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A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein. This observation prompted our group to investigate the acetyltransferase activity of the ST0452 protein. Detection of the release of coenzyme A (CoA) from acetyl-CoA and the production of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.
The nucleotide-sugar molecule is used as a substrate for the construction of the polymer structure of carbohydrates and as a starting material for the biosynthesis of modified-sugar-nucleotide conjugates; for example, TDP-glucose is the starting material for the construction of TDP-rhamnose (25). Several sugar-1-phosphate nucleotidylyltransferases (sugar-1-P NTases) involved in some biosynthetic reactions, such as glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase), N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase), and mannose-1-phosphate guanylyltransferase, have been identified from many bacteria, including pathogens, as well as from eukaryotes (5, 10, 11, 17, 25, 27, 29). However, only limited information on the archaeal enzyme with sugar-1-P NTase activity has been obtained.
Our group has previously reported the first thermostable enzyme with sugar-1-P NTase activity from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7 (31). The sugar-1-P NTase activity of the ST0452 protein, which was primarily identified by sequence similarity with the Escherichia coli RmlA protein, indicated the utilization of Glc-1-P and GlcNAc-1-P as a sugar-1-P substrate and all deoxynucleoside triphosphates (dNTPs) and UTP as nucleoside triphosphate (NTP) substrates, but glucosamine-1-phosphate (GlcN-1-P) was not utilized as a substrate. It was also reported that the GlcNAc-1-P UTase activity of the ST0452 protein was improved by the introduction of site-directed mutagenesis (30).
The identification of GlcNAc-1-P UTase activity on the ST0452 protein suggests that the protein may have activities and features similar to those of the bacterial GlmU enzyme (19). However, the ST0452 protein exhibits only a low level of sequence identity (less than 20% identity) with E. coli GlmU (data not shown). The phylogenetic analysis of the ST0452 protein and related proteins indicates that the ST0452 protein and homologous proteins form a group independent of other related enzymes identified as RmlA or GlmU (Fig. (Fig.11).
In spite of the low level of sequence similarity of the ST0452 protein with the bacterial GlmU, GlcNAc-1-P UTase activity was detected on the protein. Twenty-four incomplete repeats of the hexapeptide repeat motif sequences with the left-handed parallel beta-helix (LβH) structural feature (22) (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X were also detected at the C-terminal region of the ST0452 protein (Fig. (Fig.2).2). This motif sequence has been identified in enzymes with acetyl- or acyltransferase (22), succinyltransferase (1), and carbonic anhydrase (12) activities. Sequences with this motif are detected at the C terminus of all three archaea-related genes shown in Fig. Fig.1,1, Saci0619, MJ1101, and SSO0745, but there is no information on the function or activities of Saci0619 and SSO0745. For MJ1101, activities were detected but detailed analysis was not performed (18). Therefore, we hypothesized that the acetyltransferase activity was also present on the ST0452 protein.
In this work, we analyzed the acetyltransferase activity of the recombinant ST0452 protein and confirmed the GlcN-1-P acetylation activity by two independent experiments, i.e., detection of conversion from acetyl coenzyme A (acetyl-CoA) to CoA and construction of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc). The substrate specificity analyses indicated that the ST0452 protein was capable of utilizing galactosamine-1-phosphate (GalN-1-P) as the substrate for the acetyltransferase activity and N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as the substrate for the sugar-1-P NTase activity of the recombinant ST0452 protein. This is the first report describing a bifunctional enzyme with both GlcN-1-P and GalN-1-P acetyltransferase activities plus GlcNAc-1-P and GalNAc-1-P NTase activities and a high degree of stability; therefore, industrial applications of this enzyme are expected.
All dNTPs, NTPs, sugar phosphate molecules, nucleotide-sugar molecules, and the thiol reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma Chemical Co. (St. Louis, MO). S. tokodaii strain 7 (JCM10545) was obtained from the Japan Collection of Microorganisms (JCM). E. coli strain BL21-Codon Plus(DE3)-RIL, which was used for the expression of recombinant protein, was obtained from Stratagene (La Jolla, CA).
Expression and purification of the recombinant ST0452 protein were carried out according to a previously described procedure (31). The purified protein was stored at 4°C. The protein concentration was determined by use of a bicinchoninic acid protein assay reagent kit (Pierce Biotechnology, Rockford, IL).
The acetyltransferase activity was primarily measured from the amount of CoA produced. This assay is based on the premise that the amount of CoA converted from acetyl-CoA by acetyltransferase activity is equal to the amount of acetylated amino-sugar-1-phosphate molecules produced (8, 19, 26). The amount of CoA was measured by using Ellman's reaction (23).
The reaction was performed in 10 μl of an acetyltransferase reaction mixture containing 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 2 mM acetyl-CoA, 2 mM amino-sugar-1-P or amino-sugar-6-P, and 50 ng of the recombinant ST0452 protein. After 2 min preincubation at 80°C, the reaction was started by the addition of the recombinant ST0452 protein and progressed at 80°C. To stop the reaction, 40 μl of a solution containing 50 mM Tris-HCl (pH 7.5) and 6.4 M guanidine hydrochloride was added to the reaction mixture. After addition of 50 μl of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.5 mM DTNB solution, the absorbance at 412 nm (A412) was measured (23). The amounts of CoA released were calculated from a standard curve. The molar absorption coefficient for the standard CoA molecule, 10,364 M−1 cm−1, was used for calculating the amount of CoA produced during the acetyltransferase reaction.
Detection of GlcNAc-1-P produced by the acetyltransferase activity is based on the observation that the sugar-1-P NTase activity of the ST0452 protein is capable of catalyzing only GlcNAc-1-P, a product of the acetyltransferase reaction, and not GlcN-1-P, a substrate of the acetyltransferase reaction. When UTP is added to the acetyltransferase reaction mixture and GlcNAc-1-P is produced from GlcN-1-P by the acetyltransferase activity of the ST0452 protein, the sugar-1-P NTase activity of the ST0452 protein synthesizes UDP-GlcNAc. For detection of GalNAc-1-P production, a similar system was utilized.
The coupling reaction was started with 10 μl of the acetyltransferase reaction mixture containing 0.1 mM UTP at 80°C. After 2 min incubation, 100 μl of 500 mM KH2PO4 was added to the reaction mixture to stop the reaction. The final product, UDP-GlcNAc or UDP-N-acetyl-d-galactosamine (UDP-GalNAc), was measured and quantified mainly according to the method described by Schulz et al. (24). In this method, the elution positions for UDP-GlcNAc and UDP-GalNAc showed individual peaks. A 50-μl aliquot of the solution was analyzed on a LaChrom Elite high-pressure liquid chromatography (HPLC) system (Hitachi High-Technologies, Tokyo, Japan) with a CarboPac PA1 column (0.4 by 25 cm; Dionex, Sunnyvale, CA). For this analysis, two mobile-phase solutions were used (solution A, which consisted of 15 mM NaOH, and solution B, which consisted of 50 mM NaOH along with 1 M sodium acetate). The separation was performed by using an isocratic procedure: 15 min of 45% solution A and 55% solution B, after which the proportion of solution B was changed from 55% to 100% for 15 min. The flow rate was maintained at 1 ml/min. Monitoring for the product of the reaction, UDP-GlcNAc or UDP-GalNAc, was done by determination of the A254.
The reverse direction of the GlcNAc-1-P or GalNAc-1-P NTase reaction of the ST0452 protein, producing UTP plus GlcNAc-1-P or GalNAc-1-P from UDP-GlcNAc or UDP-GalNAc, was performed in 10 μl of a reaction solution containing 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.5 mM pyrophosphate, and different concentrations, from 0 to 200 mM, of nucleotide-sugar molecules (UDP-GlcNAc or UDP-GalNAc) with 5 ng of the purified recombinant ST0452 protein. After progression of the reaction, monitoring for the product, UTP, was done by HPLC, according to the protocol described in a previous report (31).
A thermostable ST0452 protein, originally identified as a Glc-1-P TTase from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7, exhibited only approximately 20% amino acid sequence identity with the bacterial GlmU protein. However, the repeat motif sequences with the LβH structural feature were detected at the C terminus of the ST0452 protein. We therefore attempted to determine whether the ST0452 protein possessed acetyltransferase or similar activity.
At first, the increase in the absorbance at 412 nm was monitored for measuring the conversion to CoA from acetyl-CoA as the progression of acetyltransferase activity. When the acetyltransferase reaction solution was incubated at 80°C in the absence of the ST0452 protein, the A412 was not increased, revealing that no conversion of acetyl-CoA to CoA occurred (Fig. (Fig.3,3, open squares). Conversely, an increase in absorbance was clearly detected when the ST0452 protein was added to the reaction mixture (Fig. (Fig.3,3, closed circles). This observation indicates that conversion from acetyl-CoA to the CoA molecule specifically occurred depending on the presence of the ST0452 protein. However, the exact compound produced by this acetyltransferase reaction was not identified by this experiment. To identify the product of the acetyltransferase reaction, the following coupling reaction was carried out.
The previous analysis showed that the sugar-1-P NTase activity of the ST0452 protein could not utilize GlcN-1-P, the expected substrate for the acetyltransferase reaction, but could accept as the substrate GlcNAc-1-P, the expected product of the acetyltransferase reaction analyzed in this work (31). If the acetyltransferase reaction of the ST0452 protein converts GlcN-1-P to GlcNAc-1-P, UDP-GlcNAc will be produced in the presence of UTP by the sugar-1-P NTase activity of the ST0452 protein as the coupling activity. For confirmation of the product of the acetyltransferase activity of the recombinant ST0452 protein, the coupling reaction with the sugar-1-P NTase activity of the ST0452 protein was performed in a reaction mixture containing 2 mM UTP and 2 mM GlcN-1-P in the absence and presence of 1 mM acetyl-CoA.
When the reaction was performed without acetyl-CoA, only the peak for UTP was detected (Fig. (Fig.44 A). Conversely, when acetyl-CoA was added to the acetyltransferase reaction mixture, which included UTP, a peak for the nucleotide sugar was detected (Fig. (Fig.4B).4B). The retention time in the elution of the product was identical to that of the standard UDP-GlcNAc used (Fig. (Fig.4D).4D). This result indicates that the acetyltransferase activity of the ST0452 protein had converted GlcN-1-P to GlcNAc-1-P with the specific activity being 75.34 ± 0.71 μmol/min/mg protein (Table (Table1),1), which was determined by measurement of the amount of CoA converted.
The results from two independent experiments, the detection of CoA and UDP-GlcNAc, confirmed the enzymatic activities of the ST0452 protein for acetylation of the GlcN-1-P molecule.
To investigate what type of amino-sugar-1-phosphate molecule is acceptable as a substrate for the acetyltransferase activity of the ST0452 protein, different types of phosphor amino-sugar molecules, listed in Table Table1,1, were analyzed. When glucosamine-6-phosphate (GlcN-6-P) was used as the substrate, no conversion to CoA from acetyl-CoA was detected (Fig. (Fig.3,3, open circles, and Table Table1).1). This observation indicates that the acetyltransferase activity of the ST0452 protein was not capable of utilizing the GlcN-6-P as a substrate. In the eukaryotic UDP-GlcNAc biosynthesis pathway, only GlcN-6-P is acetylated into GlcNAc-6-P (3, 16). Conversely, in the bacteria-type UDP-GlcNAc biosynthesis pathway, acetyltransferase activity can convert only GlcN-1-P to GlcNAc-1-P (15). The property for the acetyltransferase activity of the ST0452 protein, the acceptance of GlcN-1-P and the nonacceptance of GlcN-6-P, is almost identical to that of the known bacterial GlcN-1-P acetyltransferase. This observation reveals that a bacteria-type UDP-GlcNAc biosynthesis pathway from fructose-6-phosphate (Fru-6-P) is present in S. tokodaii. Because the ST0452 protein was capable of catalyzing the last two reactions of the bacteria-type four-step biosynthesis pathway of UDP-GlcNAc from Fru-6-P, the ST0452 protein plays an important role for the bacteria-type UDP-GlcNAc biosynthesis pathway in this archaeon.
In contrast to GlcN-6-P, when GalN-1-P was used as the substrate in the acetyltransferase reaction of the ST0452 protein, a significant amount of CoA was produced: 38.4 ± 0.25 μmol/min/mg protein of CoA production, approximately 51% of the maximum GlcN-1-P acetyltransferase activity, was detected for the GalN-1-P substrate (Table (Table1).1). To explore whether GalN-1-P is converted to GalNAc-1-P by the acetyltransferase activity of the ST0452 protein, the sugar-nucleotide molecule produced in the reaction solution containing 2 mM UTP was analyzed. When acetyl-CoA was not added to the reaction mixture, no sugar-nucleotide peak was detected (Fig. (Fig.4A).4A). However, once acetyl-CoA was added to the reaction mixture, a peak with the same elution time as that of the standard UDP-GalNAc was detected (Fig. (Fig.4C).4C). This observation reveals that the ST0452 protein actually catalyzed the acetyltransferase reaction producing GalNAc-1-P from GalN-1-P. To our knowledge, this is the first report of the thermostable of GalN-1-P acetyltransferase activity.
During analysis of the product of the GalN-1-P acetyltransferase activity of the ST0452 protein, UDP-GalNAc was detected only when acetyl-CoA and UTP were added to the reaction solution (Fig. (Fig.4C).4C). This observation indicates that the ST0452 protein possesses GalNAc-1-P UTase activity. For confirmation of the GalNAc-1-P UTase activity, detection of UDP-GalNAc production from UTP and GalNAc-1-P is the most reliable method. However, since GalNAc-1-P is not commercially available in Japan, we were not able to perform this experiment. Therefore, our approach was to detect the product of the reverse direction of this reaction: the production of GalNAc-1-P and UTP from UDP-GalNAc and pyrophosphate. Our results showed that only when the ST0452 protein was added to the reaction solution was a reaction time-dependent decrease in the amount of UDP-GalNAc and an increase in the amount of UTP observed (Fig. (Fig.5).5). This result reveals that the GalNAc-1-P UTase activity, as well as other sugar-1-P NTase activities, is actually present on the ST0452 protein. The ST0452 protein is identified as the first thermostable enzyme with both GalNAc-1-P and GlcNAc-1-P UTase activities.
There are no previous reports concerning the effects of metal ions on GlcN-1-P acetyltransferase activity. Therefore, the requirement for and effects of metal ions on the activities of the two acetyltransferases of the ST0452 protein were analyzed.
For analysis of the requirement for metal ions on the activities of the two acetyltransferases of the ST0452 protein, the activities were compared for two reaction conditions: in the presence and in the absence of 2 mM EDTA. Both the GlcN-1-P and GalN-1-P acetyltransferases of the ST0452 protein exhibited almost the same specific activities under the two different reaction conditions, without the addition of any metal ions (control) and in the presence of 2 mM EDTA (Fig. (Fig.6,6, open and hatched bars), revealing that the metal ion is nonessential for the activities of the two acetyltransferases of the ST0452 protein.
The effects of metal ions on the activities of the two acetyltransferases of the ST0452 protein were analyzed in 10 μl of a reaction solution containing 2 mM different kinds of divalent cations. The GlcN-1-P acetyltransferase activity was inhibited by all the divalent cations tested (Fig. (Fig.6,6, open bars). The order of the degree of inhibition of the GlcN-1-P acetyltransferase activity of the ST0452 protein caused by the metal ions was Zn2+ > Mn2+ > Mg2+ > Co2+ > Ca2+.
Conversely, the GalN-1-P acetyltransferase activities of the ST0452 protein were enhanced 1.2, 1.4, and 2.1 times by 2 mM Co2+, Mg2+, and Ca2+, respectively. However, when 2 mM Zn2+ or Mn2+ was added, only 25% and 20% of the control GalN-1-P acetyltransferase activity of the ST0452 protein, respectively, was detected (Fig. (Fig.6,6, hatched bars).
These results reveal that no metal ion was required for the amino-sugar-1-P acetyltransferase activity of the ST0452 protein and that the effectiveness of each divalent cation differed depending on the type of amino-sugar-1-P molecule substrate.
Double-reciprocal plots of initial velocity were prepared to determine the effect of the substrate concentration on the enzymatic activity. The Km value for each substrate was analyzed and calculated under conditions where the concentration of the second substrate was 5 to 10 times higher than its Km value.
For the GlcN-1-P acetyltransferase reaction of the ST0452 protein, the apparent Km values for GlcN-1-P and acetyl-CoA were 0.59 mM and 0.63 mM, respectively (Table (Table2).2). For the GalN-1-P acetyltransferase reaction of the ST0452 protein, those for GalN-1-P and acetyl-CoA were 1.71 mM and 0.63 mM, respectively. The calculated kcat values for the GlcN-1-P and GalN-1-P acetyltransferase reactions were 123.2 s−1 and 69.7 s−1, respectively. Comparison of these values with those for the acetyltransferase activity of the E. coli GlmU enzyme (Table (Table2)2) showed that the GlcN-1-P acetyltransferase activities of E. coli GlmU and the ST0452 protein were similar. However, for the GalN-1-P acetyltransferase activity, the ST0452 protein had a reliably higher level of activity than the E. coli GlmU (Table (Table22).
We were not able to obtain the correct kinetic parameters for the forward reaction of the ST0452 GalNAc-1-P UTase activity, because GalNAc-1-P is not commercially available in Japan. Therefore, the kinetic parameters for the reverse reaction of the GalNAc-1-P UTase activity of the ST0452 protein were obtained. The Km values for UDP-GlcNAc and UDP-GalNAc were 0.016 mM and 0.056 mM, respectively (Table (Table3).3). The kcat values for the reverse reactions of the GlcNAc-1-P and GalNAc-1-P UTase activities were 8.4 s−1 and 3.3 s−1, respectively (Table (Table3).3). These values indicate that the relative activity of GalNAc-1-P UTase is approximately 15% of that of GlcNAc-1-P UTase of the ST0452 protein.
The detection of the ST0452 protein-dependent and GlcN-1-P-dependent conversion of acetyl-CoA to CoA showed the presence of some acetyltransferase activity on the ST0452 protein. To identify the types of compounds produced by this acetyltransferase reaction, the sugar nucleotide molecule produced with the sugar-1-P NTase activity of the ST0452 protein was analyzed. The sugar-1-P NTase activity of the ST0452 protein can utilize GlcNAc-1-P but not GlcN-1-P as a substrate (31). The detection of UDP-GlcNAc after the coupling reaction (Fig. (Fig.4B)4B) allows the conclusion that the acetyl group released from acetyl-CoA is transferred to GlcN-1-P to produce GlcNAc-1-P.
GlcNAc is generally detected within the polymer structures of carbohydrate molecules as one of the most important components. The biosynthetic pathways of UDP-GlcNAc from Fru-6-P in bacteria and eukaryotes are well characterized (15, 16), but the pathway in Sulfolobus, an archaeon, remains unclear. Figure Figure77 shows two biosynthetic pathways: in the bacteria-type pathway GlcN-6-P produced from Fru-6-P is first converted to GlcN-1-P and is then acetylated, but in the eukaryote-type pathway, GlcN-6-P was directly acetylated to GlcNAc-6-P (17). The present work showed that the ST0452 protein is capable of acetylating only GlcN-1-P and not GlcN-6-P (Table (Table1).1). In addition to the already characterized GlcNAc-1-P UTase activity, this observation indicates that the ST0452 protein is capable of catalyzing the last two reactions in the bacteria-type UDP-GlcNAc biosynthetic pathway from Fru-6-P.
With regard to the genomic information for S. tokodaii, we detected one candidate for phospho-sugar mutase, which catalyzed the second reaction within the UDP-GlcNAc biosynthetic pathway from Fru-6-P (14), and two candidates for glutamine:fructose-6-phosphate amidotransferase, which catalyzed the first step of the biosynthetic pathway. The preliminary experiments for these enzymatic activities showed that these candidates exhibited the expected activities (unpublished data). These data suggest that the bacteria-type biosynthetic pathway of UDP-GlcNAc from Fru-6-P is actually at work in this archaeon.
The ST0452 homologous genes are widely distributed among most archaea (data not shown). This suggests that the bacteria-type UDP-GlcNAc biosynthetic pathway from Fru-6-P is generally present in archaea. However, the ST0452 homologous gene is not detected in most bacteria, except for the Dehalococcoides species (data not shown), which are known to be microorganisms that can degrade toxic halogen-containing organic compounds to nontoxic compounds. This analysis leads us to hypothesize that the role of the ST0452 protein is correlated with the phenotype of resistance to toxic or stress conditions in these microorganisms.
A previous analysis of the manner of inactivation of the acetyltransferase activity of the E. coli GlmU protein proposed that the cysteine residue(s) in or near the reaction center played an important role in this activity (15). Moreover, a site-directed mutagenesis experiment indicated that two cysteines, Cys307 and Cys324, were important for the acetyltransferase activity of E. coli GlmU (21). However, the ST0452 protein contains only two cysteine residues, positioned at the 44th and 109th residues within the N-terminal region that is predicted to be the sugar-1-P NTase domain. The cysteine residues corresponding to those in or near the E. coli GlmU acetyltransferase reaction center are not present in the ST0452 protein. This reveals that these cysteine residues are nonessential for the acetyltransferase activity of the ST0452 protein. An observation for Streptococcus pneumoniae GlmU that only one cysteine residue, Cys369, is located apart from the active site (13) also supports this conclusion.
The three-dimensional structure of the complex of the S. pneumoniae GlmU and acetyl-CoA (26) predicted that six amino acid residues in the protein—Asn385, Ser404, Ala422, Arg439, Lys445, and Tyr448—interacted with acetyl-CoA. All these residues except Tyr448 are conserved in the E. coli GlmU. However, only 2 amino acid residues, corresponding to Asn385 and Ser404 in S. pneumoniae GlmU, were conserved in the ST0452 protein; thus, it can be said that these two residues play an important role for binding with the acetyl-CoA molecule in the ST0452 protein.
Analyses of the effects of divalent cations on the acetyltransferase activity of the ST0452 protein showed that no divalent cations were required for this activity (Fig. (Fig.6).6). This observation was in contrast to the characteristics of the sugar-1-P NTase activity of the ST0452 protein, for which a divalent cation was essential. Furthermore, the GlcN-1-P acetyltransferase activity of the ST0452 protein was inhibited by all the divalent cations examined, and the GalN-1-P acetyltransferase activity was inhibited by two ions, Zn2+ and Mn2+. Since the mechanisms underlying these inhibitions remain unclear, more detailed analyses of this protein's structure, especially in complex with incorporated divalent cations, are required to clarify the mechanisms involved.
The most important finding of this study was that both novel GalN-1-P acetyltransferase and GalNAc-1-P UTase activities were identified on the ST0452 protein, as were GlcN-1-P acetyltransferase and GlcNAc-1-P UTase activities. In the present work, we have provided clear evidence that both the novel GalN-1-P acetyltransferase, which has not previously been identified in any organism, and the GalNAc-1-P UTase, which has not previously been identified in any thermophilic microorganism, were present on the ST0452 protein. Identification of these two activities on the same protein suggests the presence of a novel UDP-GalNAc biosynthetic pathway from GalN-1-P which differs entirely from the previously known pathway; i.e., UDP-GalNAc is synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase in bacteria and eukarya (2, 4, 6, 9, 20, 28). No gene similar to the UDP-GlcNAc 4-epimerase was detected in the genomic data for S. tokodaii strain 7. This indicates that the pathway predicted in the present work is the only pathway for producing UDP-GalNAc in this microorganism.
According to the kinetic parameters for each substrate and reaction, the overall reaction rates for the GlcNAc-1-P and GalNAc-1-P UTase activities of the ST0452 protein were 2.49 and 1.44 times greater more effective than those for the corresponding acetyltransferase activities, as shown in Table Table2.2. In particular, the Km value for the GlcNAc-1-P substrate on GlcNAc-1-P UTase activity was 73 times lower than that for the corresponding GlcN-1-P substrate on GlcN-1-P acetyltransferase activity. This observation indicates that high concentrations of amino-sugar-1-P substrates are required for the progression of the acetyltransferase reaction, but once the amino sugars are acetylated, the products easily convert to the nucleotide sugar form.
The human enzyme with GlcNAc-1-P UTase activity was identified previously to be associated with male infertility (7). Thus, the ST0452 protein can provide valuable information by serving as a model for this disorder. The extreme thermostability of the ST0452 protein shown in a previous paper and the novel activities of the ST0452 protein observed in the present work indicate that the protein has a strong potential for industrial applications.
This work was supported by a Grant-in-Aid for Scientific Research and a special grant from the Protein 3000 Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Z.Z. was supported by a postdoctoral fellowship from the New Energy and Industrial Technology Development Organization, Japan.
Published ahead of print on 16 April 2010.