Michael E. Webb

Thiamine biosynthesis in algae is regulated by riboswitches

In bacteria, many genes involved in the biosynthesis of cofactors such as thiamine pyrophosphate (TPP) are regulated by ribo switches, regions in the 5′ end of mRNAs to which the cofactor binds, thereby affecting translation and/or transcription. TPP riboswitches have now been identified in fungi, in which they alter mRNA splicing. Here, we show that addition of thiamine to cultures of the model green alga Chlamydomonas reinhardtii alters splicing of transcripts for the THI4 and THIC genes, encoding the first enzymes of the thiazole and pyrimidine branches of thiamine biosynthesis, respectively, concomitant with an increase in intracellular thiamine and TPP levels. Comparison with Volvox carteri, a related alga, revealed highly conserved regions within introns of these genes. Inspection of the sequences identified TPP riboswitch motifs, and RNA transcribed from the regions binds TPP in vitro. The THI4 riboswitch, but not the promoter region, was found to be necessary and sufficient for thiamine to repress expression of a luciferase-encoding reporter construct in vivo. The pyr1 mutant of C. reinhardtii, which is resistant to the thiamine analogue pyrithiamine, has a mutation in the THI4 riboswitch that prevents the THI4 gene from being repressed by TPP. By the use of these ribo switches, thiamine biosynthesis in C. reinhardtii can be effectively regulated at physiological concentrations of the vitamin.

Oligobenzamide proteomimetic inhibitors of the p53–hDM2 protein–protein interaction

Developing general strategies for inhibition of protein–protein interactions is a key challenge in chemical biology. Herein we describe oligobenzamide inhibitors of the p53–hDM2 protein–protein interaction.

Structural Constraints on protein self-processing in L-aspartate-alpha-decarboxylase

Aspartate decarboxylase, which is translated as a pro‐protein, undergoes intramolecular self‐cleavage at Gly24–Ser25. We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24→Ser, His11→Ala, Ser25→Ala, Ser25→Cys and Ser25→Thr mutants. Comparative analyses of the cleavage site reveal specific conformational constraints that govern self‐processing and demonstrate that considerable rearrangement must occur. We suggest that Thr57 Oγ and a water molecule form an ‘oxyanion hole’ that likely stabilizes the proposed oxyoxazolidine intermediate. Thr57 and this water molecule are probable catalytic residues able to support acid–base catalysis. The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction. The molecular mechanism of self‐processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate. Comparison of the structural features shows significant similarity to those in other self‐processing systems, and suggests that models of the cleavage site of such enzymes based on Ser→Ala or Ser→Thr mutants alone may lead to erroneous interpretations of the mechanism.

The relevance of nanoscale biological fragments for ice nucleation in clouds

Most studies of the role of biological entities as atmospheric ice-nucleating particles have focused on relatively rare supermicron particles such as bacterial cells, fungal spores and pollen grains. However, it is not clear that there are sufficient numbers of these particles in the atmosphere to strongly influence clouds. Here we show that the ice-nucleating activity of a fungus from the ubiquitous genus Fusarium is related to the presence of nanometre-scale particles which are far more numerous, and therefore potentially far more important for cloud glaciation than whole intact spores or hyphae. In addition, we quantify the ice-nucleating activity of nano-ice nucleating particles (nano-INPs) washed off pollen and also show that nano-INPs are present in a soil sample. Based on these results, we suggest that there is a reservoir of biological nano-INPs present in the environment which may, for example, become aerosolised in association with fertile soil dust particles.

Elucidating biosynthetic pathways for vitamins and cofactors

The elucidation of the pathways to the water-soluble vitamins and cofactors has provided many biochemical and chemical challenges. This is a reflection both of their complex chemical nature, and the fact that they are often made in small amounts, making detection of the enzyme activities and intermediates difficult. Here we present an orthogonal review of how these challenges have been overcome using a combination of methods, which are often ingenious. We make particular reference to some recent developments in the study of biotin, pantothenate, folate, pyridoxol, cobalamin, thiamine, riboflavin and molybdopterin biosynthesis.

Efficient N‐Terminal Labeling of Proteins by Use of Sortase

Protein labeling is a pivotal technique in molecular and cell biology. Strategies include derivatization of cysteine residues, labeling lysine or N-terminal amino groups with activated esters, periodate or PLP-mediated oxidation of the N-terminus for oxime ligation, and native-chemical ligation. Each of these methods has its own associated challenges: Selective labeling of a single cysteine residue frequently requires rounds of site-directed mutagenesis to introduce the labeling site and/or remove other cysteine residues, and selective labeling of the N-terminal amino group requires careful control of pH to ensure lysine residues are not also modified. Other modern methods for chemoselective labeling often require the introduction of specific recognition sequences or nonnatural amino acids into the protein to be labeled. In most cases, a substantial excess of the labeling reagent is necessary to ensure complete conversion to the product. We report a method for chemoselective N-terminal labeling of recombinant proteins in quantitative yield using depsipeptide substrates for the transpeptidase sortase A. The method does not require engineering of the protein sequence beyond that typically used in contemporary recombinant protein purification strategies. Unlike previous approaches, the method requires only a single N-terminal glycine residue in a sterically unhindered position, a minimal excess of the labeling reagent and substoichiometric quantities of transpeptidase. Sortase A (SrtA) catalyzes the reversible attachment of virulence factors to the cell walls of Gram positive bacteria by C-terminal modification of proteins at an LPXTG recognition sequence. The enzyme catalyzes the covalent attachment of the LPXT motif to a cysteine residue in the catalytic site to form a thioester intermediate. An N-terminal oligoglycine motif in the peptidoglycan can then react with this intermediate to covalently attach the substrate to the cell wall. SrtA has been exploited extensively for the C-terminal modification of proteins. 9] However, this method has certain constraints: the LPXTG sequence must be engineered into the protein and excess nucleophilic labeling reagent is required to push the equilibrium toward formation of product as the transpeptidase reaction is reversible (Scheme 1 a).

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B1 and B2. The cDNAs encoding functional B2 receptors have recently been cloned, but no molecular information exists at present on the B1 receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B1 and B2 types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B1 and B2 receptors. In this report, we describe oocyte expression experiments showing that the B1 receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B2 receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B2 sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B1‐type pharmacology may arise from two independent molecular mechanisms.

Expression of Functional Bradykinin Receptors in Xenopus Oocytes

Abstract: mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108‐15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 μM), with an initial inward current (10–20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 μM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 μM). mRNA from both NG108–15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately fivefold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108‐15 cells.

Triazole phosphohistidine analogues compatible with the Fmoc-strategy

Phosphorylation of histidine is essential for bacterial two-component signalling; its importance to modulation of eukaryotic protein function remains undefined. Until recently, no immunochemical probes of this post-translational modification existed, however triazole phosphonate analogues of this modified amino acid have now been applied to the generation of site-specific antibodies. The protecting group strategy used in the original report is incompatible with standard protocols for Fmoc-solid phase peptide synthesis. In this paper, we report the application of P(III) chemistry to generate the complementary dibenzyl and di-tert-butyl phosphonate esters. These forms of the triazole analogue are fully compatible with standard Fmoc-SPPS and are therefore ideal for wider application by the chemical and biochemical community.

An activator for pyruvoyl-dependent l-aspartate α-decarboxylase is conserved in a small group of the γ-proteobacteria including Escherichia coli.

In bacteria, β‐alanine is formed via the action of l‐aspartate α‐decarboxylase (PanD) which is one of the small class of pyruvoyl‐dependent enzymes. The pyruvoyl cofactor in these enzymes is formed via the intramolecular rearrangement of a serine residue in the peptide backbone leading to chain cleavage and formation of the covalently‐bound cofactor from the serine residue. This reaction was previously thought to be uncatalysed. Here we show that in Escherichia coli, PanD is activated by the putative acetyltransferase YhhK, subsequently termed PanZ. Activation of PanD both in vivo and in vitro is PanZ‐dependent. PanZ binds to PanD, and we demonstrate that a PanZ(N45A) site‐directed mutant is unable to enhance cleavage of the proenzyme PanD despite retaining affinity for PanD. This suggests that the putative acetyltransferases domain of PanZ may be responsible for activation to enhance the processing of PanD. Although panD is conserved among most bacteria, the panZ gene is conserved only in E. coli‐related enterobacterial species including Shigella, Salmonella, Klebsiella and Yersinia. These bacteria are found predominantly in the gut flora where pantothenate is abundant and regulation of PanD by PanZ allows these organisms to closely regulate production of β‐alanine and hence pantothenate in response to metabolic demand.