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J. Biol. Chem., Vol. 280, Issue 10, 9698-9705, March 11, 2005

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Identification of an Extremely Thermostable Enzyme with Dual Sugar-1-phosphate Nucleotidylyltransferase Activities from an Acidothermophilic Archaeon, Sulfolobus tokodaii strain 7^*

Zilian Zhang¶, Masanari Tsujimura¶, Jun-ichi Akutsu, Mayumi Sasaki¶, Hideji Tajima, and Yutaka Kawarabayasi||

From the National Institute of Advanced Industrial Science and Technology, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan and PSS Co., Ltd., Matsudo, Chiba 271-0064, Japan

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 L-rhamnose 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.

Received for publication, September 30, 2004 , and in revised form, December 3, 2004.

^* This work was supported by a special grant from the Protein 3000 Program of the Ministry of Education, Culture, Sports, Science, and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by postdoctoral fellowship from the New Energy and Industrial Technology Development Organization.

^|| To whom correspondence should be addressed: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan. Tel.: 81-29-861-6040; Fax: 81-29-861-6423; E-mail: kawarabayasi.yutaka{at}aist.go.jp.

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