Regulation of product chain length by isoprenyl diphosphate synthases

Abstract

An analysis of the x-ray structure of homodimeric avian farnesyl diphosphate synthase (geranyltransferase, EC 2.5.1.10) coupled with information about conserved amino acids obtained from a sequence alignment of 35 isoprenyl diphosphate synthases that synthesize farnesyl (C 15 ), geranylgeranyl (C 20 ), and higher chain length isoprenoid diphosphates suggested that the side chains of residues corresponding to F112 and F113 in the avian enzyme were important for determining the ultimate length of the hydrocarbon chains. This hypothesis was supported by site-directed mutagenesis to transform wild-type avian farnesyl diphosphate synthase (FPS) into synthases capable of producing geranylgeranyl diphosphate (F112A), geranylfarnesyl (C 25 ) diphosphate (F113S), and longer chain prenyl diphosphates (F112A͞F113S). An x-ray analysis of the structure of the F112A͞F113S mutant in the apo state and with allylic substrates bound produced the strongest evidence that these mutations caused the observed change in product specificity by directly altering the size of the binding pocket for the growing isoprenoid chain in the active site of the enzyme. The proposed binding pocket in the apo mutant structure was increased in depth by 5.8 Å as compared with that for the wild-type enzyme. Allylic diphosphates were observed in the holo structures, bound through magnesium ions to the aspartates of the first of two conserved aspartate-rich sequences (D117-D121), with the hydrocarbon tails of all the ligands growing down the hydrophobic pocket toward the mutation site. A model was constructed to show how the growth of a long chain prenyl product may proceed by creation of a hydrophobic passageway from the FPS active site to the outside surface of the enzyme.The isoprenoid biosynthetic pathway is built around a family of diphosphate esters of linear alcohols that contain increasing numbers of isoprene units. Beginning with the C 5 molecule dimethylallyl diphosphate (DMAPP), a series of C 10 (geranyl diphosphate, GPP), C 15 (farnesyl diphosphate, FPP), C 20 (geranylgeranyl diphosphate, GGPP), and higher molecular weight isoprenoid diphosphates are formed by the 1Ј-4 addition of isopentenyl diphosphate (IPP) to the growing chain. These compounds are the substrates for biosynthesis of all isoprenoid metabolites, including monoterpenes, sesquiterpenes, diterpenes, sterols, carotenoids, ubiquinones, dolichols, and prenylated proteins. Thus far, over 23,000 individual isoprenoid compounds have been identified. They form the most chemically diverse family of molecules found in nature and are widely distributed among archaea, bacteria, and eukareya.The 1Ј-4 condensation reactions are catalyzed by a family of prenyltransferases, the isoprenyl diphosphate synthases, that are highly selective for the chain lengths and double bond stereochemistries of both substrates and products. For example, yeast contains at least four distinct enzymes for chain elongation. FPP synthase (FPS) converts DMAPP to FPP for synthesis of sterols, farnesylated prot...

Cloning and Characterisation of Chlorophyll Synthase from Avena sativa

“…In the sequence of A. sativa, this region includes the residues 138 to 160. Two possibilities have been discussed for interaction of the negatively charged phosphates of polyprenyl diphosphates with the active center of polyprenyl transferases, either R and K residues as counterions (Kral et al, 1997) or acidic groups of a DDXXD motif via complexed Mg 2+ ions (Tarshis et al, 1996;Wang and Ohnuma, 1999). Indirect evidence presented in this paper shows that both possibilities apply also for chlorophyll synthase.…”

“…Interactions of Arg and Lys residues with the phosphate groups of farnesyldiphosphate were shown by X-ray analysis of the crystal structure and by site-directed mutagenesis of human farnesyl: protein transferase (Kral et al, 1997;Park et al, 1997, Long et al, 1998. On the basis of the crystal structure of farnesyldiphosphate synthase, two Arg residues were discussed either for binding the allylic diphosphate during catalysis or for pulling the diphosphate residue after lysis out of the active site (Tarshis et al, 1996). In the absence of precise structural information, we can only speculate about the function of essential Arg residues in chlorophyll synthase.…”

Biosynthesis of plant‐derived flavor compounds

“…Most of the prenyl transferases accept DMADP as the initial substrate, but they also bind GDP or FDP depending on the particular prenyltransferase (Greenhagen and Chappell, 2001;Tarshis et al, 1994Tarshis et al, , 1996Withers and Keasling, 2007). The availability of GDP and FDP are often the key factor in the production of monoterpenes and sesquiterpenes in plants.…”

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

“…This reaction is catalyzed by highly conserved prenyl transferases and the elongation stops at discrete chain lengths, depending on enzyme specificity (Ogura and Koyama, 1998). The final size of the isoprenyl product is determined by the bulk of the lateral groups of certain amino acyl residues located in a hydrophobic pocket in the enzyme, as evidenced by x-ray crystallography and kinetic studies of wild-type avian farnesyl diphosphate synthase (FPPS; EC 2.5.1.1 and EC 2.5.1.10) and mutated enzymes in which such amino acyl residues have been substituted (Tarshis et al, 1996). The active site in each one of the two subunits in the FPPS homodimer accommodates an acceptor DMAPP, and two IPP molecules are successively added to form farnesyl diphosphate (FPP;C 15 ).…”