Enhancing the fluorescence of tyr-59 in ubiquitin by blocking proton transfer.

Two highly fluorescent mutants of ubiquitin (E51Q and E51A) were produced by mutation of a single amino acid, demonstrating that excited-state proton transfer from the tyrosine residue to the carboxylate group of Glu-51 in ubiquitin is responsible for the reduced fluorescence of wild-type ubiquitin (wt-UBQ) at pH 5. E51A shows a Tm=59 degrees C at pH 1.5 and a Tm>80 degrees C at pH 5 similar to wt-UBQ, which shows that the mutation has not altered the protein structure significantly.

A highly thermostable trehalase from the thermophilic bacterium Rhodothermus marinus.

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose.

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors.

Phosphotransferase-mediated transport of the osmolyte 2-O-alpha-mannosyl-D-glycerate in Escherichia coli occurs by the product of the mngA (hrsA) gene and is regulated by the mngR (farR) gene product acting as repressor.

2-O-alpha-mannosyl-D-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100. Uptake is mediated by the P-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex.

Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment.

We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM).