A Role of the Amino Acid Residue Located on the Fifth Position before the First Aspartate-rich Motif of Farnesyl Diphosphate Synthase on Determination of the Final Product

Abstract

Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the ω-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate. Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the ω-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate. INTRODUCTIONIsoprenoids, which include over 23,000 known metabolites, are the most chemically diverse family of naturally occurring compounds. The essential and major biosynthetic step in all isoprenoid metabolism is the elongation of isoprene units by prenyltransferases (Fig. 1) (1Poulter C.D. Rilling H.C. Spurgeon S.L. Porter J.W. Biosynthesis of Isoprenoid Compounds. John Wiley & Sons, New York1982: 161-224Google Scholar). These enzymes, which consecutively mediate alkylation of isopentenyl diphosphate (IPP, C-5) 1The abbreviations used are: IPPisopentenyl diphosphateDMAPPdimethylallyl diphosphateGPPgeranyl diphosphateFPPfarnesyl diphosphateGGPPgeranylgeranyl diphosphateGFPPgeranylfarnesyl diphosphateHPPhexaprenyl diphosphate. by allylic diphosphates, are classified according to the chain length of the final product and the stereochemistry of double bond formed by the condensations. So far, a number of prenyltransferases have been determined from various organisms.For example, farnesyl diphosphate (FPP) synthase (EC 2.5.1.1) catalyzes the sequential condensations of two molecules of IPP (C-5) with dimethylallyl diphosphate (DMAPP, C-5) to give a C-15 compound with E-stereochemistry. The product, FPP, occupies a central point leading to several branches of the pathway for the synthesis of important classes of compounds, including sterols, farnesylated proteins, hemes, respiratory quinones, sesquiterpenes, and dolichols. On the other hand, geranylgeranyl diphosphate (GGPP, C-20) synthase (EC 2.5.1.29) catalyzes the condensation of IPP to give (all-E)-GGPP, which plays as a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaebacterial membrane lipids.These prenyltransferases catalyze the same sort of condensation and have a similarity in amino acid sequences (2Chen A. Kroon P.A. Poulter C.D. Protein Sci. 1994; 3: 600-607Crossref PubMed Scopus (219) Google Scholar, 3Koyama T. Obata S. Osabe M. Takeshita A. Yokoyama K. Uchida M. Nishino T. Ogura K. J. Biochem. 1993; 113: 355-363Crossref PubMed Scopus (122) Google Scholar). However, every enzyme does not catalyze a further condensation of IPP than the general ultimate product. Until now it has been left in question how the consecutive condensations precisely stop at a destined step.Recently, our group succeeded in converting FPP synthase from Bacillus stearothermophilus to GGPP synthase using chemical random mutagenesis followed by an in vivo color selection (4Ohnuma S.-i. Nakazawa T. Hemmi H. Hallberg A.-M. Koyama T. Ogura K. Nishino T. J. Biol. Chem. 1996; 271: 10087-10095Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). From the analysis of the mutations in the FPP synthases whose product specificities had become the same as GGPP synthase, we defined three amino acids that could determine the final chain length; leucine at position 34, tyrosine at position 81, and valine at position 157. In particular, the mutated enzyme that has a substitution of histidine for tyrosine at position 81, which is situated at the fifth amino acid before the first aspartate rich consensus motif, the most effectively produces GGPP. Moreover, our group also showed that, in the case of Sulfolobus acidocaldarius GGPP synthase, the amino acid at the same position also determines the chain length of the product, GGPP (5Ohnuma S.-i. Hirooka K. Hemmi H. Ishida C. Ohto C. Nishino T. J. Biol. Chem. 1996; 271: 18831-18837Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Thus, in this paper, we precisely analyze the role of the amino acid at position 81 of B. stearothermophilus FPP synthase on chain length determination.DISCUSSIONIn this study, we have demonstrated that the chain length of a final product is dramatically changed by replacement of only one amino acid at position 81, which is situated at the fifth amino acid before the first aspartate-rich motif, and have indicated that the average chain length of products is inversely proportional to the accessible surface area of the substituted amino acid.The crystal structure of avian FPP synthase has been determined to 2.6-Å resolution by Tarshis et al. (11Tarshis L.C. Yan M. Poulter C.D. Sacchettini J.C. Biochemistry. 1994; 33: 10871-10877Crossref PubMed Scopus (371) Google Scholar). The FPP synthase is composed of 13 α-helices jointed by connecting loops, and eight of them form a large central cavity. Two aspartate-rich motifs that are highly conserved among the prenyltransferase family are found on opposite walls of this cavity, and the active site of FPP synthase seems to be located in the large central cavity. In a previous report (4Ohnuma S.-i. Nakazawa T. Hemmi H. Hallberg A.-M. Koyama T. Ogura K. Nishino T. J. Biol. Chem. 1996; 271: 10087-10095Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), our group showed a considerable structural similarity between avian and B. stearothermophilus FPP synthases and indicated that the tyrosine-81 of B. stearothermophilus FPP synthase is situated on a point 11-∼12 Å apart from the first aspartate-rich motif, which has been thought to bind the diphosphate moiety of an allylic substrate via the magnesium ion (1Poulter C.D. Rilling H.C. Spurgeon S.L. Porter J.W. Biosynthesis of Isoprenoid Compounds. John Wiley & Sons, New York1982: 161-224Google Scholar, 16Marrero P.F. Poulter C.D. Edwards P.A. J. Biol. Chem. 1992; 267: 21873-21878Abstract Full Text PDF PubMed Google Scholar, 17Joly A. Edwards P.A. J. Biol. Chem. 1993; 268: 26983-26989Abstract Full Text PDF PubMed Google Scholar, 18Song L. Poulter C.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3044-3048Crossref PubMed Scopus (176) Google Scholar). The distance is similar to the length of the hydrocarbon moiety of FPP. From these data, we suggested that the aromatic ring of tyrosine-81 might prevent more chain elongations than FPP. It supports this hypothesis that an amino acid having smaller accessible surface area can form a longer final product. Moreover, the fact that a mutated GGPP synthase with an aliphatic amino acid at that position yields products slightly longer than that with a nonaliphatic amino acid with a similar accessible surface area indicates that the amino acid at position 81 directly contacts with the ω-terminal of elongating product by hydrophobic interaction. On the basis of this, we propose a simple model for the condensation and termination mechanism of the FPP synthase reaction (Fig. 4).Fig. 4Simple model for consecutive condensations of IPP by FPP synthase. First, FPP synthase accepts IPP and DMAPP. After two consecutive condensations of IPP, the ω-terminal of the formed FPP reaches the amino acid at the fifth amino acid before the first aspartate rich motif. If the side chain of the amino acid is large, further condensation is inhibited, resulting in formation of FPP (route A). On the other hand, when the side chain is small, the condensation continues to give long products such as HPP (route B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The deduced amino acid sequence analysis of cloned prenyltransferase genes shows that all prenyltransferases have a significant sequence similarity. Three prenyltransferases that catalyze consecutive condensations beyond GGPP have been cloned, hexaprenyl diphosphate synthase from Saccharomyces cerevisiae (19Ashby M.N. Edwards P.A. J. Biol. Chem. 1990; 265: 13157-13164Abstract Full Text PDF PubMed Google Scholar), heptaprenyl diphosphate synthase from B. stearothermophilus (20Koike-Takeshita A. Koyama T. Obata S. Ogura K. J. Biol. Chem. 1995; 270: 18396-18400Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and octaprenyl diphosphate synthase from E. coli (21Asai K.-i. Fujisaki S. Nishimura Y. Nishino T. Okada K. Nakagawa T. Kawamukai M. Matsuda H. Biochem. Biophys. Res. Commun. 1994; 202: 340-345Crossref PubMed Scopus (84) Google Scholar). These enzymes have alanine at the position corresponding to position 81 of B. stearothermophilus FPP synthase. These data strongly suggest that this amino acid primarily enables the enzymes to catalyze the formation of products longer than GGPP, and that other amino acid sequences of these enzymes should determine the final product to be C-30, C-35, or C-40.We have mainly been discussing the final products of mutated FPP synthases. However, product distribution was also affected by mutation. For example, Y81M produces primarily GGPP from DMAPP. On the other hand, Y81N yields three products, FPP, GGPP, and GFPP. Thus, we checked whether or not the divergence of products of each mutated enzyme is related to other factors. The divergence values were not related to the accessible surface area, the hydrophobicity (14Hopp T.P. Woods K.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3824-3828Crossref PubMed Scopus (2890) Google Scholar), conformational preference parameters for secondary structure (15Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; : 45-148PubMed Google Scholar), or specific activity for DMAPP, GPP, and FPP. It has been reported regarding some prenyltransferases that the amounts of intermediate products are affected by reaction conditions, including substrate concentration, although the destined final product is not altered. Presumably, the mutations cause additional structural changes that affect the affinity of the enzyme for reaction intermediates. Further analysis should be done to understand the reason. INTRODUCTIONIsoprenoids, which include over 23,000 known metabolites, are the most chemically diverse family of naturally occurring compounds. The essential and major biosynthetic step in all isoprenoid metabolism is the elongation of isoprene units by prenyltransferases (Fig. 1) (1Poulter C.D. Rilling H.C. Spurgeon S.L. Porter J.W. Biosynthesis of Isoprenoid Compounds. John Wiley & Sons, New York1982: 161-224Google Scholar). These enzymes, which consecutively mediate alkylation of isopentenyl diphosphate (IPP, C-5) 1The abbreviations used are: IPPisopentenyl diphosphateDMAPPdimethylallyl diphosphateGPPgeranyl diphosphateFPPfarnesyl diphosphateGGPPgeranylgeranyl diphosphateGFPPgeranylfarnesyl diphosphateHPPhexaprenyl diphosphate. by allylic diphosphates, are classified according to the chain length of the final product and the stereochemistry of double bond formed by the condensations. So far, a number of prenyltransferases have been determined from various organisms.For example, farnesyl diphosphate (FPP) synthase (EC 2.5.1.1) catalyzes the sequential condensations of two molecules of IPP (C-5) with dimethylallyl diphosphate (DMAPP, C-5) to give a C-15 compound with E-stereochemistry. The product, FPP, occupies a central point leading to several branches of the pathway for the synthesis of important classes of compounds, including sterols, farnesylated proteins, hemes, respiratory quinones, sesquiterpenes, and dolichols. On the other hand, geranylgeranyl diphosphate (GGPP, C-20) synthase (EC 2.5.1.29) catalyzes the condensation of IPP to give (all-E)-GGPP, which plays as a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaebacterial membrane lipids.These prenyltransferases catalyze the same sort of condensation and have a similarity in amino acid sequences (2Chen A. Kroon P.A. Poulter C.D. Protein Sci. 1994; 3: 600-607Crossref PubMed Scopus (219) Google Scholar, 3Koyama T. Obata S. Osabe M. Takeshita A. Yokoyama K. Uchida M. Nishino T. Ogura K. J. Biochem. 1993; 113: 355-363Crossref PubMed Scopus (122) Google Scholar). However, every enzyme does not catalyze a further condensation of IPP than the general ultimate product. Until now it has been left in question how the consecutive condensations precisely stop at a destined step.Recently, our group succeeded in converting FPP synthase from Bacillus stearothermophilus to GGPP synthase using chemical random mutagenesis followed by an in vivo color selection (4Ohnuma S.-i. Nakazawa T. Hemmi H. Hallberg A.-M. Koyama T. Ogura K. Nishino T. J. Biol. Chem. 1996; 271: 10087-10095Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). From the analysis of the mutations in the FPP synthases whose product specificities had become the same as GGPP synthase, we defined three amino acids that could determine the final chain length; leucine at position 34, tyrosine at position 81, and valine at position 157. In particular, the mutated enzyme that has a substitution of histidine for tyrosine at position 81, which is situated at the fifth amino acid before the first aspartate rich consensus motif, the most effectively produces GGPP. Moreover, our group also showed that, in the case of Sulfolobus acidocaldarius GGPP synthase, the amino acid at the same position also determines the chain length of the product, GGPP (5Ohnuma S.-i. Hirooka K. Hemmi H. Ishida C. Ohto C. Nishino T. J. Biol. Chem. 1996; 271: 18831-18837Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Thus, in this paper, we precisely analyze the role of the amino acid at position 81 of B. stearothermophilus FPP synthase on chain length determination.