The biosynthesis of saccharides is important because these diverse molecules mediate various functions from structure and storage to signaling. The biosynthesis of oligosaccharides, polysaccharides and glycoconjugates involves glycosyltransferases (EC 2.4), which transfer a sugar moiety from a nucleotide-activated donor sugar onto acceptors (Breton et al., 2006).
L-rhamnose is found widely in bacteria and plants ( Giraud & Naismith, 2000 ). It is a common component of the cell wall and the capsule of many pathogenic bacteria, and has been indicated to play an essential role in many pathogenic bacteria ( Giraud & Naismith, 2000 ). L-rhamnose is also found in the cytoplasmic membrane of archaea (Sprott et al., 1983), while pathogenic archaea have not been identified (Eckburg et al., 2003).
The nucleotide-activated L-rhamnose, dTDP-L-rhamnose, is synthesized from dTTP and glucose-1-phosphate (G-1-P) by a conserved four-step reaction. In the first reaction, RmlA (G-1-P thymidylyltransferase, EC 22.214.171.124) transfers the thymidylmonophosphate nucleotide to G-1-P. RmlB (dTDP-D-glucose 4,6-dehydratase, EC 126.96.36.199) then catalyzes oxidation of the C4 hydroxyl group of the sugar, followed by dehydration. Third, RmlC (dTDP-4-dehydrorhamnose 3,5-epimerase, EC 188.8.131.52) catalyzes an unusual double epimerization reaction at positions C3 and C5. Finally, RmlD (dTDP-4-dehydrorhamnose reductase, EC 184.108.40.206) reduces the C4 keto group to generate the final product, dTDP-L-rhamnose ( Giraud & Naismith, 2000 ).
Thermal instability of the Rml enzymes has been raised as an issue (Graninger et al., 1999, 2002). Thus far, the highest reported temperature for the dTDP-L-rhamnose synthesis reaction is around 50 ºC using thermophilic bacterial enzymes (Graninger et al., 2002; Novotny et al., 2004). The presence of dTDP-L-rhamnose has not been reported in archaea. However, putative
2. Materials and methods
2.1. Sequence analysis
2.2. Vector construction
The four genes
2.3. Preparation of supernatants containing the expressed proteins
Eight μl of each supernatant was analyzed on an E-R12.5L polyacrylamide gel (ATTO, Japan) at 30 mA constant current together with 10 μl of the broad range molecular weight standards (Bio-rad), and visualized with Coomassie Brilliant Blue R-250. The gel was photographed with a luminescent image analyzer Las-4000 mini (Ver 2.1; Fuji film, Japan). The amount of expressed protein was determined using MultiGauge software (Ver 3.2; Fuji film) in comparison to the molecular weight standards.
2.5. Reaction of the expressed protein
The reaction to detect G-1-P thymidylyltransferase activity of RmlA was performed at 80 C for 3 h in 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl2, 10 mM dTTP, 10 mM UTP and 10 mM G-1-P with the RmlA supernatant (40 μl of the supernatant, which contained 4 μg RmlA, was added to the reaction mixture for a total volume of 100 μl). UTP was also used as a substrate candidate because a product of a
The reaction to determine substrate specificity of RmlB was performed at 80 C for 3 h in 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl2, 10 mM dTDP-D-glucose, and 10 mM UDP-D-glucose with the RmlB supernatant (40 μl of the supernatant, which contained 4 μg RmlB, was added to the reaction mixture for a total volume of 100 μl). To serve as controls, this reaction was performed with the RmlC supernatant or the RmlD supernatant (40 μl of the supernatant, which contained 20 μg of RmlC or RmlD, was added to the reaction mixture for a total volume of 100 μl) instead of the RmlB supernatant. Optimal reaction temperature for RmlB was determined in the same way as just described above except that UDP-D-glucose was not included in the reaction mixture and the reaction was preformed at the indicated temperature for 2 h. The reaction to characterize the thermostability of RmlB was carried out in the same way as described above for determining the optimal temperature except that the reaction was performed at 80 C for the indicated time period.
The dTDP-L-rhamnose synthetic reaction was performed at 80 C for 3 h in 50 mM Tris-HCl buffer (pH 7.5), 2 mM MgCl2, 10 mM dTDP-D-glucose, 5 mM NADPH, and 5 mM NADH with a combination of the supernatants (4 μgs each of RmlB, RmlC and/or RmlD were added to the reaction mixture for a total volume of 100 μl). Each reaction was stopped by adding ten times the reaction volume of 500 mM KH2PO4.
2.6. HPLC of the reaction
A 50-μl aliquot of the reaction solution mixed with 500 mM KH2PO4 was subjected to HPLC using a LaChrom ELITE system with a L-2420 UV-VIS detector and a L-2130 pump (Hitachi) equipped with a Wakosil 5C18-200 column (4.6 x 250 mm; Wako, Japan). The 500 mM KH2PO4 was run as the elution buffer at a constant flow rate of 1 ml min−1. The substrates and products were detected at 254 nm, and the peak area was used for calculation of the amount.
2.7. Mass spectrometry (MS) of the reaction products
The reaction samples for MS were prepared with a smaller amount of the substrate dTDP-D-glucose (2 mM) and thus with 2 mM NADPH and 2 mM NADH to completely consume the dTDP-D-glucose (this reaction was otherwise performed in the same way as described above), as a standard sample dTDP-L-rhamnose (the predicted product; Genechem Inc., Taejon, Korea) was unable to be separated from dTDP-D-glucose by HPLC used for MS (described below).
Reaction samples, to which 500 mM KH2PO4 was not added, were separated by HPLC (LC-10AT, Shimadzu, Japan) performed on a Wakosil 5C18-200 column using a water/methanol/ acetic acid mixture (98.9/1.0/0.1, v/v/v) as the elution buffer at a flow rate of 1 ml min-1. Mass spectra of the peaks detected by the HPLC were then measured by an electrospray ionization/ion trapped mass spectrometer (LCQDECA, ThermoQuest, USA) connected to the HPLC under the following instrumental conditions: detection mode, negative; mass range,
3. Results and discussion
3.1. Putative dTDP-L-rhamnose synthesis gene cluster (
rmlABCD) from the thermophilic archaeon S. tokodaii 7
The RmlA from
3.2. Activity of RmlA
RmlA was expressed as a 35-kDa protein (data not shown), in agreement with the molecular weight (MW) of 38 kDa deduced from its nucleotide sequence. The predicted G-1-P thymidylyltransferase (RmlA) activity was assayed at 80ºC with dTTP and G-1-P, but was not detected. RmlA may thus function as the UDP-N-acetylglucosamine pyrophosphorylase/glucosamine-1-phosphate N-acetyltransferase as deduced from its sequence similarity. This deserves further investigation. A product from another
3.3. RmlB as a thermophilic dTDP-D-glucose 4,6-dehydratase
RmlB was expressed as a 35-kDa protein (Fig. 1), which corresponded to the deduced MW (Table 1). The predicted activity (Table 1) was assayed at 80ºC with dTDP-D-glucose and UDP-D-glucose (Fig. 2). UDP-D-glucose was also used as a substrate candidate because UDP-D-glucose as well as dTDP-D-glucose can be produced by the product from the another
Temperature range for the dTDP-D-glucose-utilizing activity of RmlB was measured from 60 to 99ºC, and the optimal temperature was shown to be 80ºC (Fig. 3A). Therefore, RmlB from
3.4. The dTDP-L-rhamnose synthesis reaction from dTDP-D-glucose at 80ºC catalyzed by RmlBCD
RmlC and RmlD were expressed as a 22-kDa protein and a 30-kDa protein, respectively (Fig. 1), which corresponded to their respective deduced MWs (Table 1). The dTDP-L-rhamnose synthesis reaction catalyzed by RmlBCD, suspected based on their homology (Table 1), was analyzed at 80ºC using dTDP-D-glucose and NAD(P)H as substrates (Fig. 4). A combination of RmlB plus RmlD produced peak 3 (+BCD and +BD in Fig. 4); the retention time and MS spectrum of this peak were identical to those of the standard sample dTDP-L-rhamnose. RmlB(C)D from
It is possible that peak 3 produced by the combination of RmlB plus RmlD (+BD) could have been an epimer of dTDP-L-rhamnose produced without the possible epimerase RmlC (Table 1). Addition of RmlC showed no effect on the broad
The concentrations of peak 3 (indicated from dTDP-L-rhamnose) and dTDP-D-glucose in the +BCD sample (Fig. 4) were determined to be 2.4 and 4.8 mM, respectively, showing that 52% of the added dTDP-D-glucose was used and that 46% of the dTDP-D-glucose used was converted to dTDP-L-rhamnose in the reaction. Consequently, RmlB and RmlD were estimated to show their respective activities of at least 0.33 U/mg protein.
Genes for thermostable RmlB and RmlD of the dTDP-L-rhamnose synthesis pathway were functionally identified from a thermophilic archaeon
We thank Hirofumi Sato of OMTRI for kind advice about MS and Hiromi Murakami of OMTRI for useful discussion about sugar metabolism.
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