Mechanistic studies on PseB of pseudaminic acid biosynthesis: a UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase.

UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5\u2033 stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-\u3b2-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP+ to oxidize UDP-GlcNAc at C-4\u2033, enabling dehydration. The \u3b1,\u3b2 unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6\u2033 and a proton to C-5\u2033. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5\u2033 position of product during catalysis in D2O. Likewise, PseB catalyzes solvent isotope exchange into the H-5\u2033 position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.Peer reviewed: YesNRC publication: Ye

Identification and recombinant expression of anandamide hydrolyzing enzyme from <it>Dictyostelium discoideum</it>

<p>Abstract</p> <p>Background</p> <p>Anandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH). In this study we report the identification of a FAAH homolog from <it>Dictyostelium discoideum</it> and its function to hydrolyze anandamide.</p> <p>Results</p> <p>A putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either <it>E.coli</it> or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in <it>E.coli</it> host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates.</p> <p>Conclusions</p> <p>This study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from <it>Dictyostelium discoideum</it>, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.</p

Glycosylation of bacterial and archaeal flagellins

The biosynthesis, assembly and regulation of the flagellar organelle has been extensively described over many decades and has focused primarily on the peritrichous flagella of Escherichia coli and Salmonella enterica. More recently, the characterization of flagellar systems from other bacterial and archaeal species has revealed distinct differences in flagellar composition and mode of assembly. Glycosylation of the flagellin structural protein has been identified as an important feature of numerous systems and has been shown to play an integral role in flagellar assembly or in virulence of a number of pathogenic species. This chapter focuses on the structural diversity of flagellar glycans, methods for characterization of flagellin glycoproteins and novel glycan biosynthetic pathways. the relevance of the glycosylation process to assembly as well as other novel biological roles is discussed.NRC publication: Ye

Purification and Characterization of the N-Terminal Domain of ExeA: a Novel ATPase Involved in the Type II Secretion Pathway of Aeromonas hydrophila

Aeromonas hydrophila secretes a number of degradative enzymes and toxins into the external milieu via the type II secretory pathway or secreton. ExeA is an essential component of this system and is necessary for the localization and/or multimerization of the secretin ExeD. ExeA contains two sequence motifs characteristic of the Walker superfamily of ATPases. Previous examination of substitution derivatives altered in these motifs suggested that ATP binding or hydrolysis is required for ExeAB complex formation and subsequent secretion function. To directly examine ExeA function, the N-terminal cytoplasmic domain of ExeA with the addition of a C-terminal hexahistidine tag (cytExeA) was overproduced in Escherichia coli and purified by metal chelate affinity and anion-exchange chromatographic techniques. Purified preparations of cytExeA exhibited ATPase activity in the presence of several divalent cations, Mg(2+) being the preferred cation, with an optimum reaction temperature of ∼37 to 42°C and an optimum pH of 7 to 8. cytExeA exhibited an apparent K(m) for Mg-ATP of 0.22 mM and a V(max) of 0.72 nmol min(−1) mg(−1) of protein. cytExeA displayed low specificity for nucleoside triphosphate substrates and was significantly inhibited by F-type ATPase inhibitors. Gel filtration analyses of cytExeA, ExeA, and ExeAB indicated that ExeA dimerizes and forms a very large complex with ExeB. These findings support a model whereby ExeAB utilizes energy derived from ATP hydrolysis to facilitate the correct localization and multimerization of the ExeD secretin

Protein Glycosylation in Campylobacter jejuni: Partial Suppression of pglF by Mutation of pseC▿

Campylobacter jejuni has systems for N- and O-linked protein glycosylation. Although biochemical evidence demonstrated that a pseC mutant in the O-linked pathway accumulated the product of pglF in the N-linked pathway, analyses of transformation frequencies and glycosylation statuses of N-glycosylated proteins indicated a partial suppression of pglF by pseC

Synthesis of 4-acetamidohexoses in bacteria: structural insights from the bacillosamine and nonulosonic acid pathways

Many hexose sugars in bacteria undergo a variety of modifications, including oxidation/reduction, amination and acetylation, as part of biosynthesis into their final biologically-active forms. Enzymes that catalyze these reactions normally utilize nucleotide-linked sugar substrates, utilizing the nucleotide as an 'ancient handle' to bind and orient the sugar within the enzymes' active site. We and others have focused efforts on elucidating structure-function relationships for a subset of such biosynthetic enzymes, those associated with the synthesis of trideoxy-diacetamidohexoses, and the nonulsonate sugars subsequently derived from them. With structural information combined with site-directed mutagenesis, enzymatic analysis and molecular modeling, these studies have been essential to understanding the chemistry of how these enzymes bind their substrates and effect catalysis. Enzymes having different folds, such as N-acetyltransferases, can utilize different scaffolds to attain the same 4-acetamido-sugar product. In the case of dehydratases/epimerases and aminotransferases, the enzymes have a conserved structure, but utilize subtle differences within their active site to confer substrate binding and the nature of the final product. These studies depict the structural relationships between these enzymes, while at the same time high-lighting important differences that are beginning to reveal their function at the molecular level.Peer reviewed: YesNRC publication: Ye

The CMP-legionaminic acid pathway in campylobacter : Biosynthesis involving novel GDP-linked precursors

The sialic acid-like sugar 5,7-diacetamido-3,5,7,9- tetradeoxy-D-glycero-D-galacto-nonulosonic acid, or legionaminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire\u2019s disease, contain an \u3b12,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.Peer reviewed: YesNRC publication: Ye

The engineering of bacteria bearing azido-pseudaminic acid modified flagella

We have demonstrated that by feeding nonmotile mutant C. jejuni bacteria with a neutral azide-labelled pseudaminic acid precursor we can restore their ability to generate functional flagella. The presence of azido-pseudaminic acid on the surface of the flagella provides a bio-orthogonal chemical handle that can be used to modify the flagellar proteins.Peer reviewed: YesNRC publication: Ye