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J Biol Chem, Vol. 274, Issue 50, 35407-35414, December 10, 1999

Biosynthesis of Mannosylglycerate in the Thermophilic Bacterium Rhodothermus marinus
BIOCHEMICAL AND GENETIC CHARACTERIZATION OF A MANNOSYLGLYCERATE SYNTHASE^*

Lígia O. Martins^§, Nuno Empadinhas^¶, Joey D. Marugg^¶, Carla Miguel^¶, Célia Ferreira^¶, Milton S. da Costa^¶, and Helena Santos^**

From the  Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780 Oeiras, Portugal and the ^¶ Departamento de Bioquímica and Centro de Neurociências de Coimbra, Universidade de Coimbra, 3000 Coimbra, Portugal

The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinus is proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo ¹³C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed with D-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and D-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed in Escherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose and D-glycerate. This is the first report of the characterization of a mannosylglycerate synthase.

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^* This work was supported in part by the European Community Biotech Program Extremophiles as Cell Factories, BIO4-CT96-0488, and by PRAXIS XXI and FEDER, Portugal PRAXIS/2/2.1/BIO/1109/95.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF173987.

^§ Supported by Postdoctoral Fellowship BPD/6049/95 from PRAXIS XXI.

Supported by invited Scientist Fellowship BCC/11978 from PRAXIS XXI.

^** To whom correspondence should be addressed: Instituto de Tecnologia Química e Biológica, Apartado 127, 2780 Oeiras, Portugal. Tel.: 351-1-4469800; Fax: 351-1-4428766; E-mail: santos@itqb.unl.pt.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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