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1 IUBMB Life, 58(1): 39 46, January 2006 Research Communication Inhibitory Activity of Chlorogenic Acid on Enzymes Involved in the Fatty Acid Synthesis in Animals and Bacteria Bing-Hui Li, Xiao-Feng Ma, Xiao-Dong Wu and Wei-Xi Tian Department of Biology, Graduate University of Chinese Academy of Sciences, Beijing, P. R. China Summary It was found that chlorogenic acid inhibited in vitro animal fatty acid synthase (FAS I) and the b-ketoacyl-acp reductase (FabG) from Escherichia coli in a concentration-dependent manner with respective IC50 of 94.8 and 88.1 mm. The results of Lineweaver- Burk plots indicated that chlorogenic acid inhibited competitively the binding of NADPH to FAS I, while left those of acetyl-coa and malonyl-coa unaffected. Further kinetic studies showed that chlorogenic acid blocked the activity of FAS I mainly by inhibiting the b-ketoacyl reductase domain, which catalyzed the same reaction as that done by FabG in the fatty acid synthesis. The b-ketoacyl reduction reactions accomplished by both FAS I and FabG required nucleotide cofactor, NADPH. Furthermore, the Lineweaver-Burk and Yonetani-Theorell analyses implicated that chlorogenic acid filled competitively in the binding-pocket of NADPH in the b-ketoacyl reductase domain of FAS I. The similar results were also obtained from the inhibition of FabG by chlorogenic acid. As observed in these results, the inhibitions of FAS I and FabG by chlorogenic acid were highly related to the interference of the inhibitor with NADPH, which was possibly due to the similarity between chlorogenic acid and some portion of NADPH, maybe the section consisting of the two ribose groups. IUBMB Life, 58: 39 46, 2006 Keywords Fatty acid synthase; FabG; chlorogenic acid; inhibition; kinetic. INTRODUCTION The de novo synthesis of fatty acids occurs by a serial of ubiquitously biochemical transformations that are necessary to all cells. Recently interest in human and bacterial fatty acid biosynthesis is increasing due to association of the pathway with many human diseases, and so some participating enzymes could be afforded as drug targets (1). Received 22 November 2005; accepted 2 December 2005 Address correspondence to: Prof. Wei-Xi Tian, Department of Biology, Graduate University of Chinese Academy of Sciences, P. O. Box 3908, Beijing , P. R. China. Tel: þ Fax: þ tianweixi@gucas.ac.cn The architecture of fatty acid synthase (FAS 1 ) takes two forms in nature. The associated system, FAS I, consists of a single large polypeptide that contains multiple active sites, i.e., acetyl/malonyl transferase, b-ketoacyl synthase, b-ketoacyl reductase, b-hydroxyacyl dehydratase, enoyl reductase and thioesterase, and which performs all of the elongation steps in the pathway (2, 3). This system mainly exists in animals. The dissociated system, FAS II, consists of separate proteins, and each protein carries out a different catalytic step in the pathway (4). The latter system is found in bacteria and plants. FAS synthesizes fatty acids, mainly palmitate, from the substrates acetyl-coa (Ac-CoA), malonyl-coa (Mal-CoA) and NADPH. At the beginning, acetyl moiety is transferred to the acyl carrier protein (ACP) to undergo the following reactions using Mal-CoA as the C 2 -elongating unit. In the FAS elongation cycle, the growing acyl chain is carried by ACP to a series of four enzymes (4). First, the b-ketoacyl synthase elongates the acyl-acp Cn acyl chain to a Cn þ 2 b-ketoacyl form. Next, the b-keto group is reduced by the NADPH-dependent b-ketoacyl reductase, and the resulting b-hydroxy intermediate is then dehydrated by the b-hydroxyacyl dehydratase to an enoyl-acp. Finally, the reduction of the enoyl chain by the enoyl reductase produces an acyl-acp with an elongated Cn þ 2 acyl chain which is ready to reenter the cycle. Elongation ends when the acyl chain either is used for phospholipids synthesis or grows to the maximum length dictated by the length of the active-site tunnel in the synthase, and then long-chain acyl is hydrolyzed by thioesterase to free fatty acid (3 6). All of the chemical reactions are the same and most of the active site sequences in FAS I and II are related (3, 7). Recently FAS I was regarded as a potential target for some human diseases, such as obesity and cancer (8, 9), and has aroused many researchers interest. So far, some FAS I inhibitors have been reported, including cerulenin (10), C75 (a synthetic compound using cerulenin as template) (9), thiolactomycin and its structural analogues (11), some natural ISSN print/issn online Ó 2006 IUBMB DOI: /

2 40 LI ET AL. polyphenols (12 15), and so on, some of which showed indeed antiobesity and anticancer effects at the animal- and cell-level (8, 16 20). Among the enzymes involved in FAS II, the b-ketoacyl-acp reductase (FabG) is ubiquitously expressed in bacteria, highly conserved across species, and indispensable to the growth of bacteria, because it is the only enzyme responsible for the keto reduction (21). Therefore, FabG possibly represents an ideal target for the development of new antibiotics (22, 23). However, the FabG inhibitors are so scarce that they deserve to be exploited. Chlorogenic acid, the ester of caffeic acid with quinic acid, is one of the most abundant polyphenols in human diet and has been reported to decrease the incidence of chemical carcinogenesis in several animal models of cancer, as well as to suppress the growth of bacteria (24, 25). However, the molecular mechanisms for its anti-carcinogenic and antibacterial properties are poorly understood. In this investigation, we found that chlorogenic acid could inhibit the fatty acid synthesis, which possibly gave a clue for the elucidation of such mechanisms of action. The main aim of this study is to elucidate the inhibitory mechanism of FAS I and bacterial FabG by chlorogenic acid. MATERIALS AND METHODS Materials Ac-CoA, Mal-CoA, NADPH, NADP þ, chlorogenic acid, caffeic acid, and quinic acid were purchased from Sigma- Aldrich. All other reagents were local products with purity of analytical grade. Preparation of FASs and Substrates FAS I was obtained from Fowl (duck). The preparation, storage and use of FAS I were performed as described previously (26). Briefly, the FAS I preparation was homogeneous on polyacrylamide gel electrophoresis in the presence and absence of SDS. The enzyme and substrate concentrations were determined by spectrophotometry with the following extinction coefficients: FAS I, M 71 cm 71 at 279 nm; Ac-CoA, M 71 cm 71 at 259 nm, ph 7.0; Mal-CoA, M 71 cm 71 at 260 nm, ph 6.0; NADPH, M 71 cm 71 at 340 nm and M 71 cm 71 at 259 nm, ph 9.0. Assay of FAS I Activity The overall FAS I activity was determined using an Amersham Pharmacia Ultrospec 4300 pro UV-Vis spectrophotometer at 378C by following the decrease of NADPH at 340 nm. The reaction mixture contained 100 mm potassium phosphate buffer, ph 7.0, 1 mm EDTA, 1 mm dithiolthreitol, 3 mm Ac-CoA, 10 mm Mal-CoA, 35 mm NADPH, and 5 10 mg FAS I in a total volume of 2.0 ml. The reaction was initiated by the addition of enzyme, and the initial rate was used to calculate the enzymatic activity (26). The b-ketoacyl and enoyl reduction activities were determined at 378C by measuring the change of absorption at 340 nm. The ketoacyl reduction reaction mixture (2 ml) contained 40 mm ethyl acetoacetate, 35 mm NADPH, 1 mm EDTA, 1 mm dithiolthreitol and 5 10 mg FAS I in 100 mm phosphate buffer, ph 7.0. The enoyl reduction reaction mixture (2 ml) contained 40 mm ethyl crotonate, 35 mm NADPH, 1 mm EDTA, 1 mm dithiolthreitol and 80 mg of FAS I in 10 mm phosphate buffer, ph 6.3 (27). Preparation and Assay of FabG The engineered strain producing FabG was generous gifts from Dr Charles Rock (St. Jude Children Hospital, USA.). FabG was prepared as described previously (28 30). Briefly, NH 2 -terminally His-tagged FabG were expressed in E. coli strain BL21 (DE3) and purified by nickel chelation affinity chromatography. The preparation was homogeneous on polyacrylamide gel electrophoresis (PAGE) in the presence and absence of SDS. Protein was stored in 50% glycerol at 7208C. FabG activity was determined using an Amersham Pharmacia Ultrospec 4300 pro UV-Vis spectrophotometer at 378C by measuring the change of absorption at 340 nm. The reduction reaction mixture contained 100 mm phosphate buffer, ph 7.0, 40 mm ethyl acetoacetate, 50 mm NADPH, and mg FabG in a total volume of 2.0 ml. The reaction was initiated by the addition of enzyme. Inhibition Studies Reversible inhibitions were measured by adding the inhibitor to the reaction system before the enzyme initiated the reaction. Inhibitors were dissolved in 50% dimethyl sulfoxide (DMSO) and then added to the reaction mixture described above. Reactions with 50% DMSO solvent alone were used as controls. It was found that the final low concentration of DMSO (under 0.5% (V/V)) did not interfere with FabG and FAS I activity. Time-dependent inhibitions were determined by taking aliquots to measure the residual activity at the indicated time intervals after the enzyme solutions were mixed with the inhibitor. The 50% DMSO solvent without any inhibitor was used as the control. In these experiments, DMSO (under 1% (V/V)) did not affect FAS I activity during several hours. RESULTS AND DISCUSSION Chlorogenic acid, ingested regularly with the human diet, is the ester of caffeic acid with quinic acid (Fig. 1A). In our investigation, we measured the effects of chlorogenic acid, caffeic acid and quinic acid on FAS I. As a result, chlorogenic acid showed a concentration-dependent inhibition of the overall reaction of FAS I and approximately 94.8 mm chlorogenic acid could inhibit 50% activity of FAS I (Fig. 1B), while caffeic acid and quinic acid only blocked

3 INHIBITORY ACTIVITY OF CHLOROGENIC ACID ON ENZYMES 41 Figure 1. Inhibitory effects of chlorogenic acid on FAS I. (A) The structure of chlorogenic acid. (B) The activity of FAS I was measured in the presence of various concentrations of chlorogenic acid. The inhibition of the overall reaction of FAS I (.); inhibition of the b-ketoacyl reduction reation ( ); and inhibition of the enoyl reduction reaction of FAS I (~). Each datum is the mean from three experiments. Error bars show standard deviations. weakly the overall reaction of FAS I, and the IC50s were more than 1000 mm (data not shown). The further kinetic mechanism for the inhibition of the overall reaction of FAS I by chlorogenic acid was determined by holding the concentration of chlorogenic acid at a series of fixed values, and measuring the effect of increasing one substrate on the initial reaction rate. Lineweaver-Burk plots of the results yielded three families of straight lines for three substrates of FAS I (Fig. 2). The lines shown in Fig. 2A and B, for Ac-CoA and Mal-CoA, intersect on the X-axis, indicating that chlorogenic acid was a typical noncompetitive inhibitor of FAS I against Ac-CoA or Mal-CoA. In contrast, the inhibition of FAS I overall reaction by chlorogenic acid was competitive with respect to the nucleotide cofactor, NADPH, since the lines for NADPH had a common intercept on the Y-axis (Fig. 2C). The results suggested that chlorogenic acid probably interfered with activity by binding to the binding-pocket of NADPH to prevent the binding of NADPH, whereas the binding of Ac- CoA or Mal-CoA was not influenced by chlorogenic acid. Both the b-ketoacyl reductase and enoyl reductase of FAS I entailed NADPH (3), and so the two partial reactions were also assayed for the inhibition by chlorogenic acid. As shown in Fig. 1B, chlorogenic acid exhibited activity against the b- ketoacyl reduction with an IC50 of mm, which was nearly the same as that for the overall reaction of FAS I. Interestingly, although chlorogenic acid was regarded as an inhibitor of the enoyl-acp reductase (FabI) in FAS II system (31), it did not show a considerable inhibition of the enoyl reduction involved in FAS I (Fig. 1B). This suggested that chlorogenic acid mainly suppressed the b-ketoacyl reductase domain to occlude FAS I. Further results showed that chlorogenic acid inhibited the b-ketoacyl reduction of FAS I competitively with respect to NADPH (Fig. 2D), implicating that chlorogenic acid possibly competed with NADPH for the same active site in the b- ketoacyl reductase domain. The dissociation constant for chlorogenic acid binding with the b-ketoacyl reductase calculated from the secondary plot (Fig. 2D inset) was 73.7 mm, which was approximate to that, 54.1 mm, from the inhibition of FAS I overall reaction (Fig. 2C inset). This also offered evidence for the conclusion that chlorogenic acid possibly dominantly acted on the b-ketoacyl reductase domain to inhibit the overall reaction of FAS I. NADP þ was the oxidized product of NADPH by the b- ketoacyl reductase, and so it could inhibit competitively the b- ketoacyl reductase by binding at the active site where NADPH normally bound (32). To further determine the binding site of chlorogenic acid, the combined effects of two inhibitors, chlorogenic acid and NADP þ, on the b-ketoacyl reductase of FAS I were measured. This method involves assaying the initial velocity of the enzyme at different combinations of the two inhibitors. The effects of two inhibitors on the velocity of an enzymatic reaction can be generally described by the following equation: 1 ¼ 1 1 þ ½IŠ þ ½JŠ þ ½IŠ½JŠ v ij v 0 K i K j ak i K j ð1þ;

4 42 LI ET AL. Figure 2. Lineweaver-Burk plot for inhibition of FAS I by chlorogenic acid. The overall reaction of FAS I was measured in Panel A, B and C. (A) Ac-CoA was the variable substrate. The concentrations of chlorogenic acid were: 0 mm (.), mm ( ), mm (~), and mm ( ). The inset is the plot of the concentration of chlorogenic acid versus the slopes. (B) Mal-CoA was the variable substrate. The concentrations of chlorogenic acid were: 0 mm (.), mm ( ), mm (~), and mm ( ). The inset is the plot of the concentration of chlorogenic acid versus the slopes. (C) NADPH was the variable substrate. The concentrations of chlorogenic acid were: 0 mm (.), mm ( ), mm (~), and mm ( ). The inset is the plot of the concentration of chlorogenic acid versus the slopes. (D) The b-ketoacyl reduction of FAS I was measured. NADPH was the variable substrate. The concentrations of chlorogenic acid were: 0 mm (.), mm ( ), mm (~), and mm ( ). The inset is the plot of the concentration of chlorogenic acid versus the slopes. Each datum is the mean from 2 5 experiments. Error bars show standard deviations. where v ij is the initial velocity in the presence of both inhibitors, v 0 is the maximal velocity in the absence of any inhibitor, K i and K j are the dissociation constants for inhibitors I and J, respectively, and a is an interaction term that defines the effect of the binding of one inhibitor on the affinity of the second inhibitor (33). If the two inhibitors bind in mutually exclusive fashion, a will be infinite (a ¼?), and in the Yonetani-Theorell method, the most popular way of

5 INHIBITORY ACTIVITY OF CHLOROGENIC ACID ON ENZYMES 43 evaluating the interaction between two enzyme inhibitors, it will be observed that plotting 1/v ij against [I] at fixed [J] would result in parallel straight lines (34). In this case, I and J respectively represent NADP þ and chlorogenic acid. As a result, the Yonetani-Theorell plot of experimental data yielded a family of parallel straight lines, as shown in Fig. 3, which suggested that chlorogenic acid and NADP þ mutually exclusively bound to the same site on the enzyme, that is to say, chlorogenic acid could bind competitively at the NADP þ (or NADPH)-binding pocket in the b-ketoacyl reductase, which was consistent with the result of the Lineweaver-Burk plot (Fig. 2D). When [I] ([NADP þ ]) is 0 mm (a ¼?), Equation 1 is changed into the following expression: Y intercept ¼ 1 1 þ ½JŠ ð2þ: n 0 K j The dissociation constant for chlorogenic acid, K j, can be obtained from the plot of the Y-intercepts versus [J] (Fig. 3 inset) by this equation: K j ¼ Y intercept 0 Slope 0 ð3þ: Y intercept 0 and Slope 0 are respectively the intercept and slope of the straight line in Fig. 5 inset. The obtained K j value of 91.2 mm was kinetically comparable to that, 54.1 mm, from Fig. 2C or 73.7 mm, from Fig. 2D inset. FAS I includes two identical multifunctional polypeptide chains, each containing six discrete functional domains with enzymatic activity (2), while the fatty acid synthesis in bacteria entails a series of separate enzymes, and each enzyme carries out a different catalytic step in the pathway (4). Among these enzymes, FabG is responsible for the reduction of the b-keto group (21), and is functionally equal to the b-ketoacyl reductase domain of FAS I. Fig. 4 showed that FabG was also suppressed by chlorogenic acid with an IC50 of 88.1 mm. The kinetic study further showed that chlorogenic acid was a competitive inhibitor of FabG against NADPH (Fig. 5), and the dissociation constant, 31.3 mm, for chlorogenic acid was calculated from the secondary plot of the slopes of these lines versus chlorogenic acid concentration (Fig. 5 inset). It suggested that chlorogenic acid possibly bound to the NADPH-binding site of FabG. All these inhibitory kinetic behaviors of FabG were similar to those of the b-ketoacyl reductase domain of FAS I, and both inhibitions by chlorogenic acid were highly associated with NADPH, indicative of that chlorogenic acid could, or partially, simulate NADPH to bind to the enzyme. Figure 3. Yonetani-Theorell plot for the combined inhibition of the b-ketoacyl reductase domain of FAS I by NADP þ and chlorogenic acid. The activity of the b-ketoacyl reduction of FAS I against different concentrations of NADPþ at the fixed concentration of chlorogenic acid was measured. The concentrations of chlorogenic acid were respectively: 0 mm (.), 25 mm ( ), and 50 mm (~). The inset is the plot of the Y- intercepts versus chlorogenic acid concentrations. Each datum is the mean from three experiments. Error bars show standard deviations. Figure 4. Inhibitory effects of chlorogenic acid on FabG. The activity of FabG was measured in the presence of various concentrations of chlorogenic acid. Each datum is the mean from three experiments. Error bars show standard deviations.

6 44 LI ET AL. Figure 5. Lineweaver-Burk plot for inhibition of FabG by chlorogenic acid. Concentrations of ethyl acetoacetate were fixed at 40 mm. NADPH was the variable substrate. The concentrations of chlorogenic acid were: 0 mm (.), mm ( ), mm (~), and mm ( ). The inset is the plot of the concentration of chlorogenic acid versus the slopes. Each datum is the mean from 2 5 experiments. Error bars show standard deviations. Among the reported FAS inhibitors, epigallocatechin gallate (EGCG) and analogues could also inhibit both FAS I and FabG, and the inhibitions were related to NADPH (15, 35). EGCG inhibited the b-ketoacyl reductase of FAS I competitively against NADPH with IC50 of about 100 mm, and almost showed the same inhibitory activity as chlorogenic acid (15). From their molecular structures (Fig. 6), some similarities were observed. Chlorogenic acid could resemble the phenyl ring and galloyl moiety of EGCG, the effective portion as a FAS inhibitor (35), and both inhibitors are possibly similar to some part of NADPH, maybe the two ribose groups. All of them are rich in hydroxyl groups and oxygen atoms, and could bind to the acceptor by forming hydrogen bonds. Possibly, the structural resemblances made chlorogenic acid and EGCG bind competitively at the NADPH-binding site on the enzyme. In addition, EGCG was able to inhibit irreversibly the b-ketoacyl reductase of FAS I, and the ester bond of EGCG played a critical role in the inactivation (15). Chlorogenic acid also contained a similar ester bond, but it was found that after 1 mm chlorogenic acid was mixed with FAS I, it did not show the obviously irreversible inhibition within 2 h (data not shown). It was possibly caused by the improper position of the ester bond in the chlorogenic acid. Recently, EGCG and analogues were regarded as novel leading compounds of FAS inhibitors (14, 15, 35), therefore chlorogenic acid, as a mechanical analogue of EGCG, could offer some structural information for the future development of specific potent inhibitors of fatty acid synthesis in bacterial and human systems. Figure 6. The structures of chlorogenic acid, EGCG and NADPH. Chlorogenic acid and EGCG possibly acts as a NADPH mimetic to exert their effects on FAS I and FabG. Chlorogenic acid is similar to the portion of in the box of EGCG and NADPH.

7 INHIBITORY ACTIVITY OF CHLOROGENIC ACID ON ENZYMES 45 Although chlorogenic acid has been reported to have anticancer and antibacterial effects (24, 25), little is known about the molecular mechanisms through which chlorogenic acid inhibits carcinogenesis and the growth of bacteria. Based on the current results, these effects afforded by chlorogenic acid probably was, as least partially, related to the inhibition of FAS I in cancer cells and of those enzymes involved in the bacterial fatty acid synthesis, such as FabI (31) and FabG, because the inhibition of fatty acid synthesis could lead to the suppression of the growth of cancer cells and bacteria (9, 35). Although chlorogenic acid did not show very potently inhibitory activity against fowl FAS and FabG from Escherichia coli, considering that the susceptibilities of fatty acid synthases of various sources to chlorogenic acid were different, chlorogenic acid, a plant secondary metabolic and common in human foods, still exhibited clinical or preventive potential in the treatment of some relative human diseases. ACKNOWLEDGEMENTS This work was supported by Grants and from the China National Natural Science Foundation. We thank Dr Charles Rock (St. Jude Children Hospital, USA.) for providing the generous gift of the engineered strain producing FabG. REFERENCES 1. Price, A. C., Zhang, Y. M., Rock, C. O., and White, S. W. (2001) Structure of beta-ketoacyl-[acyl carrier protein] reductase from Escherichia coli: negative cooperativity and its structural basis, Biochemistry 40, Smith, S., Witkowski, A., and Joshi, A. K. (2003) Structural and functional organization of the animal fatty acid synthase. Prog. Lipid Res. 42, Smith, S. (1994) The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. FASEB J. 8, Rock, C. O., and Cronan, J. E. (1996) Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis. Biochim. Biophys. Acta 1302, Wakil, S. J. (1989) Fatty acid synthase, a proficient multifunctional enzyme. Biochemistry 28, Rock, C. O., Goelz, S. E., and Cronan, J. E., Jr. (1981) Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to sn-glycerol 3-phosphate. J. Biol. Chem. 256, Rock, C. O., and Jackowski, S. (2002) Forty years of bacterial fatty acid synthesis. Biochem. Biophys. Res. Commun. 292, Loftus, T. M., Jaworsky, D. E., Frehywot, G. L., Townsend, C. A., Ronnett, G. V., Lane, M. D., and Kuhajda, F. P. (2000) Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288, Kuhajda, F. P., Pizer, E. S., Li, J. N., Mani, N. S., Frehywot, G. L., and Townsend, C. A. (2000) Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proc. Natl. Acad. Sci.USA 97, Vance, D., Goldberg, I., Mitsuhashi, O., and Bloch, K. (1972) Inhibition of fatty acid synthetases by the antibiotic cerulenin. Biochem. Biophys. Res. Commun. 48, McFadden, J. M., Medghalchi, S. M., Thupari, J. N., Pinn, M. L., Vadlamudi, A., Miller, K. I., Kuhajda, F. P., and Townsend, C. A. (2005) Application of a flexible synthesis of (5R)-thiolactomycin to develop new inhibitors of type I fatty acid synthase. J. Med. Chem. 48, Li, B. H., and Tian, W. X. (2004) Inhibitory effects of flavonoids on animal fatty acid synthase. J. Biochem. (Tokyo) 135, Li, B. H., and Tian, W. X. (2003) Presence of fatty acid synthase inhibitors in the rhizome of Alpinia officinarum hance. J. Enzyme Inhib. Med. Chem. 18, Wang, X., Song, K. S., Guo, Q. X., and Tian, W. X. (2003) The galloyl moiety of green tea catechins is the critical structural feature to inhibit fatty-acid synthase. Biochem. Pharmacol. 66, Wang, X., and Tian, W. (2001) Green tea epigallocatechin gallate: a natural inhibitor of fatty-acid synthase. Biochem. Biophys. Res. Commun. 288, Kim, E. K., Miller, I., Aja, S., Landree, L. E., Pinn, M., McFadden, J., Kuhajda, F. P., Moran, T. H., and Ronnett, G. V. (2004) C75, a fatty acid synthase inhibitor, reduces food intake via hypothalamic AMPactivated protein kinase. J. Biol. Chem. 279, Landree, L. E., Hanlon, A. L., Strong, D. W., Rumbaugh, G., Miller, I. M., Thupari, J. N., Connolly, E. C., Huganir, R. L., Richardson, C., Witters, L. A., Kuhajda, F. P., and Ronnett, G. V. (2004) C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism. J. Biol. Chem. 279, Wolfram, S., Raederstorff, D., Wang, Y., Teixeira, S. R., Elste, V., and Weber, P. (2005) TEAVIGO (epigallocatechin gallate) supplementation prevents obesity in rodents by reducing adipose tissue mass. Ann. Nutr. Metab. 49, Yeh, C. W., Chen, W. J., Chiang, C. T., Lin-Shiau, S. Y., and Lin, J. K. (2003) Suppression of fatty acid synthase in MCF-7 breast cancer cells by tea and tea polyphenols: a possible mechanism for their hypolipidemic effects. Pharmacogenomics J. 3, Brusselmans, K., De Schrijver, E., Heyns, W., Verhoeven, G., and Swinnen, J. V. (2003) Epigallocatechin-3-gallate is a potent natural inhibitor of fatty acid synthase in intact cells and selectively induces apoptosis in prostate cancer cells. Int. J. Cancer 106, Zhang, Y., and Cronan, J. E., Jr. (1998) Transcriptional analysis of essential genes of the Escherichia coli fatty acid biosynthesis gene cluster by functional replacement with the analogous Salmonella typhimurium gene cluster. J. Bacteriol. 180, Campbell, J. W., and Cronan, J. E., Jr. (2001) Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. Annu. Rev. Microbiol. 55, Heath, R. J., White, S. W., and Rock, C. O. (2001) Lipid biosynthesis as a target for antibacterial agents. Prog. Lipid. Res. 40, Tamimi, R. M., Lagiou, P., Adami, H. O., and Trichopoulos, D. (2002) Prospects for chemoprevention of cancer. J. Intern. Med. 251, Zhu, X., Zhang, H., and Lo, R. (2004) Phenolic compounds from the leaf extract of artichoke (Cynara scolymus L.) and their antimicrobial activities. J. Agric. Food Chem. 52, Tian, W. X., Hsu, R. Y., and Wang, Y. S. (1985) Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthetase of chicken liver. Inactivation by 5,5 0 -dithiobis-(2-nitrobenzoic acid) and intersubunit cross-linking of the inactivated enzyme. J. Biol. Chem. 260, Shi, Y., Luo, W., Tian, W. X., Zhang, T., and Zhou, H. M. (1998) Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate. Int. J. Biochem. Cell Biol. 30,

8 46 LI ET AL. 28. Heath, R. J., and Rock, C. O. (1995) Regulation of malonyl-coa metabolism by acyl-acyl carrier protein and beta-ketoacyl-acyl carrier protein synthases in Escherichia coli. J. Biol. Chem. 270, Heath, R. J., and Rock, C. O. (1996) Regulation of fatty acid elongation and initiation by acyl-acyl carrier protein in Escherichia coli. J. Biol. Chem. 271, Heath, R. J., and Rock, C. O. (1996) Inhibition of beta-ketoacyl-acyl carrier protein synthase III (FabH) by acyl-acyl carrier protein in Escherichia coli. J. Biol. Chem. 271, Kirmizibekmez, H., Calis, I., Perozzo, R., Brun, R., Donmez, A. A., Linden, A., Ruedi, P., and Tasdemir, D. (2004) Inhibiting activities of the secondary metabolites of Phlomis brunneogaleata against parasitic protozoa and plasmodial enoyl-acp Reductase, a crucial enzyme in fatty acid biosynthesis. Planta. Med. 70, Tian, W. X., Jiang, R. F., Wu, H. B., Shi, Y. H., and Wang, Y. H. (1994) The substrate inhibition by NADPH and kinetics of fatty acid synthase from duck liver. Chinese Biochem. J. 10, Copeland, R. A. (2000) Enzymes: a practical introduction to structure, mechanism, and data analysis. In Reversible Inhibitors (2nd ed)., pp , John Wiley & Sons, New York. 34. Martinez-Irujo, J. J., Villahermosa, M. L., Mercapide, J., Cabodevilla, J. F., and Santiago, E. (1998) Analysis of the combined effect of two linear inhibitors on a single enzyme. Biochem. J. 329 (Pt 3), Zhang, Y. M., and Rock, C. O. (2004) Evaluation of epigallocatechin gallate and related plant polyphenols as inhibitors of the FabG and FabI reductases of bacterial type II fatty-acid synthase. J. Biol. Chem. 279,