Frank M. Raushel

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The bacterium is commensal in poultry, and the ingestion of contaminated or undercooked chicken products can result in rather severe cases of nausea, abdominal pain, and diarrhea. Protecting the bacterium from the host immune system is a thick polysaccharide coat known as the capsular polysaccharide. The repeating disaccharide unit in the undecorated capsular polysaccharide from the HS:19 serotype of C. jejuni consists of N-acetyl-d-glucosamine (GlcNAc) and the serinol amide of d-glucuronic acid (GlcA). It was demonstrated that the GT2 glycosyltransferase from the N-terminal domain of HS19.08 catalyzes the transfer of d-glucuronic acid from UDP-GlcA to C3 of the GlcNAc moiety at the nonreducing end of the growing polysaccharide chain. The carboxylate of the added GlcA is then amidated by the ATP-dependent ligase activity of the C-terminal domain of HS19.11 using (S)-serinol-phosphate. The phosphorylated serinol amide is subsequently dephosphorylated by the catalytic activity of HS19.09. In the final step of the series, the GT2 glycosyltransferase from the N-terminal domain of HS19.11 catalyzes the transfer of GlcNAc from UDP-GlcNAc to C4 of the amidated glucuronic acid. This series of reactions has been exploited to enzymatically synthesize and isolate defined oligomers containing 2, 3, 5, and 7 monomeric units, and procedures were developed for the synthesis of oligomeric mixtures ranging from 9 to 15 monomeric units.

Chemical potentials (molar Gibbs energies) are usually extrapolated to the remote physical–chemical reference state and then stored. Subsequent use under in vivo conditions requires a similarly substantial, reverse extrapolation, again with significant potential errors. In order to shrink both extrapolations drastically and thereby enhance both biological meaning and accuracy, we propose a transformation to a more biological reference state: pH = 7, pMg = 3, 99.5% water, with 1 m m each of the additional ‘precursors’ inorganic phosphate, sulfate, ammonium, and bicarbonate, and with twin temperatures 37 and 25 °C, ionic strength 0.15 m and m m as concentration unit. These precursors substitute for reference compounds alien to biology such as H ₂ at 1 bar, and solid graphite, sulfur, and phosphorus. The standard chemical potentials are herewith increased by the magnitudes of the chemical potentials of protons, Mg ²⁺ , water, and the four precursors, each multiplied by the number of corresponding atoms in the molecule. This defines standard ‘metabolic potentials’. We make these potentials findable and accessible as 1360 collated standard chemical potentials for 320 compounds of biochemical interest at the twin metabolic reference states. We do this for 3 reference pH's: We present the metabolic reference state as a convenient anchor , not a universal intracellular milieu. All datasets must continue to report the actual experimental state ( T , pH, pMg, I , osmolarity, concentrations), yet aim at (also) reporting parameter values for this anchor state; we supply algorithms to transform between states. This preserves interoperability across diverse organelles, media and between enzymology and chemical engineering, while facilitating reuse.

The critical need to decontaminate surfaces from highly toxic chemical warfare agents (CWA) and the complexity of interfacial domains underscore the challenges associated with developing efficient surface cleanup technologies. To address these challenges and needs, we describe a light-switchable surface targeting microrobotic decontamination platform for accelerated biocatalytic degradation of surface-bound CWA. Biohybrid microrobots, based on Chlamydomonas reinhardtii algae, functionalized with the organophosphate-degrading enzyme phosphotriesterase (PTE), are shown to display unique light-switchable surface adhesion and gliding behaviors to offer an extremely efficient and fast surface decontamination. These biohybrid algae-PTE microrobots reversibly target hard-to-reach contaminated surfaces under blue light irradiation, while red light triggers their transition back to the bulk solution. In the surface-bound state, PTE-functionalized algae can glide collectively across the contaminated surface toward enhanced degradation efficiency. Upon completing the surface decontamination, the algae-PTE microrobots are easily released from the surface by switching to red irradiation. The applicability of the light-switchable microrobotic platform is demonstrated using a variety of contaminated surfaces, including fabrics, skin, and irregular solid surfaces, displaying surface-decontamination efficiencies higher than 90% within 5 min. Such highly efficient and rapid light-switchable surface targeting biocatalytic microrobotic platform holds considerable promise for diverse environmental surface remediation applications.

The exterior surface of the human pathogen Campylobacter jejuni is coated with a capsular polysaccharide (CPS) that helps protect it from the host immune system. In C. jejuni NCTC 11168 the repeating linear polysaccharide is composed of d-ribose, N-acetyl-d-galactosamine and d-glucuronic acid that is further amidated with either ethanolamine or serinol. The CPS is also decorated with d-glycero-l-gluco-heptose and methyl phosphoramidate. We have now shown that the polymerization of the undecorated CPS requires the sequential activity of six unique enzymes that must act in concert with one another. The catalytic activity of these six enzymes enabled a robust synthetic strategy to be developed to facilitate the assembly and isolation of specific oligosaccharides of up to 10 units in length. Modifications to this strategy enabled the isolation of mixtures containing oligosaccharides containing at least 19 monomeric units. The oligosaccharides were isolated by anion exchange chromatography and chemically characterized using ESI mass spectrometry and ¹H NMR spectroscopy. These oligosaccharides will enable the reaction mechanisms for the decoration of the capsular polysaccharides to be determined and may facilitate the development of glycoconjugate vaccines.

Campylobacter jejuni is a Gram-negative human pathogen and is the most common cause of gastroenteritis in the United States and Europe. C. jejuni expresses a capsular polysaccharide (CPS) that enables the evasion of the host immune response and adherence to host epithelial cells. The various subspecies (serotypes) of C. jejuni are distinguished by their unique CPS repeating units. The repeating trisaccharide in the HS:41 serotype was previously found to contain l-arabinofuranose, 6-deoxy-d-altro-heptofuranose, and a third sugar, which is either d-fucofuranose or 6-deoxy-l-altrofuranose. Genome neighborhood and sequence similarity networks were employed to identify five candidate genes for the biosynthesis of d-fucofuranose and 6-deoxy-l-altrofuranose. Here, it was demonstrated that the biosynthetic pathways for both sugars are initiated by the formation of CDP-d-glucose from CTP and d-glucose-1-phosphate as catalyzed by HS41.21. This product is dehydrated by an NAD⁺-dependent 4,6-dehydratase (HS41.20) that produces CDP-4-keto-6-deoxy-d-glucose. The third enzyme (HS41.19) was shown to catalyze the inversion of stereochemistry at C3 and C5 using CDP-4-keto-6-deoxy-d-glucose as the substrate. The fourth enzyme (HS41.18) is an NADPH-dependent C4-reductase that catalyzes the formation of CDP-d-fucopyranose from CDP-4-keto-6-deoxy-d-glucose in the absence of the 3,5-epimerase but catalyzes the formation of CDP-6-deoxy-l-altropyranose in the presence of the epimerase. The last enzyme (HS41.17) in the pathway was shown to catalyze the FADH₂-dependent interconversion of CDP-d-fucopyranose and CDP-6-deoxy-l-altropyranose to CDP-d-fucofuranose and CDP-6-deoxy-l-altrofuranose, respectively. The overall synthesis of the two possible products is governed by the catalytic activity of the epimerase, since the C4-reductase and pyranose-furanose mutase are not affected by the stereochemistry at C5.

Campylobacter jejuni is the leading cause of food poisoning in the United States. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) that helps protect the organism from the host immune system. In the HS:2 serotype of strain C. jejuni NCTC 11168, the minimal repeating trisaccharide consist of d-ribose, N-acetyl-d-galactosamine (GalNAc) and the serinol amide of d-glucuronic acid. Here we demonstrate that the C-terminal domain of Cj1432 (residues 574-914) is responsible for the transfer of d-ribose-5-P from phosphoribosyl pyrophosphate (PRPP) to C5 of the d-GalfNAc moiety of the growing polysaccharide chain. In the next step the middle domain of Cj1432 (residues 357-573) catalyzes the hydrolysis of phosphate from this product. The N-terminal domain of Cj1432 (residues 1-356) catalyzes the transfer of d-GlcA from UDP-d-GlcA to C2 of the d-ribose moiety and thus Cj1432 catalyzes three consecutive reactions during the biosynthesis of the capsular polysaccharide of C. jejuni. We have previously shown that the remaining three reactions required for the polymerization of the CPS are catalyzed by the bifunctional enzyme Cj1438 and Cj1435. We have now demonstrated that the minimal repeating trisaccharide of the CPS of C. jejuni NCTC 11168 requires six enzyme-catalyzed reactions with six intermediate structures. This accomplishment will now enable the large-scale cell-free enzyme-catalyzed synthesis of well-defined oligomers of the CPS that can potentially be used in the production of glycoconjugate vaccines for the prevention of infections by C. jejuni.

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in poultry and is the leading cause of gastrointestinal infections in the United States. Similar to other Gram-negative bacteria, C. jejuni possesses an extracellular carbohydrate-based capsular polysaccharide (CPS) composed of repeating units of monosaccharides bound via glycosidic linkages. The gene cluster for serotype 1 (HS:1) of C. jejuni contains 13 different genes required for the production and presentation of the CPS. Each repeating unit within the HS:1 CPS structure contains a backbone of glycerol phosphate and d-galactose. Here, the enzyme HS1.11 was shown to catalyze the formation of CDP-(2R)-glycerol from MgCTP and l-glycerol-3-phosphate. HS1.09 was found to be a multidomain protein that catalyzes the polymerization of l-glycerol-3-phosphate and d-galactose using UDP-d-galactose and CDP-(2R)-glycerol as substrates. The domain of HS1.09 that extends from residues 286 to 703 was shown to catalyze the transfer of l-glycerol-P from CDP-glycerol to the hydroxyl group at C4 of the d-galactose moiety at the nonreducing end of the growing oligosaccharide. The transfer of d-galactose to the C2 hydroxyl group of the glycerol-phosphate moiety was shown to be catalyzed with retention of configuration by the domain of HS1.09 that extends from residues 704 to 1095. Primers as short as a single d-galactoside were accepted as initial substrates. Oligosaccharide products were isolated by ion exchange chromatography and identified by high-resolution ESI-mass spectrometry and NMR spectroscopy.

The external control of catalytic activity and substrate specificity of enzymes by light has aroused great interest in the fields of biocatalysis and pharmacology. Going beyond, we have attempted to photocontrol enzyme stereoselectivity on the example of phosphotriesterase (PTE), which is capable of hydrolyzing a wide variety of racemic organophosphorus substrates where one of two enantiomers is often highly toxic. To pursue this goal, the photocaged unnatural amino acid o-nitrobenzyl-l-tyrosine (ONBY) was incorporated by genetic code expansion at the large subsite of the active center, together with additional mutations at the small subsite. The stereoselectivities of the resulting PTE variants were tested with the achiral control substrate paraoxon and four different racemic substrates, which contained a p-nitrophenol leaving group in combination with either methyl-phenyl, ethyl-phenyl, methyl-cyclohexyl, or ethyl-cyclohexyl substituents. Comparison of the enantioselectivities (k _cat/K _M for S_p divided by k _cat/K _M for R_p) before and after decaging of ONBY using irradiation revealed the desired photoinduced inversion of enantioselectivity for three of the variants: PTE_I106A-H257ONBY exhibited a 43-fold stereoselectivity switch for the methyl-phenyl substrate, PTE_I106A-F132A-H257ONBY a 184-fold stereoselectivity switch for the methyl-cyclohexyl substrate, and PTE_I106A-F132A-S308A-H257ONBY a 52-fold and a 57-fold stereoselectivity switch for the methyl-cyclohexyl and the ethyl-cyclohexyl substrates. A computational analysis including molecular dynamics simulations and docking showed that a complicated interplay between steric constraints and specific enzyme-substrate interactions is responsible for the observed effects. Our findings significantly broaden the scope of possibilities for the spatiotemporal control of enantioselective transformations using light in biocatalytic systems.

Campylobacter jejuni is the leading cause of food poisoning in Europe and North America. The exterior surface of this bacterium is encased by a capsular polysaccharide that is attached to a diacyl glycerol phosphate anchor via a poly-Kdo (3-deoxy-d-manno-oct-2-ulosinic acid) linker. In the HS:2 serotype of C. jejuni NCTC 11168, the repeating trisaccharide consists of d-ribose, N-acetyl-d-glucosamine, and d-glucuronate. Here, we show that the N-terminal domain of Cj1432 (residues 1-356) is responsible for the reaction of the C2 hydroxyl group from the terminal d-ribose moiety of the growing polysaccharide chain with UDP-d-glucuronate as the donor substrate. This discovery represents the first biochemical identification and functional characterization of a glycosyltransferase responsible for the polymerization of the capsular polysaccharide of C. jejuni. The product of the reaction catalyzed by the N-terminal domain of Cj1432 is the substrate for the reaction catalyzed by the C-terminal domain of Cj1438 (residues 453-776). This enzyme catalyzes amide bond formation using the C6 carboxylate of the terminal d-glucuronate moiety and (S)-serinol phosphate as substrates. It is also shown that Cj1435 catalyzes the hydrolysis of phosphate from the product catalyzed by the C-terminal domain of Cj1438. These results demonstrate that amide decoration of the d-glucuronate moiety occurs after the incorporation of this sugar into the growing polysaccharide chain.