We characterized the enzyme corresponding to N

We characterized the enzyme corresponding to N1. FPP was the most effective substrate as allylic diphosphate, and the activities with GPP and DMAPP were 50% and 11% that with FPP, respectively. The analysis of the prenols resulting from the treatment of prenyl diphosphates with EX527 phosphatase () revealed geranylgeraniol in the reaction with FPP, and farnesol in the reaction with GPP (). Thus, the reaction products of the enzyme corresponding to N1 were geranylgeranyl diphosphate (GGPP) in the reaction of IPP with FPP, and FPP in the reaction of IPP with GPP. Both GGPP and FPP were produced in the reaction of IPP with DMAPP. This enzyme required magnesium ion for its activity, and its highest activity was observed at 1 mM Mg. No detergent was necessary for the enzyme activity; however, the addition of 0.05% (w/v) Triton X-100 activated the enzyme by 1.5-fold. The addition of 0.1% (w/v) or a higher concentration of Triton X-100 was rather inhibitory to the enzyme reaction. These results revealed that the enzyme corresponding to N1 was a short-chain prenyl diphosphate synthase, the reaction product of which is FPP or GGPP. This enzyme was further purified by Butyl-Toyopearl, Mono-Q, and Superose-12 column chromatographies. The specific activity of the fraction from the Superose-12 column was 13.1-fold that of the fraction from the DEAE-Toyopearl column (). The molecular mass of the enzyme was 65±8 kDa, as estimated by Superose-12 chromatography. Octaprenyl diphosphate was produced in the reaction of IPP with FPP by both the enzymes corresponding to N2 and N3. The enzyme corresponding to N3 is considered to be same as that corresponding to B in from its elution profile in the chromatography, and to be octaprenyl diphosphate synthase that was described previously (). The enzyme corresponding to N2 is also considered to be octaprenyl diphosphate synthase, the isoform of that corresponding to N3. The Mono-Q chromatography of the enzyme corresponding to N2 gave a single peak, and the chromatography of the enzyme corresponding to N3 also gave another peak. Thus, no interconversion between the enzymes corresponding to N2 and N3 was observed, unlike that observed for the isoforms of mammalian FPP synthases that are interconvertible with each other during column chromatography (, ). The enzymes corresponding to N1 and N2 were described for the first time in this study. Either one or both of these enzymes are considered to also occur in the wild-type strain, because a corresponding peak was observed in the DEAE-Toyopearl column chromatogram of cell-free homogenate of W3110. The enzyme corresponding to N1 was the only enzyme that catalyzed the synthesis of FPP from IPP and DMAPP in the -null mutant. This enzyme is considered to be mainly responsible for the condensation of IPP with DMAPP in the homogenate of the mutant (), although both octaprenyl diphosphate synthase and undecaprenyl diphosphate synthase have a weak activity for the condensation of IPP with DMAPP. The only gene of that is significantly homologous with is (). It is unlikely that the newly found enzyme contains part of the polypeptide derived from because the mutant used in this study lacked more than two thirds of (). The molecular mass of the enzyme corresponding to N1, 65±8 kDa, is similar to those of octaprenyl diphosphate synthase, which is a dimer of a 35,217-Da polypeptide encoded by (), and of undecaprenyl diphosphate synthase, which is a dimer of a 28,444-Da polypeptide encoded by (). The identification of the polypeptide component of this enzyme will clarify whether a component of this enzyme is derived from , or another gene that have not been characterized yet.
Introduction Over the last 4decades, nitrogen-containing bisphosphonates (N-BPs) such as zoledronic acid (ZOL), have become the gold standard of treatment for post-menopausal osteoporosis as well as Paget's disease of bone (PDB) and tumour-associated bone diseases [1], [2], [3], [4].