CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.

Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.

Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.

The critical role of a conserved lysine residue in periplasmic nitrate reductase catalyzed reactions.

Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.

Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

Effect of WQ-3334 on Campylobacter jejuni carrying a DNA gyrase with dominant amino acid substitutions conferring quinolone resistance.

INTRODUCTION: Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of quinolones-resistant Campylobacter jejuni poses a significant challenge. The predominant mechanism of quinolones-resistance in this bacterium involves point mutations in the gyrA, resulting in amino acid substitution from threonine to isoleucine at 86th position, representing more than 90% of mutant DNA gyrase, and aspartic acid to asparagine at 90th position. WQ-3334, a novel quinolone, has demonstrated strong inhibitory activity against various bacteria. This study aims to investigate the effectiveness of WQ-3334, and its analogues, WQ-4064 and WQ-4065, with a unique modification in R1 against quinolones-resistant C. jejuni. METHODS: The structure-activity relationship of the examined drugs was investigated by measuring IC50 and their antimicrobial activities were accessed by MIC against C. jejuni strains. Additionally, in silico docking simulations were carried out using the crystal structure of the Escherichia coli DNA gyrase. RESULT: WQ-3334 exhibited the lowest IC50 against WT (0.188 ± 0.039 mg/L), T86I (11.0 ± 0.7 mg/L) and D90 N (1.60 ± 0.28 mg/L). Notably, DNA gyrases with T86I substitutions displayed the highest IC50 values among the examined WQ compounds. Moreover, WQ-3334 demonstrated the lowest MICs against wild-type and mutant strains. The docking simulations further confirmed the interactions between WQ-3334 and DNA gyrases. CONCLUSION: WQ-3334 with 6-amino-3,5-difluoropyridine-2-yl at R1 severed as a remarkable candidate for the treatment of foodborne diseases caused by quinolones-resistant C. jejuni as shown by the high inhibitory activity against both wild-type and the predominant quinolones-resistant strains.

The flavodoxin FldA activates the class Ia ribonucleotide reductase of Campylobacter jejuni.

Campylobacter jejuni is a microaerophilic zoonotic pathogen with an atypical respiratory Complex I that oxidizes a flavodoxin (FldA) instead of NADH. FldA is essential for viability and is reduced via pyruvate and 2-oxoglutarate oxidoreductases (POR/OOR). Here, we show that FldA can also be reduced by FqrB (Cj0559), an NADPH:FldA reductase. An fqrB deletion mutant was viable but displayed a significant growth defect. FqrB is related to flavoprotein reductases from Gram-positive bacteria that can reduce NrdI, a specialized flavodoxin that is needed for tyrosyl radical formation in NrdF, the beta subunit of class 1b-type (Mn) ribonucleotide reductase (RNR). However, C. jejuni possesses a single class Ia-type (Fe) RNR (NrdAB) that would be expected to be ferredoxin dependent. We show that CjFldA is an unusually high potential flavodoxin unrelated to NrdI, yet growth of the fqrB mutant, but not the wild-type or a complemented strain, was stimulated by low deoxyribonucleoside (dRNS) concentrations, suggesting FldA links FqrB and RNR activity. Using purified proteins, we confirmed the NrdB tyrosyl radical could be regenerated in an NADPH, FqrB, and FldA dependent manner, as evidenced by both optical and electron paramagnetic resonance (EPR) spectroscopy. Thus, FldA activates RNR in C. jejuni, partly explaining its essentiality.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Transcription of Cystathionine ß-Lyase (MetC) Is Repressed by HeuR in Campylobacter jejuni, and Methionine Biosynthesis Facilitates Colonocyte Invasion.

A previously identified transcriptional regulator in Campylobacter jejuni, termed HeuR, was found to positively regulate heme utilization. Additionally, transcriptomic work demonstrated that the putative operons CJJ81176_1390 to CJJ81176_1394 (CJJ81176_1390-1394) and CJJ81176_1214-1217 were upregulated in a HeuR mutant, suggesting that HeuR negatively regulates expression of these genes. Because genes within these clusters include a cystathionine ß-lyase (metC) and a methionine synthase (metE), it appeared HeuR negatively regulates C. jejuni methionine biosynthesis. To address this, we confirmed mutation of HeuR reproducibly results in metC overexpression under nutrient-replete conditions but did not affect expression of metE, while metC expression in the wild type increased to heuR mutant levels during iron limitation. We subsequently determined that both gene clusters are operonic and demonstrated the direct interaction of HeuR with the predicted promoter regions of these operons. Using DNase footprinting assays, we were able to show that HeuR specifically binds within the predicted -35 region of the CJJ81176_1390-1394 operon. As predicted based on transcriptional results, the HeuR mutant was able to grow and remain viable in a defined medium with and without methionine, but we identified significant impacts on growth and viability in metC and metE mutants. Additionally, we observed decreased adherence, invasion, and persistence of metC and metE mutants when incubated with human colonocytes, while the heuR mutant exhibited increased invasion. Taken together, these results suggest that HeuR regulates methionine biosynthesis in an iron-responsive manner and that the ability to produce methionine is an important factor for adhering to and invading the gastrointestinal tract of a susceptible host. IMPORTANCE As the leading cause of bacterium-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health. Investigating colonization factors that allow C. jejuni to successfully infect a host furthers our understanding of genes and regulatory elements necessary for virulence. In this study, we have begun to characterize the role of the transcriptional regulatory protein, HeuR, on methionine biosynthesis in C. jejuni. When the ability to synthesize methionine is impaired, detrimental impacts on growth and viability are observed during growth in limited media lacking methionine and/or iron. Additionally, mutations in the methionine biosynthetic pathway result in decreased adhesion, invasion, and intracellular survival of C. jejuni when incubated with human colonocytes, indicating the importance of regulating methionine biosynthesis.

Turnover Chemistry and Structural Characterization of the Cj0843c Lytic Transglycosylase of Campylobacter jejuni.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

Functional Characterization of Two PLP-Dependent Enzymes Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni.

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.

Functional and Structural Characterization of the UDP-Glucose Dehydrogenase Involved in Capsular Polysaccharide Biosynthesis from Campylobacter jejuni.

Campylobacter jejuni is a pathogenic organism that can cause campylobacteriosis in children and adults. Most commonly, campylobacter infection is brought on by consumption of raw or undercooked poultry, unsanitary drinking water, or pet feces. Surrounding the C. jejuni bacterium is a coat of sugar molecules known as the capsular polysaccharide (CPS). The capsular polysaccharide can be very diverse among the different strains of C. jejuni, and this diversity is considered important for evading the host immune system. Modifications to the CPS of C. jejuni NCTC 11168 include O-methylation, phosphoramidylation, and amidation of glucuronate with either serinol or ethanolamine. The enzymes responsible for amidation of glucuronate are currently unknown. In this study, Cj1441, an enzyme expressed from the CPS biosynthetic gene cluster in C. jejuni NCTC 11168, was shown to catalyze the oxidation of UDP-α-d-glucose into UDP-α-d-glucuronic acid with NAD+ as the cofactor. No amide products were found in an attempt to determine whether the putative thioester intermediate formed during the oxidation of UDP-glucose by Cj1441 could be captured in the presence of added amines. The three-dimensional crystal structure of Cj1441 was determined in the presence of NAD+ and UDP-glucose bound in the active site of the enzyme (Protein Data Bank entry 7KWS). A more thorough bioinformatic analysis of the CPS gene cluster suggests that the amidation activity is localized to the t-terminal half of Cj1438, a bifunctional enzyme that is currently annotated as a sugar transferase.

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria.

The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown "route I" nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni.

Kinetic consequences of the endogenous ligand to molybdenum in the DMSO reductase family: a case study with periplasmic nitrate reductase.

The molybdopterin enzyme family catalyzes a variety of substrates and plays a critical role in the cycling of carbon, nitrogen, arsenic, and selenium. The dimethyl sulfoxide reductase (DMSOR) subfamily is the most diverse family of molybdopterin enzymes and the members of this family catalyze a myriad of reactions that are important in microbial life processes. Enzymes in the DMSOR family can transform multiple substrates; however, quantitative information about the substrate preference is sparse, and, more importantly, the reasons for the substrate selectivity are not clear. Molybdenum coordination has long been proposed to impact the catalytic activity of the enzyme. Specifically, the molybdenum-coordinating residue may tune substrate preference. As such, molybdopterin enzyme periplasmic nitrate reductase (Nap) is utilized as a vehicle to understand the substrate preference and delineate the kinetic underpinning of the differences imposed by exchanging the molybdenum ligands. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic properties of these variants are discussed and compared with those of the native enzyme, providing quantitative information to understand the function of the molybdenum-coordinating residue.

Structure and mechanism of an active lipid-linked oligosaccharide flippase.

The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

Functional Characterization of Cj1427, a Unique Ping-Pong Dehydrogenase Responsible for the Oxidation of GDP-d-glycero-α-d-manno-heptose in Campylobacter jejuni.

The capsular polysaccharides (CPS) of Campylobacter jejuni contain multiple heptose residues with variable stereochemical arrangements at C3-C6. The immediate precursor to all of these possible variations is currently believed to be GDP-d-glycero-α-d-manno-heptose. Oxidation of this substrate at C4 enables subsequent epimerization reactions at C3-C5 that can be coupled to the dehydration/reduction at C5/C6. However, the enzyme responsible for the critical oxidation of C4 from GDP-d-glycero-α-d-manno-heptose has remained elusive. The enzyme Cj1427 from C. jejuni NCTC 11168 was shown to catalyze the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose in the presence of α-ketoglutarate using mass spectrometry and nuclear magnetic resonance spectroscopy. At pH 7.4, the apparent kcat is 0.6 s-1, with a value of kcat/Km of 1.0 × 104 M-1 s-1 for GDP-d-glycero-α-d-manno-heptose. α-Ketoglutarate is required to recycle the tightly bound NADH nucleotide in the active site of Cj1427, which does not dissociate from the enzyme during catalysis.

Structural Analysis of Cj1427, an Essential NAD-Dependent Dehydrogenase for the Biosynthesis of the Heptose Residues in the Capsular Polysaccharides of Campylobacter jejuni.

Many strains of Campylobacter jejuni display modified heptose residues in their capsular polysaccharides (CPS). The precursor heptose was previously shown to be GDP-d-glycero-α-d-manno-heptose, from which a variety of modifications of the sugar moiety have been observed. These modifications include the generation of 6-deoxy derivatives and alterations of the stereochemistry at C3-C6. Previous work has focused on the enzymes responsible for the generation of the 6-deoxy derivatives and those involved in altering the stereochemistry at C3 and C5. However, the generation of the 6-hydroxyl heptose residues remains uncertain due to the lack of a specific enzyme to catalyze the initial oxidation at C4 of GDP-d-glycero-α-d-manno-heptose. Here we reexamine the previously reported role of Cj1427, a dehydrogenase found in C. jejuni NTCC 11168 (HS:2). We show that Cj1427 is co-purified with bound NADH, thus hindering catalysis of oxidation reactions. However, addition of a co-substrate, α-ketoglutarate, converts the bound NADH to NAD+. In this form, Cj1427 catalyzes the oxidation of l-2-hydroxyglutarate back to α-ketoglutarate. The crystal structure of Cj1427 with bound GDP-d-glycero-α-d-manno-heptose shows that the NAD(H) cofactor is ideally positioned to catalyze the oxidation at C4 of the sugar substrate. Additionally, the overall fold of the Cj1427 subunit places it into the well-defined short-chain dehydrogenase/reductase superfamily. The observed quaternary structure of the tetrameric enzyme, however, is highly unusual for members of this superfamily.

Asparagine-84, a regulatory allosteric site residue, helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase.

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzymes.

Thiosulfate dehydrogenases (TsdAs) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic CD (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a microaerobic human pathogen, and from the purple sulfur bacterium Allochromatium vinosum In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase, respectively. The active site Heme 1 in both enzymes has His/Cys ligation in the ferric and ferrous states and the midpoint potentials (Em ) of the corresponding redox transformations are similar, -185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni, TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em , -129 mV) to His/Met (Em , +266 mV), but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s).

Chemoenzymatic Synthesis of Campylobacter jejuni Lipo-oligosaccharide Core Domains to Examine Guillain-Barré Syndrome Serum Antibody Specificities.

Guillain-Barré syndrome is often caused by Campylobacter jejuni infection that has induced antibodies to the lipo-oligosaccharide (LOS) that cross-react with gangliosides at peripheral nerves causing polyneuropathy. To examine fine specificities of anti-ganglioside antibodies and develop a more robust platform for diagnosis and disease monitoring, we developed a chemoenzymatic approach that provided an unprecedented panel of oligosaccharides composed of the inner-core of the LOS of C. jejuni extended by various ganglioside mimics. The compounds and corresponding ganglio-oligosaccharides were printed as a microarray to examine binding specificities of lectins, anti-ganglioside antibodies, and serum antibodies of GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis, disease monitoring, and immunological research.

Activity of fructose-1,6-bisphosphatase from Campylobacter jejuni.

The glycolytic pathway of the enteric pathogen Campylobacter jejuni is incomplete; the absence of phosphofructokinase means that the suppression of futile cycling at this point in the glycolytic-gluconeogenic pathway might not be required, and therefore the mechanism for controlling pathway flux is likely to be quite different or absent. In this study, the characteristics of fructose-1,6-bisphosphatase (FBPase) of C.jejuni are described and the regulation of this enzyme is compared with the equivalent enzymes from organisms capable of glycolysis. The enzyme is insensitive to AMP inhibition, unlike other type I FBPases. Campylobacterjejuni FBPase also shows limited sensitivity to other glycolytic and gluconeogenic intermediates. The allosteric cooperative control of the enzyme's activity found in type I FBPases appears to have been lost.