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

Isoprenoids, 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) (1). These enzymes, which consecutively mediate alkylation of isopentenyl diphosphate (IPP, 1 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 (2, 3). 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 (4). 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 (5). 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.

Crystal Structure of Octaprenyl Pyrophosphate Synthase from Hyperthermophilic Thermotoga maritima and Mechanism of Product Chain Length Determination

“…Apparently, this residue located at the fifth position before the first DDXXD motif is the key residue in determining the product chain length for FPPs. Predicted from the structure of FPPs, this residue is located from the first Asp-rich motif at a distance of ϳ12 Å which is similar to the length of hydrocarbon moiety of FPP (23). The chain length of the product catalyzed by these mutants is inversely proportional to the accessible surface volume of the substituted amino acid residue in the first DDXXD.…”

“…1). The corresponding residue at this position for FPPs from Bacillus stearothermophilus is a large Tyr, and when changed to smaller residue such as Gly and Ala the product chain length was increased the most (22,23). In addition to the fifth amino acid residue, sixth and eighth positions before the first DDXXD have also been shown important to control the product specificity of archael FPPs and C 20 -GGPPs (24).…”

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli

“…It has been shown that the substrate specificity of IspA can be altered by introducing a mutation in Ser81 to Phe (S81F), resulting in an enzyme that synthesizes GPP as the main product while FPP synthase activity is significantly reduced (Ohnuma et al, 1996;Reiling et al, 2004). We hypothesized that we could increase the GPP pool for monoterpene biosynthesis while maintaining a basal level of FPP for growth by introducing this mutation into E.…”

A Pathway Where Polyprenyl Diphosphate Elongates in Prenyltransferase

“…Although we examined various FPP synthases and GGPP synthases mutated at the 5th position before FARM, there was no mutated enzyme that could yield any products longer than C 30 (21)(22)(23). Therefore, we hypothesized that another amino acid upstream from FARM must block the further elongation and that the alteration of this amino acid would be essential to create from GGPP synthase and FPP synthase an enzyme that produces compounds longer than C 30 .…”

“…From the analysis of the amino acid, we demonstrated that the terminus of an elongating allylic product directly contacts with this amino acid residue, and the interaction must prevent further condensation of IPP (23).…”