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Several eukaryotic organisms have a conserved pattern of induction of the heat shock proteins or their cognates during development that is uncoupled from the heat shock response. Two of these proteins, hsp84 and hsp70, are conserved throughout evolution and are homologous to HtpG and DnaK respectively of Escherichia coli. As the most primitive form of development is sporulation, a response to nutritional deprivation, we tested the ability of E. coli cells with mutations in dnaK or htpG to respond to starvation for carbon, nitrogen or phosphate. The htpG mutants were found to respond to starvation like their wild type parents. DnaK103 mutants, however, were highly sensitive to starvation for carbon, and to a lesser extent, to starvation for nitrogen and phosphate. Analysis of proteins induced under starvation conditions on two-dimensional gels showed that the dnaK103 mutant is defective for the synthesis of some proteins under catabolic control and some starvation induced proteins which are synthesized at high levels during the stationary phase and under all starvation conditions. Furthermore, the dnaK103 mutant synthesized a unique set of proteins during starvation. Although the synthesis of some carbon starvation proteins was altered by the dnaK103 mutation, the synthesis of proteins specifically induced by nitrogen starvation was unaffected. Similarly, the dnaK103 mutant was able to utilize glutamine or arginine as sources of nitrogen at a rate approximate to the wild type parent, but it inefficiently utilized glycerol or maltose as carbon sources. Several differences in the protein pattern of the dnaK103 mutant and wild type were observed after phosphate starvation. In addition, the highly conserved Escherichia coli protein, HtpG, was overproduced and purified by column chromatography on DEAE-52 cellulose, hydroxyapatite, and Sephacryl S-200 columns. Immunoprecipitation of labeled cells with antisera specific for HtpG demonstrated that this protein is a phosphoprotein. The native molecular weight, determined by gel filtration and sedimentation analysis, was twice the molecular weight as determined by DNA sequence analysis. Thus, like its eukaryotic homologue,hsp84, HtpG is a dimer and a phosphoprotein.

In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.

International audience; In search of α-galactosidases with improved kinetic properties for removal of the immunodominant α1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of α-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454–464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galα1–3(Fucα1–2)Gal, whereas linear oligosaccharides terminated by α1,3-linked galactose such as the immunodominant xenotransplantation epitope Galα1–3Galβ1–4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific α1,3-galactosidases that act equally well on both branched blood group B and linear α1,3Gal structures. We determined by one-dimensional 1H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known α-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant α3Gal xenotransplantation epitope.