Kinetic characterization of methylthio-d-ribose-1-phosphate isomerase.

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-d-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-d-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose-ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [32P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct.

Covalent Adduct Formation in Methylthio-d-ribose-1-phosphate Isomerase: Reaction Intermediate or Artifact?

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) functions in the methionine salvage pathway by converting the cyclic aldose MTR1P to its open-chain ketose isomer methylthio-d-ribulose 1-phosphate (MTRu1P). What is particularly challenging for this enzyme is that the substrate's phosphate ester prevents facile equilibration to an aldehyde, which in other aldose-ketose isomerases is known to activate the α-hydrogen for proton or hydride transfer between adjacent carbons. We speculated that MtnA could use covalent catalysis via a phosphorylated residue to permit isomerization by one of the canonical mechanisms, followed by phosphoryl transfer back to form the product. In apparent support of this mechanism, [32P]MTR1P was found by SDS-PAGE and gel-filtration chromatography to radiolabel the enzyme. Susceptibility of this adduct to strongly acidic and basic pH and nucleophilic agents is consistent with an acyl phosphate. C160S and D240N, mutants of two conserved active-site residues, however, exhibited no difference in radiolabeling despite a reduction in activity of ∼107, leading to the conclusion that phosphorylation is unrelated to catalysis. Unexpectedly, prolonged incubations with C160S revealed up to 30% accumulation of radioactivity, which was identified by 31P and 13C NMR to be the result of a second adduct─a hemiketal formed between Ser160 and the carbonyl of MTRu1P. These results are interpreted as indirect support for a mechanism involving transfer of the proton from C-2 to C-1 by Cys160.

Characterization of the ß-KDO Transferase KpsS, the Initiating Enzyme in the Biosynthesis of the Lipid Acceptor for Escherichia coli Polysialic Acid.

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the ß-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a ß-KDO linkage.

Chemical structure and genetic organization of the E. coli O6:K15 capsular polysaccharide.

Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gram-negative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identified as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1 → 5)-α-KDOp-(2 → partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.

Interaction of Neisseria meningitidis Group X N-acetylglucosamine-1-phosphotransferase with its donor substrate.

Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.

Regulation and Molecular Basis of Environmental Muropeptide Uptake and Utilization in Fastidious Oral Anaerobe Tannerella forsythia.

Tannerella forsythia is a Gram-negative oral anaerobe associated with periodontitis. This bacterium is auxotrophic for the peptidoglycan amino sugar N-acetylmuramic (MurNAc) and likely relies on scavenging peptidoglycan fragments (muropeptides) released by cohabiting bacteria during their cell wall recycling. Many Gram-negative bacteria utilize an inner membrane permease, AmpG, to transport peptidoglycan fragments into their cytoplasm. In the T. forsythia genome, the Tanf_08365 ORF has been identified as a homolog of AmpG permease. In order to confirm the functionality of Tanf_08365, a reporter system in an Escherichia coli host was generated that could detect AmpG-dependent accumulation of cytosolic muropeptides via a muropeptide-inducible ß-lactamase reporter gene. In trans complementation of this reporter strain with a Tanf_08365 containing plasmid caused significant induction of ß-lactamase activity compared to that with an empty plasmid control. These data indicated that Tanf_08365 acted as a functional muropeptide permease causing accumulation of muropeptides in E. coli and thus suggested that it is a permease involved in muropeptide scavenging in T. forsythia. Furthermore, we showed that the promoter regulating the expression of Tanf_08365 was activated significantly by a hybrid two-component system regulatory protein, GppX. We also showed that compared to the parental T. forsythia strain a mutant lacking GppX in which the expression of AmpG was reduced significantly attenuated in utilizing free muropeptides. In summary, we have uncovered the mechanism by which this nutritionally fastidious microbe accesses released muropeptides in its environment, opening up the possibility of targeting this activity to reduce its numbers in periodontitis patients with potential benefits in the treatment of disease.