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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.

Human peptidylarginine deiminase type IV (PAD4), a Ca(2+)-dependent enzyme known to convert arginine residues to citrulline residues in histones, has been shown to be associated with the development of rheumatoid arthritis. Recently, it was noted that the human peptidylarginine deiminase type IV gene (PADI4) regulates the expression of estrogen-responsive genes by modifying the methylated arginine sites in histones H3 and H4. In this study, we demonstrated that PADI4 was expressed in MCF-7 cells and was responsive to estrogen at the transcriptional level. Using the luciferase reporter gene fused to wild-type or mutated 5'-flanking region of PADI4, we characterized that as few as 348 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Chromatin immunoprecipitation and small interfering RNA assays revealed that activator protein-1, Sp1/Sp3, and nuclear factor-Y were cis-acting factors bound to the minimal promoter of PADI4 and that they regulated gene expression in a cooperative manner. Moreover, it was indicated that estrogen stimulated PADI4 expression through binding of estrogen receptor (ER)-alpha to the upstream of the PADI4 gene and ERalpha-mediated enhancement of activator protein-1, Sp1, and nuclear factor-Y levels. These findings indicated that estrogen stimulated PADI4 expression through both of the classical and nonclassical ER-mediated pathways.