Activity Of Mutant Enzyme Research Articles

Random amplified polymorphic DNA analysis of salt-tolerant tobacco mutants generated by gamma radiation.

Salinity is one of the major problems limiting the yield of agricultural products. Radiation mutagenesis is used to improve salt-tolerant mutant plants. In this study, we aimed to improve salt-tolerant mutants of two oriental tobacco varieties. One thousand seeds of each variety (M₀) were irradiated with 100, 200, 300, and 400 Gy gamma rays by Cs-137 gamma. In the M₁ generation, 2999 single plants were harvested. The next season, these seeds were bulked and planted to obtain M₂ progeny. The seeds of 1900 M₂ plants were picked separately. Salinity tolerance was tested in the M₃ generation. Among M₃ plantlets, 10 salt-tolerant tobacco mutants were selected. According to the results of the selection studies, 100- and 200-Gy gamma radiation doses were the effective doses to obtain the desired mutants. Glutathione reductase enzyme activities of salt-tolerant tobacco mutants were determined biochemically as a stress-tolerance marker. The differences between control and salt-tolerant mutants belonging to the Akhisar 97 and İzmir Özbaş tobacco varieties were evaluated by random amplified polymorphic DNA analysis. The total polymorphism rate was 73.91%.

The activity of carp muscle-specific creatine kinase at low temperature is enhanced by decreased hydrophobicity of residue 268.

Abstract The muscle-specific forms of creatine kinase in rabbit (RM-CK) and carp (M1-CK) exhibit different temperature-dependent functional properties. Replacing the glycine at residue 268 of RM-CK with asparagine increases the enzyme's activity at 10°C. In this study, we investigated how hydrophobicity of residue 268 affects the biochemical properties of RM-CK and M1-CK at low temperature. We generated three mutants of both RM-CK and M1-CK: Asp268, Lys268, and Leu268. The secondary structures of these mutants were similar, as revealed by their circular dichroism spectra. Similar to the Asn268 mutants, the Asp268 and Lys268 mutants of RM-CK and M1-CK exhibited higher specific activities at 10°C and pH 8.0. However, no such effect was observed for the RM-CK and M1-CK Leu268 mutants. While in the presence of cryoprotectant (sucrose or trehalose), the activities of wild-type RM-CK and M1-CK mutant enzymes with a hydrophobic residue at 268 were higher, and the effect was more profound at pH 8.0. It may be inferred that water molecules affect protein conformation around residue 268, thereby influencing protein stability at low temperature.

Optimized condition for enhanced soluble-expression of recombinant mutant anabaena variabilis phenylalanine ammonia lyase.

Recently discovered Anabaena variabilis phenylalanine ammonia lyase (AvPAL) proved to be a good candidate for enzyme replacement therapy of phenylketonuria. Outstanding stability properties of a mutant version of this enzyme, produced already in our laboratory, have led us to the idea of culture conditions optimization for soluble expression of this therapeutically valuable enzyme in E. coli. In the present study, the gene encoding mutant version of AvPAL was cloned into the pET28a expression vector. Different concentrations of IPTG, induction period, growth temperature, shaking speed, as well as different types of culture media were examined with respect to the amount of recombinant protein produced and specific activity of the enzyme. Based upon our findings, maximum amount of active mutant enzyme was attained by addition of 0.5 mM IPTG at 150 rpm to the TB culture media. The yield of active enzyme at cluture tempreature of 25 °C and induction period of 18 hour was the highest. The results of this study indicated that the yield of mutant AvPAL production in E. coli can be affected mainly by culture temperature and inducer concentration.

The use of reaction timecourses to determine the level of minor contaminants in enzyme preparations

Enzyme mutagenesis is a commonly used tool to investigate the structure and activity of enzymes. However, even minute contamination of a weakly active mutant enzyme by a considerably more active wild-type enzyme can partially or completely obscure the activity of the mutant enzyme. In this work, we propose a theoretical approach using reaction timecourses and initial velocity measurements to determine the actual contamination level of an undesired wild-type enzyme. To test this method, we applied it to a batch of the Q215A/R235A double mutant of orotidine 5′-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae that was inadvertently contaminated by the more active wild-type OMPDC from Escherichia coli. The enzyme preparation showed significant deviations from the expected kinetic behavior at contamination levels as low as 0.093mol%. We then confirmed the origin of the unexpected kinetic behavior by deliberately contaminating a sample of the mutant OMPDC from yeast that was known to be pure, with 0.015% wild-type OMPDC from E. coli and reproducing the same hybrid kinetic behavior.

Aromatic amino acid mutagenesis at the substrate binding pocket of Yarrowia lipolytica lipase Lip2 affects its activity and thermostability.

The lipase2 from Yarrowia lipolytica (YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F) were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towards p-nitrophenyl palmitate (pNPC16) and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted from p-nitrophenyl laurate (pNPC12) to p-nitrophenyl caprate (pNPC10). The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

A single mutation within a Ca2 + binding loop increases proteolytic activity, thermal stability, and surfactant stability

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca2+ ion. This residue lies 15–20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate–enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca2+ and affects the packing of the Ca2+ binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.

Good Codons, Bad Transcript: Large Reductions in Gene Expression and Fitness Arising from Synonymous Mutations in a Key Enzyme

Biased codon usage in protein-coding genes is pervasive, whereby amino acids are largely encoded by a specific subset of possible codons. Within individual genes, codon bias is stronger at evolutionarily conserved residues, favoring codons recognized by abundant tRNAs. Although this observation suggests an overall pattern of selection for translation speed and/or accuracy, other work indicates that transcript structure or binding motifs drive codon usage. However, our understanding of codon bias evolution is constrained by limited experimental data on the fitness effects of altering codons in functional genes. To bridge this gap, we generated synonymous variants of a key enzyme-coding gene in Methylobacterium extorquens. We found that mutant gene expression, enzyme production, enzyme activity, and fitness were all significantly lower than wild-type. Surprisingly, encoding the gene using only rare codons decreased fitness by 40%, whereas an allele coded entirely by frequent codons decreased fitness by more than 90%. Increasing gene expression restored mutant fitness to varying degrees, demonstrating that the fitness disadvantage of synonymous mutants arose from a lack of beneficial protein rather than costs of protein production. Protein production was negatively correlated with the frequency of motifs with high affinity for the anti-Shine-Dalgarno sequence, suggesting ribosome pausing as the dominant cause of low mutant fitness. Together, our data support the idea that, although a particular set of codons are favored on average across a genome, in an individual gene selection can either act for or against codons depending on their local context.

A Novel meso -Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d -Amino Acid Synthesis

meso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP(+)-dependent enzyme which catalyzes the reversible oxidative deamination on the d-configuration of meso-2,6-diaminopimelate to produce l-2-amino-6-oxopimelate. In this study, the gene encoding a meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum was cloned and expressed in Escherichia coli. In addition to the native substrate meso-2,6-diaminopimelate, the purified enzyme also showed activity toward d-alanine, d-valine, and d-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generate d-amino acids in up to 99% conversion and 99% enantiomeric excess. Since meso-diaminopimelate dehydrogenases are known to be specific to meso-2,6-diaminopimelate, this is a unique wild-type meso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential for d-amino acid synthesis. The enzyme is the most stable meso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites of S. thermophilum meso-DAPDH different from the sequences of other known meso-DAPDHs were replaced with the conserved amino acids in other meso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile of S. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effective d-amino acid dehydrogenases by protein engineering.

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy

Deficiencies of lysosomal β-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant β-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal β-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable.

Citric acid cycle and nitrogen assimilation enzyme activities in glutamate dehydrogenase mutants of S. cerevisiae

In Saccharomyces cerevisiae reductive amination of the citric acid cycle intermediate α‐ketoglutarate to glutamate is the main pathway for nitrogen assimilation. This is catalyzed by the NADP‐dependent glutamate dehydrogenases (Gdh1p & 3p), and the reverse reaction by an NAD‐dependent enzyme (Gdh2p). It has been proposed that, in GDH‐mutant strains, either the proline utilization (PUT) or the glutamine synthetase‐glutamate synthase (GS/GOGAT) pathway is the alternative nitrogen assimilation pathway to the exclusion of the other. Using a GDH‐null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway‐specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). Growth of the null mutant was only slightly impaired on all nitrogen sources tested, confirming other pathways for nitrogen assimilation exist. Enzyme assays point to GS/GOGAT as the alternate pathway when ammonia, glutamine or proline are the sole nitrogen sources. In contrast, GOGAT activity was decreased on urea and Put2p activity was slightly increased. These results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH‐dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serve as the sole compensatory pathway. (Supported by NIH grant R15‐GM069372 to P.J.T.)

Structure-Based Redesign of GST A2-2 for Enhanced Catalytic Efficiency with Azathioprine

Glutathione transferase (GST) A2-2 is the most efficient human enzyme in the biotransformation of the prodrug azathioprine (Aza). The activation of Aza has therapeutic potential for possible use of GSTs in targeted enzyme-prodrug treatment of diseases. Based on the assumed catalytic mechanism and computational docking of Aza to the active site of the enzyme, active-site residues were selected for construction of focused mutant libraries, which were thereafter screened for Aza activity. Mutants with elevated Aza activity were identified, DNA sequenced, and the proteins purified. The two most active mutants showed up to 70-fold higher catalytic efficiency than the parental GST A2-2. The structure of the most active triple mutant (L107G/L108D/F222H) enzyme was determined by X-ray crystallography demonstrating significant changes in the topography of the active site facilitating productive binding of Aza as a substrate.

C35T mutation could slightly decrease the activity of human α-(1,2)-fucosyltransferase

Purpose of the studyThe C35T mutant α-(1,2)-fucosyltransferase (FUT1) gene had been reported related to the para-Bombay phenotype, but recently, our laboratory found that the C35T was a polymorphism in the Chinese population. This study aims at further clarifying the property of C35T mutant FUT1. Material and methodsThe mutant C35T FUT1 gene was cloned into expression vector in vitro, the mRNA expression was analyzed by real-time quantitative polymerase chain reaction (PCR), and the mutant enzyme's activity was determined in vitro. ResultsThe results showed that the frequencies of 35C and 35T alleles were 0.735 and 0.265, respectively. The FUT1 mRNA level of transfected cells with C35T recombination vector showed 99.85% of the wild-type FUT1 transfected cells. The enzyme relative activity of transfected cell lysates with C35T FUT1 recombination vector was 79.45% compared with that of the wild-type FUT1 transfected cell lysates. The Kmphenyl-gal value of enzyme encoded by C35T allele was 0.5 times higher than that of the enzyme encoded by FUT1 wild-type allele. ConclusionThese results suggested that FUT1 C35T was a polymorphism in the Chinese population and did not affect its mRNA transcription, but could slightly decrease the activity of human α-(1,2)-fucosyltransferase in vitro.

High Throughput Screening for Small Molecule Therapy for Gaucher Disease Using Patient Tissue as the Source of Mutant Glucocerebrosidase

Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

Rhodobacter sphaeroides Uses a Reductive Route via Propionyl Coenzyme A To Assimilate 3-Hydroxypropionate

3-Hydroxypropionate is a product or intermediate of the carbon metabolism of organisms from all three domains of life. However, little is known about how carbon derived from 3-hydroxypropionate is assimilated by organisms that can utilize this C(3) compound as a carbon source. This work uses the model bacterium Rhodobacter sphaeroides to begin to elucidate how 3-hydroxypropionate can be incorporated into cell constituents. To this end, a quantitative assay for 3-hydroxypropionate was developed by using recombinant propionyl coenzyme A (propionyl-CoA) synthase from Chloroflexus aurantiacus. Using this assay, we demonstrate that R. sphaeroides can utilize 3-hydroxypropionate as the sole carbon source and energy source. We establish that acetyl-CoA is not the exclusive entry point for 3-hydroxypropionate into the central carbon metabolism and that the reductive conversion of 3-hydroxypropionate to propionyl-CoA is a necessary route for the assimilation of this molecule by R. sphaeroides. Our conclusion is based on the following findings: (i) crotonyl-CoA carboxylase/reductase, a key enzyme of the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, was not essential for growth with 3-hydroxypropionate, as demonstrated by mutant analyses and enzyme activity measurements; (ii) the reductive conversion of 3-hydroxypropionate or acrylate to propionyl-CoA was detected in cell extracts of R. sphaeroides grown with 3-hydroxypropionate, and both activities were upregulated compared to the activities of succinate-grown cells; and (iii) the inactivation of acuI, encoding a candidate acrylyl-CoA reductase, resulted in a 3-hydroxypropionate-negative growth phenotype.

Bases moleculares del tratamiento en la enfermedad de Gaucher

In Gaucher's disease (GD) there is an imbalance in the monocyte-macrophage system between the rate of formation and degradation of glucosylceramide due to low activity of the lysosomal enzyme betaglucorebrosidase. Therapeutic approaches are aimed to reduce the synthesis of glucosylceramide by inhibiting ceramide-glucosyltransferase or increasing the degradation of glucosylceramide using a recombinant beta-glucorebrosidase or recovering the residual activity of mutant enzymes with pharmacological chaperones. Inhibitory molecules of ceramide-glucosyltransferase used mimic glucose or ceramide. All recombinant enzymes used in GD enzyme replacement therapy have similar kinetic parameters, but differ in their amino acid sequence, as well as, in the exposed mannose oligosaccharide chains. The mannose content and localization in the oligosaccharide chains are essential for cell uptake. Velaglucerase alpha have a high content of mannoses in their oligosaccharide chains.

Identification and functional characterization of a novel mutation P459H and a rare mutation R483W in the CYP21A2 gene in two Chinese patients with simple virilizing form of congenital adrenal hyperplasia.

Steroid 21-hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH). Clinically, 21-OHD is categorized into saltwasting, simple-virilizing (SV), and non-classical (NC) forms. It is well recognized that a good correlation exists between genotype and clinical phenotype of CAH. The aim of this study was to identify CYP21A2 gene mutations in 2 Chinese patients with SV CAH along with their parents and other family members. By direct sequencing the CYP21A2 gene, a novel mutation, P459H, was detected in 1 patient; and a previously described uncharacterized mutation, R483W, was found in another patient. The 21-hydroxylase activities were determined by measuring the converting rate of progesterone to 11-deoxycorticosterone in COS-7 cells overexpressed with these mutated proteins. Our results revealed significantly reduced enzyme activity in both mutants: residual activity of P459H and R483W towards progesterone was 6.8%±2.1 and 2.9%±1.5, respectively compared to that of the wild type. We also demonstrated the loss of 21-hydroxylase activities using a three-dimensional model of CYP21A2. Both R483W and P459H mutations are confirmed to be related to NC CAH by in vitro functional study, with phenotype variance of R483W in Tunisian and Chinese patients. This study will aid in predicting disease severity and in facilitating family genetic counseling.

Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases

The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site-directed mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. Modification of the SBS resulted in an up to three-fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates. Weakening of the SBS increased K(d) values by up to 70-fold in binding affinity tests using natural substrates. The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such structural elements can increase the efficiency of these single domain enzymes on their natural substrates.

Structural and Biochemical Elucidation of Mechanism for Decarboxylative Condensation of β-Keto Acid by Curcumin Synthase

The typical reaction catalyzed by type III polyketide synthases (PKSs) is a decarboxylative condensation between acyl-CoA (starter substrate) and malonyl-CoA (extender substrate). In contrast, curcumin synthase 1 (CURS1), which catalyzes curcumin synthesis by condensing feruloyl-CoA with a diketide-CoA, uses a β-keto acid (which is derived from diketide-CoA) as an extender substrate. Here, we determined the crystal structure of CURS1 at 2.32 Å resolution. The overall structure of CURS1 was very similar to the reported structures of type III PKSs and exhibited the αβαβα fold. However, CURS1 had a unique hydrophobic cavity in the CoA-binding tunnel. Replacement of Gly-211 with Phe greatly reduced the enzyme activity. The crystal structure of the G211F mutant (at 2.5 Å resolution) revealed that the side chain of Phe-211 occupied the hydrophobic cavity. Biochemical studies demonstrated that CURS1 catalyzes the decarboxylative condensation of a β-keto acid using a mechanism identical to that for normal decarboxylative condensation of malonyl-CoA by typical type III PKSs. Furthermore, the extender substrate specificity of CURS1 suggested that hydrophobic interaction between CURS1 and a β-keto acid may be important for CURS1 to use an extender substrate lacking the CoA moiety. From these results and a modeling study on substrate binding, we concluded that the hydrophobic cavity is responsible for the hydrophobic interaction between CURS1 and a β-keto acid, and this hydrophobic interaction enables the β-keto acid moiety to access the catalytic center of CURS1 efficiently.