant, homologous BSDLs (from human and cod), and to investigate their hydrolysis of different fatty acid esters at different assay conditions affecting the physicochemical phase of the substrate. Depending on assay conditions, large variations were seen in the hydrolysis rate for esters of different fatty acids. The two enzymes displayed similar fatty acid specificity patterns, with small, but significant differences that were maintained at various assay conditions. Compared to the human enzyme, the cod enzyme showed a preference for hydrolysis of long-chain polyunsaturated fatty acyl esters (up to 22 carbons in length). On the other hand, the human enzyme hydrolysed esters of shorter chain saturated fatty acids at significantly higher rates compared to the cod enzyme. Changing physicochemical factors affecting the substrate phase induced large changes in fatty acid specificity that affected both enzymes in similar manners. It is concluded that though the aliphatic chains of the fatty acids may not be recognized by the enzymes, these chains indirectly affect the conformation or interfacial availability of the carboxyl ester bond in the substrate, and the enzymes show minor specificities for variations in these structures.     

The first plant type III polyketide synthase that catalyzes formation of aromatic heptaketide

A cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned from rhubarb (Rheum palmatum). A recombinant enzyme expressed in Escherichia coli accepted acetyl-CoA as a starter, carried out six successive condensations with malonyl-CoA and subsequent cyclization to yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino acid sequence identity with chalcone synthases (CHSs), and maintains almost identical CoA binding site and catalytic residues conserved in the CHS superfamily enzymes. Further, homology modeling predicted that the 43-kDa protein has the same overall fold as CHS. This provides new insights into the catalytic functions of type III PKSs, and suggests further involvement in the biosynthesis of plant polyketides.     

Molecular analysis of the role of tyrosine 224 in the active site of Streptomyces coelicolor RppA, a bacterial type III polyketide synthase

Streptomyces coelicolor RppA (Sc-RppA), a bacterial type III polyketide synthase, utilizes malonyl-CoA as both starter and extender unit substrate to form 1,3,6,8-tetrahydroxynaphthalene (THN) (therefore RppA is also known as THN synthase (THNS)). The significance of the active site Tyr(224) for substrate specificity has been established previously, and its aromatic ring is believed to be essential for RppA to select malonyl-CoA as starter unit. Herein, we describe a series of Tyr(224) mutants of Sc-RppA including Y224F, Y224L, Y224C, Y224M, and Y224A that were able to catalyze a physiological assembly of THN, albeit with lower efficiency, challenging the necessity for the Tyr(224) aromatic ring. Steady-state kinetics and radioactive substrate binding analysis of the mutant enzymes corroborated these unexpected results. Functional examination of the Tyr(224) series of RppA mutants using diverse unnatural acyl-CoA substrates revealed the unique role of malonyl-CoA as starter unit substrate for RppA, leading to the development of a novel stericelectronic constraint model.     

定点突变提高虎杖聚酮合酶的苯亚甲基丙酮合酶活性北大核心CSCD

虎杖(Polygonum cuspidatum)聚酮合酶(polyketide synthase 1,PcPKS1)同时具有查尔酮合酶(chalcone synthase,CHS)及苯亚甲基丙酮合酶(benzylidene acetone synthase,BAS)催化活性,能够催化生成聚酮类化合物柚皮素查尔酮和苯亚甲基丙酮,进而催化合成黄酮类或覆盆子酮等具有多种生物学活性的化合物。本研究通过分析虎杖PcPKS1与掌叶大黄(Rheum palmatum)BAS、拟南芥(Arabidopsis thaliana)CHS等家族成员的序列以及酶催化位点的构象,确定可能影响酶功能的3个氨基酸位点:Thr133、Ser134、Ser339。采用定点突变对PcPKS1进行分子修饰,成功获得2个突变体并进行相关体外酶促反应,高效液相色谱(high performance liquid chromatography,HPLC)产物分析结果表明,在pH 7.0和pH 9.0的体外酶促条件下,突变体T133LS134A和S339V维持BAS和CHS双功能活性,且BAS活性显著高于原PcPKS1。本研究为利用PcPKS1进行基因工程调节黄酮类和覆盆子酮化合物的生物合成提供理论依据。