L-Rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of Lrhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100°C, with a half-life of 60 min at 95°C and 180 min at 80°C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing ␣-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not ␣-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.
The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.
We have developed an integrated automation system for genetic analysis and gene manipulation. The system, SX-8G Plus, is equipped with an 8-nozzle dispensing unit, a thermal cycler, a cooled reagent reservoir, four tip storage racks, four microplate platforms, buffer reservoirs, an agarose gel electrophoresis unit, a power supply, a pump for exchanging electrophoresis buffer, and a CCD camera. Automation of nucleic acid extraction and purification, the most difficult step in automating genetic analysis and gene manipulation, was realized using magnetic beads with Magtration Technology, which we have previously developed for automating the handling of paramagnetic beads. Using this system, we could perform the automated separation and purification of DNA fragments by agarose gel electrophoresis starting from sample loading. The system would enable the automation of almost all procedures in genetic analysis and gene manipulation.
UDP-GlcNAc, an activated and essential form of GlcNAc which is an important component in the polysaccharide structure of most organisms, is synthesized from GlcNAc-1-P and UTP by GlcNAc-1-P UTase. We previously reported the identification of the extremely thermostable ST0452 protein, which has dual sugar-1-P NTase activities (Glc-1-P TTase and GlcNAc-1-P UTase activities) from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Detailed analyses of the protein indicated that the activity is slightly lower than that of bacteria. For industrial applications, activity needs to be increased without decreasing thermostability. Therefore, to enhance this activity, we introduced mutations into the amino acid residues located within the predicted reaction centre by targeted mutagenesis. All 12 mutant ST0452 proteins showed no decrease in thermostability. Among them, six mutant proteins were found to have increased GlcNAc-1-P UTase activity under optimal reaction conditions with sufficient substrates or an appropriate metal ion. Our results indicate that targeted mutagenesis is a powerful technique for in vitro production of a thermostable enzyme with enhanced activity. The results of this study also indicate that the space for the metal ion is important for selecting the type of metal ion and also affects the rate of the reaction.
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. 1). 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. Twe...
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