Martin E. Tanner

A prototype of a new inductively coupled plasma time-of-flight mass spectrometer providing temporally resolved, multi-element detection of short signals generated by single particles and droplets

A prototype inductively coupled plasma time-of-flight mass spectrometer (ICPTOFMS) for time resolved measurements of transient signals in the microsecond regime is described in this work. Analytical figures of merit for the prototype are given for both liquid nebulization and single droplet introduction and are compared to a conventional quadrupole-based ICPMS using the same ICP source and vacuum interface. Quasi-simultaneous detection at a time resolution of 33 μs of the prototype ICPTOFMS allows multi-isotope monitoring of short signals (200–500 μs duration) generated from individual droplets and particles. The capabilities of the instrument for the analysis of single nanoparticles are studied using microdroplets consisting of a multi-element standard solution and containing 114 nm Au particles. The detection efficiencies for Ag and Au, calculated from the response of individual droplets and particles, are similar to those of the quadrupole-based instrument and amount to 1.3 × 10−6 ions per atom and 3.1 × 10−6 ions per atom, respectively. The sizes of the smallest detectable Ag, Au and U metallic nanoparticles are estimated to be 46 nm, 32 nm and 22 nm, respectively. Furthermore, time shifts of the signals of different elements within single droplets were observed. These new results demonstrate the advantage of the temporal resolution of the instrument for studying processes taking place in the plasma on the μs-time scale.

Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci IRREVERSIBLE INHIBITION BY UDP-CHLOROACETOL

UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 μM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5′-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 × 103 mM−1 min−1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5′-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.

The synthesis and stability of aziridino-glutamate, an irreversible inhibitor of glutamate racemase

Abstract Aziridino-glutamate (2-(2-carboxyethyl)aziridine-2-carboxylic acid, (±) 4 ) was synthesized by heating α-fluoromethylglutamate in base. In neutral solution, 4 was shown to cyclize to the γ-lactone 5 with a half life of 4 minutes. Aziridino-glutamate was shown to irreversibly inactivate glutamate racemase by alkylating an active site cysteine residue. Electrospray mass spectrometry was used to establish that a covalent bond had formed and that this bond protects one of the enzymes two cysteine residues from reacting with iodoacetate under denaturing conditions.

Simultaneous Mass Quantification of Nanoparticles of Different Composition in a Mixture by Microdroplet Generator-ICPTOFMS

This work investigated the potential of a high temporal resolution inductively coupled plasma time-of-flight mass spectrometer (ICPTOFMS) in combination with a microdroplet generator (MDG) for simultaneous mass quantification of different nanoparticles (NPs) in a mixture. For this purpose, a test system containing certified Au NPs, well characterized Ag NPs, and core-shell NPs composed of an Au core and an Ag shell was employed. Thanks to the full spectra coverage and rapid simultaneous detection of the TOFMS, the element composition of individual particles can be determined. The pure Ag NPs and the core-shell NPs could be differentiated despite the same mass of Ag they contain. Calibration with monodisperse droplets consisting of standard solutions allowed for the mass quantification of NPs without the use of NP certified materials. On the basis of this mass quantification, the sizes of NPs originating from the same aqueous suspension were simultaneously determined with an accuracy of 7-12%. The size-equivalent limits of detection estimated with the 3*σ criterion were 13 nm for Au and 16 nm for Ag. Estimation of the LODs using Poisson statistics resulted in 19 and 27 nm, respectively. In addition, the 30 μs temporal resolution of the ICPTOFMS allowed studying interactions of NPs with the ICP based on their transient MS signals. The results demonstrated a difference in vaporization behavior of the core-shell NPs and solutions and indicated that vaporization of the Ag shell takes place prior to the Au core.

Mechanistic aspects of enzymatic carbohydrate epimerization

Carbohydrate epimerases are enzymes that catalyze an inversion of stereochemistry at a stereogenic center in a sugar. This review focuses on the mechanistic strategies that are employed by this diverse family of enzymes, and is divided into the following sections: deprotonation/reprotonation, transient oxidation, cofactor independent, and mutarotation. The review is comprehensive up to Oct. 2001 and contains 216 references.

Mechanism of Dimethylallyltryptophan Synthase: Evidence for a Dimethylallyl Cation Intermediate in an Aromatic Prenyltransferase Reaction

Dimethylallyltryptophan synthase is an aromatic prenyltransferase that catalyzes an electrophilic aromatic substitution reaction between dimethylallyl diphosphate (DMAPP) and L-tryptophan. The synthase is found in a variety of fungi, where it catalyzes the first committed step in the biosynthesis of the ergot alkaloids. The enzymatic reaction could follow either a dissociative mechanism involving a discrete dimethylallyl cation intermediate or an associative mechanism in which the indole ring directly displaces diphosphate in a single step. In this work, positional isotope exchange (PIX) experiments are presented in support of a dissociative mechanism. When [1-(18)O]-DMAPP is subjected to the synthase reaction and recovered starting material is analyzed, 15% of the (18)O-label is found to have scrambled from a bridging to a nonbridging position on the alpha-phosphorus. Kinetic isotope effect studies show that steps involved in the formation of the arenium ion intermediate are rate-determining, and therefore the scrambling occurs during the lifetime of the dimethylallyl cation/diphosphate ion pair. Similarly, when the unreactive substrate analogue, 6-fluorotryptophan, was employed, complete scrambling of the (18)O-label in DMAPP was observed. To our knowledge, this is the first observation of PIX in any prenyltransferase reaction, and it provides strong evidence supporting the existence of a carbocation intermediate.

The NeuC Protein of Escherichia coli K1 Is a UDP N-Acetylglucosamine 2-Epimerase

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.

The Engineering of Bacteria Bearing Azido-Pseudaminic Acid-Modified Flagella

Catch a tiger by the tail: 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.

PseG of pseudaminic acid biosynthesis: A UDP-sugar hydrolase as a masked glycosyltransferase

The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon α-keto acid, pseudaminic acid. The presence of this posttranslational modification is absolutely required for assembly of functional flagella. Since motility is required for colonization, pseudaminic acid biosynthesis represents a virulence factor in these bacteria. Pseudaminic acid is generated from UDP-N-acetylglucosamine in five biosynthetic steps. The final step has been shown to involve the condensation of 2,4-diacetamido-2,4,6-trideoxy-l-altrose (6-deoxy-Altdi-NAc) with phosphoenolpyruvate as catalyzed by the enzyme pseudaminic acid synthase, NeuB3. The 6-deoxy-AltdiNAc used in this process is generated from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This manuscript describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be kcat = 27 s–1 and Km = 174 μm. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C-1 and to utilize a C–O bond cleavage mechanism during catalysis. These results, coupled with homology comparisons, suggest that the closest ancestors to the hydrolase are members of the metal-independent GT-B family of glycosyltransferases that include the enzyme MurG.

5'-Methylthioadenosine nucleosidase is implicated in playing a key role in a modified futalosine pathway for menaquinone biosynthesis in campylobacter jejuni

Menaquinone (vitamin K2) serves as an electron carrier in the electron transport chain required for respiration in many pathogenic bacteria. Most bacteria utilize a common menaquinone biosynthetic pathway as exemplified by Escherichia coli. Recently, a novel biosynthetic pathway, the futalosine pathway, was discovered in Streptomyces. Bioinformatic analysis strongly suggests that this pathway is also operative in the human pathogens Campylobacter jejuni and Helicobacter pylori. Here, we provide compelling evidence that a modified futalosine pathway is operative in C. jejuni and that it utilizes 6-amino-6-deoxyfutalosine instead of futalosine. A key step in the Streptomyces pathway involves a nucleosidase called futalosine hydrolase. The closest homolog in C. jejuni has been annotated as a 5′-methylthioadenosine nucleosidase (MTAN). We have shown that this C. jejuni enzyme has MTAN activity but negligible futalosine hydrolase activity. However, the C. jejuni MTAN is able to hydrolyze 6-amino-6-deoxyfutalosine at a rate comparable with that of its known substrates. This suggests that the adenine-containing version of futalosine is the true biosynthetic intermediate in this organism. To demonstrate this in vivo, we constructed a C. jejuni mutant strain deleted for mqnA2, which is predicted to encode for the enzyme required to synthesize 6-amino-6-deoxyfutalosine. Growth of this mutant was readily rescued by the addition of 6-amino-6-deoxyfutalosine, but not futalosine. This provides the first direct evidence that a modified futalosine pathway is operative in C. jejuni. It also highlights the tremendous versatility of the C. jejuni MTAN, which plays key roles in S-adenosylmethionine recycling, the biosynthesis of autoinducer molecules, and the biosynthesis of menaquinone.