ChemBioChem

The cover picture shows 3D models of the pyranose rings of UDP-sugar metabolites that were identified by examining the PseB reaction from Helicobacter pylori by using NMR spectroscopy. PseB plays an important role in the biosynthesis of pseudaminic acid (Pse)—a sialic acid-like sugar that is found on the flagella of H. pylori. Pse is essential for bacterial motility and as such is a virulence factor. Since, it is not found in humans, agents that interfere with Pse biosynthesis could offer therapeutic potential. Several bacteria produce sugars that are attractive therapeutic targets. However, their biosynthetic pathways can involve multiple enzymatic reactions that produce unstable metabolites in minute quantities. Examination of the WbjB and WbjC reactions from Pseudomonas aeruginosa, the PglF reaction from C. jejuni and the PseB reaction from C. jejuni and H. pylori with NMR clarified biosynthetic pathways led to the identification of unstable metabolites that had not been previously observed. Further details can be found in the article by D. McNally et al. on p. 1865 ff.

Creating new catalytic function in proteins. Anchoring an organometallic moiety within a protein affords artificial metalloenzymes for enantioselective catalysis. Both chemical and genetic tools can be applied in the optimization of such systems, which lie at the interface between homogeneous and enzymatic catalysis. This minireview presents the latest developments in the field of artificial metalloenzymes.

Getting to heme. A system for the incorporation of unnatural amino acid into cytochrome c₃ by site-specific viologen modification has been established. A viologen was linked to the side chain of the unnatural amino acid, and cytochrome c₃ accepted an electron through the linked viologen.

A methodology allowing the differentiation of azido groups in protected oligosaccharides was optimized. It allowed the first synthesis of tetrasaccharides 1 and 2 containing both N-acetylated and N-unsubstituted glucosamine, as well as glucuronic and/or iduronic acid residues. These tetrasaccharides were then examined for their ability to interact with 10E4, an antibody recognizing early lesions in the brain of scrapie prion-infected mice.

A 23-fold enhancement of fluorescence is observed upon RabGGTase-mediated protein prenylation by NBD-FPP. We propose that the chaperone of prenylated Rab GTPases, REP, which harbors the conjugated prenyl moieties, functions as a fluorescence amplifier and leads to intermolecular fluorescence enhancement. This reaction was characterized and used to develop a fluorescent prenylation assay that can be adapted for a high-throughput format.

Bigger and bigger. A novel, isothermal DNA detection method has been developed that contains two successive amplifications and integrates both a protein enzyme and a DNA enzyme. The current detection limit is 1 pM.

UDP-hexos-4-ulose sugars occupy a central role in the biosynthesis of lipopolysaccharides, capsular polysaccharides, and antibiotics in bacteria. Little is known about these sugars as they are labile compounds that are produced in minute quantities within the bacterial cell. Examination of the PglF, PseB, WbjB, and WbjC reactions directly with NMR lead to the first detailed structural description of UDP-hexos-4-ulose sugars in various bacteria.

Using the yeast expression system of ATX, a 6-methylsalicylic acid synthase from Aspergillus terreus, reconstitution of active catalytic reaction centers was examined by coexpression of inactive N-terminal and C-terminal deletion mutants. By a series of coexpression experiments, the interdomain (ID) region of 122 amino acids was identified, between the dehydratase and ketoreductase domains, which is required for the subunit–subunit interaction of ATX.

Phages infecting the polySia-encapsulated human pathogenE. coli K1 are equipped with capsule-degrading tail spikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. The X-ray crystallographic structure of endosialidase has been reported but it remains unclear how polySia interacts with the active site. Here we report STD and trNOE NMR experiments that investigate the binding mode of polySia DP5 at a molecular level.

Knowledge gap reduced. Although this enzyme is responsible for the two-electron reduction of nitric oxide to nitrous oxide, our knowledge of the sensitive, membrane-bound nitric oxide reductase is less than that of the other enzymes of the denitrification pathway. In this communication, we report the first successful direct electrochemical experiments to reveal relevant catalytic aspects of this interesting and important enzyme.

Tryptamine trial. Chemical microarrays have been used to identify ligands that recognize the surface of a variety of pathogenic organisms. In each case, the highest affinity ligand is tryptamine, which is shown to recognize cell surfaces when multivalently displayed on a microarray surface or on a water-soluble polymer.

Propagation of conformational dynamics in rubredoxin is characterized by interchanging a set of residues for which the increase in the hydrogen exchange-active local fluctuations of the hyperthermophile-derived hybrid is faithfully mirrored by a corresponding decrease for the complementary hybrid derived from a less thermostable rubredoxin.

RNA-templated chemistry to discriminate pathogenic bacteria. A new variant of FRET quenched autoligation (QUAL) probes is described; in the new design, electrophilic QUAL probes contain both a quencher and fluorescein or a fluorescein-tetramethylrhodamine FRET pair; RNA-templated ligation with nucleophilic phosphorothioate probes yields a green or red signal with a single excitation. We test the probes in discrimination of pathogenic strains of bacteria.

Hepatitis C virus (HCV) infection is associated with changes in host-cell lipid metabolism. Here we describe a new approach for detecting HCV RNA using two-photon fluorescence (TPF), and HCV-associated changes in cellular lipids using coherent anti-Stokes Raman scattering (CARS) microscopy. By combining the two types of microscopy with a common laser source, we visualized both phenomena simultaneously and profiled cellular lipids and subcellular localization of RNA in real time.

The ins and outs of ring cleavage. Directed evolution studies on extradiol catechol dioxygenase MhpB from Escherichia coli by error-prone PCR have yielded two mutants (R215W and K273R, see image) that were found to produce a mixture of extradiol and intradiol oxidative ring-cleavage products.

REplacement with Partial Ligand Alternatives through Computational Enrichment is a structure-guided design method that permits the stepwise and iterative introduction of nonpeptidic and drug-like fragments into a peptide ligand. Here REPLACE is exemplified in the redesign of cyclin groove inhibitors of the cyclin-dependent kinase 2–cyclin A complex.

Unlucky for some. Leukemia and lymphoma cell lines are especially susceptible to the apoptosis-inducing effects of compound 13-D. This compound has no effect on noncancerous cell types or on other cancer cell lines.

The genetic way. Protein analytes can be detected by stochastic sensing in which the modulation of a current flowing through a single protein pore is observed. In previous work, proteins have been detected with pores to which ligands were appended by targeted chemical modification. We now report the first genetically encoded stochastic sensor element for detecting a protein: an α-hemolysin pore containing a single copy of a protein kinase inhibitor sequence. This development should facilitate the rapid screening of kinase inhibitors.

Shaping up. Molecular modelling of benzaldehyde lyase from Pseudomonas fluorescens and benzoylformate decarboxylase from Pseudomonas putida showed that the differences in the shape of the two binding sites can explain the experimentally observed differences in activity, substrate specificity and stereoselectivity between the two enzymes. The model also demonstrated the molecular basis of effects observed with previously reported mutations.

The chain gang. Several unusual features of biosynthesis on the mycolactone (see scheme) polyketide synthase (PKS) challenge conventional views on the control of chain growth. Our studies on recombinant ketoreductase domains derived from the PKS provide insight into both substrate recognition and stereocontrol by this system.

A synthetic antimalarial protein presenting a native-like fold. A virosomally formulated, synthetic EGF-like domain from P. falciparum MSP-1, which is shown by NMR to adopt a native-like fold, elicits antibodies in mice that cross-react with the native protein on mature schizonts. This opens a medicinal chemistry-oriented approach to optimization of the antigenicity of the protein as a potential malaria vaccine candidate.

The AT and ACP catalytic domains of the type I iterative polyketide synthase (PKS) involved in norsolorinic acid biosynthesis in A. parasiticus were cloned and expressed. These were active in PKS assays with type II enzymes of actinorhodin biosynthesis from S. coelicolor, thus indicating the close functional similarity of PKS proteins from fungi and bacteria.

Chirality takes a back seat. The unnatural disaccharide L-trehalose is an equally remarkable cryopreservant of yeast cells as D-trehalose, whereas the achiral meso-trehalose is far less effective (see bar graph). Mirror-image carbohydrates can be useful for probing biological functions that depend on relative rather than absolute stereochemistry.

Sugar-substituted. A route has been developed for the synthesis of a light-sensitive oligonucleotide in which the light-sensitive functionality is attached at the sugar moiety rather than to the backbone or the nucleobase (see scheme). The utility of this modified nucleotide in photoaffinity labeling of human DNA polymerase β has been demonstrated.

It takes two. Oligodeoxynucleotide duplexes containing two or more consecutive 1,8-naphthyridine C-nucleoside (Na-N^O) and imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (Im-O^N) pairs at their sticky ends were markedly stabilized thermally. Furthermore, it was demonstrated that this duplex acted as a potent NF-κB decoy molecule.

Unique potential for Sec-targeted labeling. While dithiol or selenolthiol tags offer the promise of highly useful capabilities for one-step purification of tagged proteins over PAO sepharose, we consider the most important result in this study to be the superior targeting of Sec components in selenolthiol motifs with electrophilic agents, and—specifically—labeling with ¹¹C.

Metabolic chess. Larvae of the cabbage white butterfly (Pieris rapae), important pests in Europe and North America, feed exclusively on plants chemically defended by the glucosinolate–myrosinase system. We studied how P. rapae larvae overcome this defence system by investigating the metabolic fate of one particular glucosinolate by using feeding experiments with isotopic tracers. (Photograph of P. rapae larvae feeding on Tropaeolum majus by Danny Keßler.)

Glowing, glowing, gone. 5-Phenyl-indolyl-2′-deoxyriboside triphosphate (5-PhITP) is a fluorescent, non-natural nucleotide that is selectively incorporated opposite an abasic site. The observed rate constant in fluorescence quenching increases as a function of 5-PhITP concentration.

Insertion of lysophosphatidylcholine (LPC) or arachidonic acid (AA) into the cell membrane outer leaflet induces significant effects on the exocytosis process at chromaffin cells. That LPC favors the events (frequency, kinetics) while AA disfavors them is because of their shapes. Moreover, the amount of catecholamines released during the process depends on the inserted compound.

Chemical rescue of the cavity mutant of OxdB. The enzymatic activity of the H282G mutant of phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB) was rescued by imidazole or pyridine derivatives that acted as the exogenous proximal ligand (see scheme). By changing the electron-donation ability of the exogenous ligand with different substituents, the enzymatic activity could be regulated systematically.

Suspected nongenotoxic carcinogens with benzenoid structures have apoptosis inhibitory activity in C. elegans. This subclass of benzenoid chemicals might promote tumorigenesis by suppressing apoptosis. These findings establish C. elegans as an alternative animal model for assaying the antiapoptotic activity of tumor-promoting chemicals.

Searching for SAM: A 55 kb gene cluster for the biosynthesis of furanonaphthoquinone I (see scheme) and prenylated phenazines has been identified and analyzed. The biosynthetic functions of a prenyltransferase gene and two methyltransferase genes were confirmed experimentally. The cluster contains five genes that together encode all the enzymatic functions required for S-adenosylmethionine (SAM) biosynthesis.