Reversible derivatization of sugars with carbobenzyloxy groups and use of the derivatives in solution-phase enzymatic oligosaccharide synthesis.

Simple protocols for attaching and detaching carbobenzyloxy (Cbz) groups at the reducing end of sugars was developed. Briefly, lactose was converted into its glycosylamine, which was then acylated with carbobenzyloxy chloride in high overall yield. The obtained lactose Cbz derivative was used in sequential glycosylations using glycosyltransferases and nucleotide sugars in aqueous buffers. Isolation of the reaction products after each step was by simple C-18 solid-phase extraction. The Cbz group was removed by catalytic hydrogenolysis or catalytic transfer hydrogenation followed by in situ glycosylamine hydrolysis. In this way, a trisaccharide (GlcNAc-lactose), a human milk tetrasaccharide (LNnT), and a human milk pentasaccharide (LNFPIII) were prepared in a simple and efficient way.

Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease.

The micro aerophilic pathogen Helicobacter mustelae synthesizes an oxygen-labile, iron-containing urease (UreA2B2) in addition to its standard nickel-containing enzyme (UreAB). An apoprotein form of the iron urease was prepared from ureA2B2-expressing recombinant Escherichia coli cells that were grown in minimal medium. Temperature-dependent circular dichroism measurements of holoprotein and apoprotein demonstrate an enhancement of thermal stability associated with the UreA2B2 metallocenter. In parallel to the situation reported for nickel activation of the standard urease apoprotein, incubation of UreA2B2 apoprotein with ferrous ions and bicarbonate generated urease activity in a portion of the nascent active sites. In addition, ferrous ions were shown to be capable of reductively activating the oxidized metallocenter. Resonance Raman spectra of the inactive, aerobically-purified UreA2B2 holoprotein exhibit vibrations at 495cm(-1) and 784cm(-1), consistent with ν(s) and ν(as) modes of an Fe(III)OFe(III) center; these modes undergo downshifts upon binding of urea and were unaffected by changes in pH. The low-frequency mode also exhibits an isotopic shift from 497 to 476cm(-1) upon (16)O/(18)O bulk water isotope substitution. Expression of subunits of the conventional nickel-containing Klebsiella aerogenes urease in cells grown in rich medium without nickel resulted in iron incorporation into a portion of the protein. The inactive iron-loaded species exhibited a UV-visible spectrum similar to oxidized UreA2B2 and was capable of being reductively activated under anoxic conditions. Results from these studies more clearly define the formation and unique properties of the iron urease metallocenter.

Iron-containing urease in a pathogenic bacterium.

Helicobacter mustelae, a gastric pathogen of ferrets, synthesizes a distinct iron-dependent urease in addition to its archetypical nickel-containing enzyme. The iron-urease is oxygen-labile, with the inactive protein exhibiting a methemerythrin-like electronic spectrum. Significantly, incubation of the oxidized protein with dithionite under anaerobic conditions leads to restoration of activity and bleaching of the spectrum. Structural analysis of the oxidized species reveals a dinuclear iron metallocenter bridged by a lysine carbamate, closely resembling the traditional nickel-urease active site. Although the iron-urease is less active than the nickel-enzyme, its activity allows H. mustelae to survive the carnivore's low-nickel gastric environment.

Characterization of NikR-responsive promoters of urease and metal transport genes of Helicobacter mustelae.

The NikR protein is a nickel-responsive regulator, which in the gastric pathogen Helicobacter pylori controls expression of nickel-transporters and the nickel-cofactored urease acid resistance determinant. Although NikR-DNA interaction has been well studied, the Helicobacter NikR operator site remains poorly defined. In this study we have identified the NikR operators in the promoters of two inversely nickel-regulated urease operons (ureAB and ureA2B2) in the ferret pathogen Helicobacter mustelae, and have used bioinformatic approaches for the prediction of putative NikR operators in the genomes of four urease-positive Helicobacter species. Helicobacter mustelae NikR bound to the ureA2 promoter to a sequence overlapping with the -35 promoter region, leading to repression. In contrast, NikR binding to a site far upstream of the canonical sigma(80) promoter in the H. mustelae ureA promoter resulted in transcriptional induction, similar to the situation in H. pylori. Using H. pylori NikR operators and the newly identified H. mustelae NikR operators a new consensus sequence was generated (TRWYA-N(15)-TRWYA), which was used to screen the genomes of four urease-positive Helicobacter species (H. mustelae, H. pylori, H. acinonychis and H. hepaticus) for putative NikR-regulated promoters. One of these novel putative NikR-regulated promoters in H. mustelae is located upstream of a putative TonB-dependent outer membrane protein designated NikH, which displayed nickel-responsive expression. Insertional inactivation of the nikH gene in H. mustelae resulted in a significant decrease in urease activity, and this phenotype was complemented by nickel-supplementation of the growth medium, suggesting a function for NikH in nickel transport across the outer membrane. In conclusion, the H. mustelae NikR regulator directly controls nickel-responsive regulation of ureases and metal transporters. The improved consensus NikR operator sequence allows the prediction of additional NikR targets in Helicobacter genomes, as demonstrated by the identification of a new nickel-repressed outer membrane protein in H. mustelae.

Bacterial homologue of human blood group A transferase.

A bacterial version of human blood group A transferase was identified and found to be able to accept five naturally existing H-antigen core structures as good substrates, demonstrating its versatility for synthesis of blood group A antigens. Furthermore, this enzyme was applied in the engineering of bacterial cell surface polysaccharides by remodeling blood group B mimicry into blood group A mimicry.

Inverse nickel-responsive regulation of two urease enzymes in the gastric pathogen Helicobacter mustelae.

The acidic gastric environment of mammals can be chronically colonized by pathogenic Helicobacter species, which use the nickel-dependent urea-degrading enzyme urease to confer acid resistance. Nickel availability in the mammal host is low, being mostly restricted to vegetarian dietary sources, and thus Helicobacter species colonizing carnivores may be subjected to episodes of nickel deficiency and associated acid sensitivity. The aim of this study was to investigate how these Helicobacter species have adapted to the nickel-restricted diet of their carnivorous host. Three carnivore-colonizing Helicobacter species express a second functional urea-degrading urease enzyme (UreA2B2), which functions as adaptation to nickel deficiency. UreA2B2 was not detected in seven other Helicobacter species, and is in Helicobacter mustelae only expressed in nickel-restricted conditions, and its expression was higher in iron-rich conditions. In contrast to the standard urease UreAB, UreA2B2 does not require activation by urease or hydrogenase accessory proteins, which mediate nickel incorporation into these enzymes. Activity of either UreAB or UreA2B2 urease allowed survival of a severe acid shock in the presence of urea, demonstrating a functional role for UreA2B2 in acid resistance. Pathogens often express colonization factors which are adapted to their host. The UreA2B2 urease could represent an example of pathogen adaptation to the specifics of the diet of their carnivorous host, rather than to the host itself.