Biosynthesis of mannosylglycerate in the thermophilic bacterium Rhodothermus marinus. Biochemical and genetic characterization of a mannosylglycerate synthase.
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 (13)C 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.
Martins
L O
LO
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780 Oeiras, Portugal.
Empadinhas
N
N
Marugg
J D
JD
Miguel
C
C
Ferreira
C
C
da Costa
M S
MS
Santos
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D005988:Glyceric Acids / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008358:Mannose / Q000031:analogs & derivatives* / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013816:Thermodynamics
D005988:Glyceric Acids; D011994:Recombinant Proteins; C402688:mannosylglycerate; D008364:Mannosyltransferases; C402687:mannosylglycerate synthase; D008358:Mannose