Conversion of Product Specificity of Archaebacterial Geranylgeranyl-diphosphate Synthase

Abstract

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO 2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH 3 , which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from ϳ1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.Prenyltransferases, which are essential enzymes in isoprenoid biosynthesis, catalyze the consecutive condensation of isopentenyl diphosphate (IPP) 1 with allylic diphosphates to synthesize linear prenyl diphosphates with various chain lengths. These enzymes are classified according to the products with the longest chain length and the geometry of the double bonds that are formed by the condensations. So far, a number of prenyltransferases have been found in various organisms and characterized (1). For example, farnesyl-diphosphate synthase, which is a key enzyme in the biosynthesis of steroids, prenylquinones, farnesylated proteins, and dolichols, catalyzes the condensations of IPP with dimethylallyl diphosphate (DMAPP; C 5 ) and with geranyl diphosphate (GPP; C 10 ) to give farnesyl diphosphate (FPP; C 15 ) as an ultimate product. Geranylgeranyl-diphosphate (GGPP; C 20 ) synthase, whose product is a precursor of carotenoids, geranylgeranylated proteins, chlorophylls, and ether-linked lipids of archaebacteria, utilizes DMAPP, GPP, and FPP as allylic substrates to give an amphipathic molecule containing four isoprene units, GGPP (Fig.

Mechanism of Product Chain Length Determination and the Role of a Flexible Loop in Escherichia coliUndecaprenyl-pyrophosphate Synthase Catalysis

“…X-ray structural (8) and site-directed mutagenesis studies (9 -12) of farnesyl-pyrophosphate synthase (FPPs) have shown that the first aspartate-rich motif binds the allylic substrate, whereas the second DDXXD binds IPP via Mg 2ϩ . Mutagenesis studies indicate that the 5th amino acid residue (Phe-77) upstream from the first DDXXD plays a critical role in controlling the chain length of the final product formed in the reaction catalyzed by E-type geranylgeranyl-pyrophosphate synthase from archaebacterium (13). By substituting this large residue with the smaller Ser, product synthesis was shifted from the production of C 20 product to C 25 and C 30 products (14).…”

“…Mutagenesis studies indicate that the 5th amino acid residue (Phe-77) upstream from the first DDXXD plays a critical role in controlling the chain length of the final product formed in the reaction catalyzed by E-type geranylgeranyl-pyrophosphate synthase from archaebacterium (13). By substituting this large residue with the smaller Ser, product synthesis was shifted from the production of C 20 product to C 25 and C 30 products (14). Double (F77G/L74G) and triple (F77G/L74G/I71G) mutants were observed to catalyze the formation of even longer chain products (15).…”

Interaction with the Small Subunit of Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Geranylgeranyl Diphosphate Synthase to Produce Geranyl Diphosphate

“…Evaluation of the crystal structure of homodimeric avian FPP synthase (7), coupled with prenyltransferase sequence alignment data, has led to the directed mutagenesis of FPP synthase to alter the contour of the active site, thereby generating mutant enzymes that synthesize GGPP (8) or GPP (9,10). A random chemical mutagenesis approach has also provided an altered FPP synthase capable of producing GGPP (11) and has yielded homodimeric GGPP synthase mutants capable of producing FPP (5) and polyprenols greater than C 20 (12). This work eventually led to the direct mutagenesis of an archaeal GGPP synthase so as to produce FPP by the modified enzyme (13).…”

Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans

“…It will be interesting to know how esis of farnesyl diphosphate (farnesyl-PP) synthase from birds we can interpret these observations to uncover the significance [16] and B. stearothermophilus [17], and of geranylgeranyl in the diversity of Q species. length determinant of prenyl diphosphate synthases [18]. How- ever, the identity of the amino acids that function to determine addition to obtaining information about their structure and funcNote.…”