Chemical biology

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Chemical biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in chemical biology. Current Opinion in Chemical Biology 2000, 4:125–134 Contents (chosen by) 125 Interaction, assembly and processing (Conn and Kappock) 126 Biocatalysis and biotransformation (Pohl) 127 Bio-inorganic chemistry (Cammack) 128 Combinatorial chemistry (Conn and Hall) 130 Next generation therapeutics (Projan) 130 Analytical techniques (Cass) 131 Mechanisms (Stewart) 132 Model systems (Cousins, Sanders) 133 Biopolymers (Flitsch, Lowden and Newman)

• ••

of special interest of outstanding interest

Interaction, assembly and processing Selected by M Morgan Conn Amherst College, Amherst, Massachusetts, USA

“Synapsable” DNA double helices: self-selective modules for assembling DNA superstructures. Fahlman RP, Sen D: J Am Chem Soc 1999, 121:11079-11085. • Significance: The relative rigidity of a DNA double helix and the structural possibilities of interstrand base pairing make DNA a possibly interesting material from which to construct well-ordered objects. Findings: The authors describe the use of interstrand guanine quartets (G-quartets) between regions containing G·G mismatches to assemble a pair of double helices into a stable dimer. This motif is proposed as an alternative to the base-pairing motif used in previous DNA junctions. In this paper, the authors show that differently sized runs of G·G mismatches preferentially dimerise with strands containing mismatch runs of identical length. This specificity should open the path for the construction of complicated three-dimensional DNA arrays. DNA computing on surfaces. Liu Q, Wang L, Frutos AG, Condon AE, Corn RM, Smith LM: Nature 2000, 403:175-179. •• Significance: This work shows that it is possible to use DNA hybridization techniques to solve more difficult mathematical problems. This technique shows some promise to solve massively complex problems more efficiently than traditional computing. Findings: DNA computing was used to solve a satisfiability problem that requires several statements to be simultaneously true, (i.e. that [(w OR x OR y) AND (w OR not y OR z) AND (not x OR y) AND (not w OR not y)] be simultaneously true). DNA oligomers corresponding to all possible combinations of the binary variables (0 or 1 corresponding to false or true, respectively) were laid on a solid surface. The oligomers that satisfied the first condition (w OR x OR y) were annealed to the library of all answers. DNA that remain unannealed (i.e. those

sequences that did not satisfy the first condition) was decomposed by Escherichia coli exonuclease I. Double-stranded DNA was denatured and the procedure repeated with oligomers for the remaining conditions. At the end of the sequence, the DNA remaining was amplified by polymerase chain reaction and sequenced. The number of cycles required to solve this problem scales linearly with the number of conditions in the AND sequence, rather than exponentially as is seen in traditional computational analysis of these problems. Inhibition of human telomerase in immortal human cells leads to progressive telomer shortening and cell death. Herbert BS, Pitts AE, Baker SI, Hamilton SE, Wright WE, Shay JW, Corey DR: Proc Natl Acad Sci USA 1999, 96:14276-14281. • Significance: Human telomerase is proposed to immortalize cancer cells by maintaining the chromosome ends (telomers), which otherwise progressively shorten as cells divide and age. This paper validates telomerase as a target for anticancer therapies. Findings: Using antisense oligonucleotides (peptide nucleic acids or 2′-OMeRNA) targeted against the RNA component of telomerase in immortalized human mammary epithelial (HME) cells, the authors describe progressive and reversible telomer shortening leading to cell death. These effects where shown to be caused by specific sequence matching rather than generally toxic effects of the inhibitors used. Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. Yahoo IMA: J Am Chem Soc 1999, 121:5856-5859. •• Significance: The aryl hydrocarbon receptor (AhR) is a transcription regulator that is bound by polycyclic aromatic hydrocarbons (PAHs) and activates the expression of a number of metabolic enzymes that convert the PAH to mutagenic products. This paper provides the first direct proof of AhR’s mediating role in carcinogenicity. Findings: Homozygous AhR-deficient mice (AhR(–/–)) were shown to be resistant to tumor formation as elicited by subcutaneous (male) or topical (female) application of benzo[a]pyrene. AhR-heterozygous (Ahr(+/–)) and AhR-positive (AhR(+/+)) mice developed these tumors rapidly. There was no evident difference between the three strains of mice, beyond the absence of AhR expression. Selected by Joe Kappock Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

A molecular basis for nitric oxide sensing by soluble guanylate cyclase. Zhao Y, Brandish PE, Ballou DP, Marletta MA: Proc Natl Acad Sci USA 1999, 96:14753-14758. • Significance: Intercellular nitric oxide (NO) signals are received by soluble guanylate cyclase (sGC), a heterodimeric protein composed of a heme-containing NO receiver domain (in subunit β1) and a catalytic domain that is activated after NO binding. NO is sufficiently reactive to cast doubt on whether its intercellular transit could be direct, or might have to be carrier-mediated. This paper presents evidence supporting the feasibility of direct transfer and a new mechanism for NO-dependent sGC activation.

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Findings: NO binds to sGC at a nearly diffusion-controlled rate that is a factor of ~10 higher than the rate of NO binding to potential hemoprotein competitors. The initial NO–sGC adduct is an inactive six-coordinate ferrous–nitrosyl complex. Subsequent iron–histidine bond breaking — conversion to a five-coordinate species — is required for cyclase activation. Intriguingly, the rate of this activation depends upon the binding of a second NO. A receiver-only fragment [β1(1–385)] rapidly forms the five-coordinate species, suggesting that cyclase activation is correlated with a protein conformational change in intact sGC. A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Bánfi B, Maturana A, Jaconi S, Arnaudeau S, Laforge T, Sinha B, Ligeti E, Demaurex N, Krause KH: Science 2000, 287:138-142. • Significance: Voltage-gated H+ channels, which are important for mammalian cell pH regulation, have not yet been cloned. A subunit of phagocyte NADPH oxidase, gp91phox, was suspected of being an H+ channel, but gp91phox-deficient cells still contained normal H+ currents. This paper reports the first molecular characterization of an H+ channel and clarifies its relationship to NADPH oxidase. It reveals an interesting relationship between electron and proton transfers apparently carried out by different products of the same gene. Findings: The NOH-1 gene produces two transcripts that share the first five exons: a six exon transcript and a thirteen exon transcript that is homologous to gp91phox. The short transcript (NOH-1S) produces a four-helix integral membrane protein, which is expressed in a limited number of tissues. NOH-1S lacks the prosthetic-group-binding regions required for gp91phox function but retains a histidine-rich motif, postulated to function in voltage-gating. NOH-1S-expressing cells contain a channel with the anticipated characteristics. Direct protein-protein coupling enables cross-talk between dopamine D5 and γ-aminobutyric acid A receptors. Liu F, Wan Q, Pristupa ZB, Yu XM, Wang YT, Niznik HB: Nature 2000, 403:274-280. • Significance: Dopamine receptors are encoded by five genes (D1–D5) whose functions have not been adequately distinguished. The subfamily of D1-like receptors (D1 and D5) preferentially couple to Gs proteins, ultimately regulating adenylyl cyclase and phosphorylation pathways. A D1-like dopamine receptor is found in neurons that contain the ligandgated γ-aminobutyric acid A (GABAA) receptor, suggesting a functionally specific interaction. The paper describes this interaction and a new signalling pathway in which receptors of different types attenuate each others activities. Findings: In cells coexpressing the dopamine D5 receptor and the GABAA receptor, dopamine leads to inhibition of GABAA receptor function, and GABA inhibits dopamine-stimulated, D5-mediated adenylyl cyclase activity. The two receptor types are colocalized in neurons and interact directly. A specific, agonistdependent interaction between D5 and the γ2 subunit of the GABAA receptor is mediated by the carboxy-terminal domain of D5, which differs most from the D1 receptor. D1 receptor neither affects nor binds GABAA receptor, indicating that the D5 receptor selectively downregulates GABAA receptor function. Redox signaling in chloroplasts: cleavage of disulfides by an iron-sulfur cluster. Dai S, Schwendtmayer C, Schürmann P, Ramaswamy S, Eklund H: Science 2000, 287:655-658.

•• Significance: The conversion of ‘inorganic’ reducing equivalents generated by membrane-bound proteins into the universal ‘organic’ reducing currency, the two cysteine thiols in thioredoxin (Trx), is of considerable functional importance for redox-regulated enzymes and processes. In plants, where photoreduction by photosystem I is a key part of the Calvin cycle, two electrons from two reduced ferredoxin (Fd) molecules are transmuted into one reduced Trx by a pathway that includes the adaptor protein ferredoxin:thioredoxin reductase (FTR). This paper presents a new mechanism for this conversion, suggested by the structure of FTR and its essential Fe4S4 cluster. Findings: FTR is only 10 Å thick in the region of the Fe4S4 cluster, which separates putative Fd-binding and Trx-binding surfaces. Direct, cluster-mediated reduction of a surface disulfide near the FTR cluster would account for the unusual redox properties of FTR. Trx reduction is proposed to occur by disulfide exchange in a complex of Trx, FTR, and Fd. The structure would allow the first, oxidized Fd to be exchanged for a second Fd without disruption of the FTR:Trx interaction; that is, FTR or the FTR:Trx complex can transiently store a single reducing equivalent. This pathway is quite different from Trx reduction in bacteria and animals, which requires the NADPH-dependent flavoenzyme Trx reductase.

Biocatalysis and biotransformation Selected by Nicola Pohl Stanford University, Stanford, USA

β-carotene) biosynthetic Engineering the provitamin A (β pathway into (carotenoid-free) rice endosperm. Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I: Science 2000, 287:303-305. •• Significance: Vitamin A deficiency is a significant health problem in countries that consume large quantities of rice. The production of provitamin A in rice endosperm by the transformation of this biosynthetic pathway into rice plants offers a possible solution to this deadly vitamin deficiency. Findings: Agrobacterium-mediated transformation of the entire β-carotene biosynthetic pathway (phytoene synthase, plant and bacterial phytoene desaturases, lycopene β-cyclase) with appropriate transit peptide sequences into rice embryos produced rice seeds that were yellow from β-carotene. The plants were fertile and appeared to manufacture enough provitamin A to meet nutritional requirements. Cloning and heterologous expression of the epothilone gene cluster. Tang L, Shah S, Chung L, Carney J, Katz L, Khosla C, Julien B: Science 2000, 287:640-642. •• Significance: The macrolide epothilone is a potential replacement for Taxol; however, the natural producer is too inefficient to provide an economical supply of this compound for further testing. The cloning and heterologous expression of the entire polyketide synthase gene cluster in a faster-growing and better characterized bacterial strain opens the door to a supply of both the natural and engineered forms of epothilone. Findings: The epothilone biosynthetic gene cluster was cloned, fully sequenced, and then heterologously expressed in Streptomyces coelicolor. A P450 epoxidase responsible for the final epoxidation step of 12,13-desoxyepothilone was cloned, expressed in Escherichia coli, purified and assayed. This desoxyepothilone is more biologically active than the final compound and can be produced in high yields by elimination of the epoxidase gene.

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A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of ε-rhodomycinone to rhodomycin D. Olano C, Lomovskaya N, Fonstein L, Roll JT, Hutchinson CR: Chem Biol 1999, 6:845-855. • Significance: Deoxysugars are crucial for the biological activity of a variety of polyketide natural products. The facile introduction of plasmid-borne deoxysugar biosynthetic genes along with a plasmid bearing other biosynthetic genes of an anthracycline polyketide results in the isolation of an appropriately glycosylated polyketide product. This plasmid-borne gene system will greatly facilitate in vivo structure/function studies of these enzyme pathways as well as the combinatorial biosynthesis of possible new drugs. Findings: The genes for glucose-1-phosphate thymidyltransferase, TDP-glucose-4,6-dehydratase and antibiotic resistance genes were cotransformed with the biosynthetic genes responsible for rhodomcyin D biosynthesis in the heterologous host Streptomyces lividans. The effect of three of these genes (dnmW, dnmZ, and dnmM) on anthracycline production was also assessed. Enzymatic synthesis of neoglycopeptide building blocks. Ramos D, Rollin P, Klaffke W: Angew Chem Int Ed 2000, 39:396-398. • Significance: Glycopeptides have become important tools in the study of carbohydrate functions in cellular and pathogenic processes. The synthesis of monovalent and multivalent glycopeptides, however, is limited by difficulties in the efficient chemical or enzymatic glycosylation of peptides. This combined chemoenzymatic approach promises a route to the facile synthesis of small libraries of glycopeptides. Findings: Allylamine was reacted with a variety of disaccharides to form β-glycosides. Photochemical addition of cysteamine provided a linker between the sugar and an enzymatically reactive terminal amine. Subsequent enzymatic coupling of this amine with a dipeptide using a commercially available transglutaminase provided a neoglycopeptide. Various linkers were also tested with this enzyme. Convenient enzymatic resolution of alcohols using highly reactive, nonharmful acyl donors, 1-ethoxyvinyl esters. Kita Y, Takebe Y, Murata K, Naka T, Akai S: J Org Chem 2000, 65:83-88. • Significance: Lipases and esterases are often used in organic solvents for the chiral resolution of alcohols in organic synthesis. The most commonly used esterification reagent in these processes, methyl vinyl ester, suffers from slow reaction times and by-product reactions with the enzyme. The reported one-pot 1-ethoxyvinyl ester procedure allows a greater variety of lipase-mediated acylations with reasonable enantiomeric excesses (>75%) and 2–3 times faster reaction times than those of presently used protocols. Findings: Kinetic resolutions of twelve racemic secondary alcohols and one desymmetrization of a meso-bicyclic diol with four different lipases are presented along with a one-pot synthesis of ethoxyvinyl esters from carboxylic acids and enzymatic resolution. Alternative modular polyketide synthase expression controls macrolactone structure. Xue Y, Sherman DH: Nature 2000, 403:571-575. •• Significance: The combinatorial biosynthesis of polyketides via genetic engineering enables the production of a wide variety of structural forms within this therapeutically valuable class

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of compounds. The authors have discovered the molecular determinants of a growth-medium-induced switch between a 12-membered and 14-membered macrolactone that provides insight not only into polyketide synthase (PKS) quaternary structure but also into the means by which organisms could respond quickly to varying environmental conditions. Findings: Isolation of an 85 rather than 110 KDa form of the last module (PikAIV) of the methymycin/pikromycin PKS in different growth media suggested controlled gene expression complementation experiments with various truncated PikAIV modules. An alternate ribosomal binding site and start codon was found ~600 bases into the PikAIV gene, thereby providing a second protein translation site under certain condition. Expression of this truncated form is responsible for 12-membered macrolactone formation.

Bio-inorganic chemistry Selected by Richard Cammack King’s College London, London, UK

Photoassembly of the manganese cluster and oxygen evolution from monomeric and dimeric CP47 reaction center photosystem II complexes. Buchel C, Barber J, Ananyev G, Eshaghi S, Watt R, Dismukes C: Proc Natl Acad Sci USA 1999, 96:14288-14293. •• Significance: The oxygen-evolving complex of plant photosynthesis uses a tetranuclear manganese cluster, which assembles spontaneously in a light-driven process. The assembly has now been demonstrated in a cell-free system, and has been shown to take place without the need for accessory proteins, in contrast to the assembly of metal centres in other proteins. Findings: A protein complex, lacking the chlorophyll-protein CP43, was isolated from spinach photosystem II. After removal of manganese, the soluble complex was shown to reconstitute in the same way as occurs in the chloroplast membrane. The reconstitution required manganese, calcium, chloride and light, and produced a Mn4CaClx cluster capable of photooxidation of water. Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Qi ZH, Hamza I, O’Brian MR: Proc Natl Acad Sci USA 1999, 96:13056-13061. • Significance: In bacterial cells, the synthesis of porphyrin is regulated according to the availability of iron. The transcriptional regulator Irr is involved in a new type of regulation by the concentration of heme. When heme binds to Irr, the protein becomes unstable and is degraded. Findings: Addition of heme to cells of Bradyrhizobium japonicum facilitated the degradation of the Irr protein. The protein was shown to bind heme. Mutations to the heme-binding site made the protein stable in the presence of iron, and suppressed the synthesis of porphyrin by the cells. A short Fe-Fe distance in peroxodiferric ferritin: control of Fe substrate versus cofactor decay? Hwang J, Krebs C, Huynh BH, Edmondson DE, Theil EC, PennerHahn JE: Science 2000, 287:122-125. • Significance: The peroxodiferric complex is an intermediate in the mineralization of iron by the iron-storage protein ferritin. The Fe–Fe distance of 0.253 nm is consistent with a unique triply bridged structure, which distinguishes this centre from those seen in dinuclear iron hydroxylases. Findings: The Fe–Fe distance, measured by X-ray absorption spectroscopy, is less than any known µ-carboxylato diferric

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complex. It indicates a small Fe–O–O angle, which would favour the release of H2O2 and the formation of µ-oxo or µ-hydroxo FeIII2biomineral precursors, in preference to O–O bond cleavage, as occurs in the hydroxylases.

Combinatorial chemistry Selected by M Morgan Conn Amherst College, Amherst, Massachusetts, USA

Incorporation of two different nonnatural amino acids independently into a single protein through extension of the genetic code. Hohsaka T, Ashizuka Y, Sasaki H, Murakami H, Sisido M: J Am Chem Soc 1999, 121:12194-12195. •• Significance: The ability to incorporate multiple unnatural amino acids into proteins would allow unparalleled opportunities to study and manipulate protein structure and function. Findings: The authors report for the first time the incorporation of two unnatural amino acids into a protein (streptavidin) using an expanded genetic code. The unnatural amino acids were specified using four-base codons matched with tRNAs containing four-base anticodons. No cross-reactivity was observed between the two four-base codons. Incorporation was modest, yielding unnatural protein with only a 9% yield relative to the natural protein in the Escherichia coli in vitro translation system. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain. Jaeger L, Write MC, Joyce GF: Proc Natl Acad Sci 1999, 96:14712-14717. • Significance: The selection of ribozymes can be hampered by the requirement to select both structure and function from a necessarily limited pool of random sequences. This work shows that it is possible to assemble ribozymes with complex function and structure from smaller domains. Findings: A structural domain found in group I ribozymes was used as a folding motif to support a randomized RNA library. The RNA pool was selected for its ability to ligate an oligonucleotide to itself. The resultant ribozymes isolated after only 10 rounds of selection were highly active. The sequences bore no resemblance to previous ribozymes (class I ligase) selected from completely randomized RNA libraries, despite displaying the identical ability to form 3′,5′-phosphodiester bonds. Non-unity molecular heritability demonstrated by continuous evolution in vitro. Schmitt T, Lehman N: Chem Biol 1999, 6:857-869. • Significance: This paper demonstrates that a complex genetic characteristic (non-uniform correlation between phenotype and genotype) can be demonstrated by a simple molecular system. Findings: The authors used serial continuous evolution of a pre-existing ligase ribozyme to select for sequences under increasingly reduced Mg2+ concentration. No purification steps were performed during the transfer of material from one set of conditions to another to avoid introducing additional selection criteria. Over the course of the selection, the initial ribozyme (E100(#3)) was replaced in the pool of molecules by a sequence with six altered bases (B16(#19)). The new ribozyme was found to be no more active as a catalyst than the original sequence. Rather, the authors hypothesise, B16(#19) folds more efficiently into an active conformation directly upon transcription, whereas E100(#3) requires complete denaturation and renaturation to do so. This result highlights the difficulty in isolating only the desired property when additional selection criteria are introduced by each experimental manipulation in an in vitro selection experiment.

Creating a protein-based element of inheritance. Li L, Lindquist S: Science 2000, 287:661-664. •• Significance: The ability of proteins to transmit ‘information’ from one generation to the next muddles the RNA- world hypothesis of evolution, in which nucleic acids form both the phenotype and genotype of prebiotic chemistry. Findings: The authors found that yeast proteins capable of selfpropagating conformational changes have a prion domain that appears solely capable of conveying prion function. A rat transcriptional regulating protein was expressed in yeast as a fusion product with the prion domain from the yeast Sup35 protein. This fusion protein (amino-terminal region, middle region, glucocorticoid receptor; NMGR) functioned in vivo normally to activate β-galactosidase expression, unless subjected to experimental conditions that also cause Sup35 to convert to its insoluble, inactive form. The changes in NMGR were heritable — cells expressing active NMGR produced inactive colonies when mated with those expressing insoluble NMGR. This is presumably attributable to the self-propagating tendencies of the insoluble proteins. Selected by Dennis Hall University of Alberta, Edmonton, Alberta, Canada

Combinatorial synthesis of four-helix bundle hemoproteins for tuning of cofactor properties. Rau HK, DeJonge N, Haehnel W: Angew Chem Int Ed 2000, 39:250-253. • Significance: It is very difficult to control and optimize cofactor properties of de novo hemoproteins. Therefore, combinatorial methods for the synthesis of proteinaceous cofactors and the screening of hemoproteins for their redox potential are important in biomimetic chemistry. Findings: A parallel library of four-helix bundle proteins was elaborated by spot synthesis on a cellulose membrane. Protein design involved two sets of helical antiparallel peptides containing heme-binding histidine residues. A set of eight amino acids was used, mostly hydrophobic ones, and a few polar residues, to randomize 5 positions on helix A and four on helix B. According to molecular modeling, these specific residues form the hydrophobic shield of the heme unit. A total of 462 modular peptides resulted from the different combinations of helices. The Fe(III)-protoporphyrin IX was loaded by soaking the membrane in a solution of the cofactor. The membrane-bound hemoproteins were evaluated by UV/visible spectroscopy and screened for their midpoint redox potential. A large range of potentials resulted even for subtle variation in amino acid composition. This combinatorial approach was successful in finding proteins that tune the heme to a midpoint potential significantly more positive than previously designed ones. Chemical genetic and genomic approaches reveal a role for copper in specific gene activation. Stockwell BR, Hardwick JS, Tong JK, Schreiber SL: J Am Chem Soc 1999, 121:10662-10663. •• Significance: The combined use of small-molecule libraries and cell-based assays constitutes a powerful technique to study gene function. This work provides a nice demonstration that the use of combinatorial libraries and genomics techniques can help characterize genes such as those involved in ion homeostasis. Findings: A commercial unbiased library of 16,000 structurally diverse compounds was screened in a whole cell assay for the activation of a reporter gene for transforming growth factor (TGF)β signaling. Of four active compounds, two were shown to act as

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TGF-β-like molecules as demonstrated by their effect in reportergene activation and inhibition of DNA synthesis. Using a transcriptional profiling study of Saccharomyces cerevisiae, only one of the two compounds, 2,2´-(methylimino)bis(8-quinolinol), showed an effect by increasing the expression of five genes. Three of these genes are known to be involved in metal ion homeostasis and the two uncharacterized ones are therefore hypothesized to have a similar role. By testing the effect of various ions on the reporter activity in the presence of the bis(8-quinolinol) compound it was found that copper suppressed reporter activity. Furthermore, Cu(II) alone activates the reporter specifically. This and other results are consistent with the property of bis(8-quinolinol) compounds to chelate copper (and also Zn(II) and Fe(III) ions). In response to copper depletion, yeast responds by activating the above three genes involved in ion homeostasis. Coupling rational design with libraries leads to the production of an ATP selective chemosensor. Schneider SE, O’Neil SN, Anslyn EV: J Am Chem Soc 2000, 122:542-543. •• Significance: The application of combinatorial methods to a proven scaffold (i.e. a lead) is a powerful way to improve the affinity and selectivity of artificial receptors for small molecules. This approach was demonstrated with the discovery of a selective chemosensor for ATP. Findings: A bis(guanidinium) core structure known to provide tight but unselective binding of nucleosides via anionic recognition was combinatorialized with two homogeneous tripeptide chains. A 4913 member library elaborated by split-and-pool synthesis on a Tentagel-based support was screened with N-methylanthraniloyl-labeled ATP. Roughly 15% of all beads afforded highly fluorescent ‘hits’. Several of these peptides were cleaved from the beads and sequenced. Three fluorescently labeled receptors and a negative control were resynthesized and further studied for their ATP chemosensing properties. Ultimately, one sequence (Ser–Tyr–Ser) was found to have a binding constant ten-times higher than the underivatized bis(guanidinium) core structure. Furthermore, AMP and GTP did not respond to the chemosensor, suggesting that both the triphosphate–binding guanidinium units and the base-binding tripeptide chains are required. Preparation of designer resins via living free radical polymerization of functional monomers on solid support. Hodges JC, Harikrishnan LS, Ault-Justus S: J Comb Chem 2000, 2:80-88. • Significance: Because solid supports are of crucial importance in combinatorial chemistry, there is a continuing need for new methods that enable the preparation of new types of resins whose properties can be easily tailored to specific uses. The titular method allows the derivatization of standard cross-linked beaded polystyrene and is effective at preparing electrophilic scavenging resins. Findings: Standard Merrifield resin was converted to a TEMPOmethyl resin acting as a solid-supported radical initiator. The latter resin reacts by nitroxide-mediated living polymerization with a variety of styrene-based and acrylate-based monomers to provide ‘Rasta’ beads: a new type of larger beads with longer straight chains coming out from the backbone phenyl groups. This solvent-free method is amenable to monomers containing electrophilic groups, which typically do not tolerate aqueous suspension polymerization. This way, resin properties such as solvent compatibility, loading, and internal architecture can be controlled. The authors applied the process to the preparation of

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a high-loading isocyanate resin useful in solution-phase synthesis as a scavenger for primary and secondary amines. Visualizing functional group distributions in solid-support beads by using optical analysis. McAlpine SR, Schreiber SL: Chem Eur J 1999, 5:3528-3532. • Significance: Little is known about the precise microstructure and distribution of site functionalization at the interior of resin beads. By affecting chemical reactivity and solid-phase screening, the nature and properties of solid supports are crucial in solid-phase synthesis. A better knowledge on the interior functionalization of common types of solid supports is desirable. Findings: The interior functionalization of four types of aminofunctionalized resins — ArgoPore, porous glass, standard polystyrene (1–2% cross-linked), and Tentagel — were studied by optical analysis. The amino sites of the resins were derivatized with a rhodamine dye. Using the optical analysis technique, slices of each bead type were obtained. The fluororescence intensity values, plotted as a function of bead diameter, were compared. The macroporous resins (ArgoPore and glass) showed a relatively even functional group distribution. The gel type resins (polystyrene and Tentagel), however, showed a largely uneven distribution. This observation has implications in solid-phase synthesis. These resins have a significant proportion of sites congested at the outer bead surface. This can limit access of reagents in the interior sites and thus corroborates the slower reaction rates observed for these sites. NOE pumping. 2. A high-throughput method to determine compounds with binding affinity to macromolecules by NMR. Chen A, Shapiro MJ: J Am Chem Soc 2000, 122:414-415. • Significance: There is a continuing need for high-throughput methods to identify tight-binding ligands of biomolecules from mixtures of small molecules. Such methods can be very useful to screen combinatorial libraries for drug lead discovery. Findings: Reverse nuclear Overhauser effect (NOE) pumping (RNP), is a new technique that increases sensitivity and reduces experiment time compared with NOE pumping. It suppresses signals from the receptor while allowing the detection of any signals transferred from the binding ligands to the receptor. In this paper, the technique was demonstrated by analyzing the interaction of human serum albumin with several binding, unbranched fatty ligands, and with glucose, a non-binding compound. The signal integral of the binding ligand is significantly reduced compared with that of the non-binding compound. One advantage of this technique is the relatively low concentration of receptor required and its potential in high-throughput automation. Monitoring the solid-phase synthesis of depsides and depsipeptides. A color test for hydroxyl groups linked to resin. Kuisle O, Lolo M, Quiñoá E, Riguera R: Tetrahedron 1999, 55:14807-14812. • Significance: Qualitative color tests, such as the nynhydrin test for the detection of resin-bound amino groups, are very useful in solid-phase synthesis to monitor reaction progress. They are the equivalent of thin layer chromatography for solutionphase chemistry. A reliable color test for alcohols, as described in this paper, will prove useful in solid-phase synthesis. Findings: A color test for resin-bound primary, secondary, and tertiary alcohols is described. The test involves the formation and displacement of a tosylate derivative by p-nitrobenzylpyridine followed by conversion to a colored pyridinium salt with

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base, providing violet to pink colored beads. Amino and carboxyl groups give a negative outcome, whereas low concentrations of alcohols can be detected visually. As exemplified in the esterification of Wang resin, this method is sensitive enough to monitor loading completion of hydroxylbased supports. Therein, the test was also applied to the synthesis of various sequences of depsides and depsipeptides.

Next generation therapeutics Selected by Steven Projan Wyeth-Ayerst Research, Pearl River, New York, USA

Drug target validation: lethal infection blocked by inducible peptide. Tao J, Endler P, Connelly G, Lim A, Zhang J, King M, Li T, Silverman JA, Schimmel PR, Tally FP: Proc Natl Acad Sci USA 2000, 97:783-786. • Significance: Many potential antibacterial targets are being revealed by a myriad of genomic and molecular genetic discovery programs. Although gene ‘knockout’ experiments can address the essential nature of a given target, they do not demonstrate that the inhibition of a given target can have a therapeutic effect in a bacterial infection. The authors of this paper have combined two techniques (phage display and inducible expression) to address both the ability to target a given gene product and to eventually develop an assay to find inhibitors of that putative target. This work describes a general method for antibacterial target validation. Findings: Using phage display, the authors first identified specific binders to prolyl-tRNA synthetase, one of these inhibited the in vitro aminoacylation of tRNA by the enzyme with a Ki of 250 nM. The authors then fused a coding sequence for the most active peptide inhibitor (called ‘Pro-3’) to a sequence coding for glutathione-S-transferase (GST) and put it under the transcriptional control of a tetracycline inducible promoter in Escherichia coli. Induction was accomplished using anhyrdotetracycline (which functions as an inducer but has no antibacterial activity). Expression of the Pro-3/GST fusion in culture demonstrated a marked inhibition of bacterial growth. The authors then successfully protected mice from an intraperitoneal challenge with a virulent strain of E. coli carrying the Pro-3/GST expression system, but only when expression of the Pro-3/GST fusion was induced. It should be noted that once a specific peptide binder has been validated in vivo (as with Pro3 and prolyl-tRNA synthetase) then a ‘function blind’ ligand displacement assay can be formatted to identify small molecular inhibitors of the target. Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Hazuda DJ, Felock P, Witmer M, Wolfe A, Stillmock K, Grobler JA, Espeth A, Gabryelski L, Schleif W, Blau C, Miller MD: Science 2000, 287:646-650. AND

Structure and absolute stereochemistry of HIV-1 integrase inhibitor integric acid. A novel eremophilane sesquiterpenoid produced by a Xylaris sp. Singh SB, Zink D, Polishook J, Valentino D, Shafiee A, Silverman K, Felock P, Teran A, Vilella D, Hazuda D, Lingham RB: Tetrahedron Lett 1999, 40:8775-8779. •• Significance: To date, therapeutic agents used to treat HIV-1 infections have targeted either reverse transcriptase or protease. Despite the efficacy of highly active anti-retroviral therapy (HAART) in driving down viral load in infected individuals, resistance to the current anti-HIV agents eventually arises. Althought HIV integrase is essential for viral replication and several

inhibitors of integrase have been described previously, none of these inhibitors block viral replication in infected cells in culture. Findings: The first of these two articles describes a series of diketo acids that are HIV integrase inhibitors and do prevent HIV replication in infected cells. These compounds appear to specifically inhibit the catalysis of strand transfer (a reaction in which the 3′ end of the viral DNA is joined covalently to cellular DNA). The best of these compounds was shown to have an IC50 of 50 nM in an in vitro strand transfer reaction and a 1.0 µM IC50 in a viral infectivity assay. This IC50 values for replication inhibition is probably too high for these compounds to be considered as potential therapeutic agents but the values do provide a proof of principle that integrase is a ‘drugable’ target. The second paper describes a natural product derived from Xylaria that also inhibited integrase activity: it blocks at least two of the steps of integration including strand transfer, but has an IC50 of 3–10 µM. Technical comment: activation and inhibition of the staphylococcal agr system. Novick RP, Ross HF, Figuerefo AMS, Abramochkin G, Muir T. Response by: Balaban N, Sngh B, Goldkorn T, Rasooly A, Torresand JV, Uziel O: Science 2000, 287:391a. • Significance: The ‘accessory gene regulator’ (agr) system of Staphylococcus aureus encodes an autocrine, quorum-sensing regulatory system that controls the expression of a large number of virulence factors. Previously, Balaban et al. (Science 1998, 280:438.) reported that a 38 kD protein (RAP), produced by an agr-null strain (RN6911), functioned as the agr activator and that this protein had immunoprotective activity in a murine abscess model. In addition, Balaban et al. reported that a linear heptapeptide (RIP), presumably derived from RAP, inhibited agr activation and inhibited infectivity in the murine abscess model. These findings are at odds with previous work by Novick and colleagues demonstrating that the activator/inhibitor peptides are seven or eight amino acids in length, contain a five-membered thiolactone ring and are uniformly encoded by agrD. Given the interest in the development of novel anti-staphylococcal agents and staphylococcal vaccines these conflicting results are of more than passing interest. Findings: Novick et al. reported an inability to replicate the work of Balaban et al., failing to demonstrate agr-inducing activity with preparations of RN6911 supernatant or inhibition of agr by the heptapeptide, RIP. In response Balaban et al. take issue with the preparation and/or handling of the material by Novick et al. and suggest that the β-lactamase reporter system used by Novick et al. is lacking in sensitivity compared with the northern blots used by Balaban et al. Although the handling of the respective samples may be a legitimate difference in the work described by both groups, it should be pointed out that the use of reporter-gene-enzyme assays tend to be both more sensitive and more reproducible than northern blots of bacterial mRNA. The failure of RIP to inhibit in the experiments Novick et al. is in direct contrast to the continued success of RIP to inhibit agr expression in vitro and virulence in vivo in the experiments of Balaban et al. Resolution of these disparate findings will now require studies in other, independent laboratories.

Analytical techniques Selected by Tony Cass Imperial College of Science, Technology and Medicine, London, UK

Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays. Steemers FJ, Ferguson JA, Walt DR: Nat Biotechnol 2000, 18:91-94.

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•• Significance: DNA microarrays have been developed in several formats and to further improve their performance advances need to be made in sample volume, ‘upstream’ sample handling and probe density. In this paper an optical fibre array is combined with molecular beacons to address these issues. Findings: Molecular beacons with probe sequences corresponding to variants of the cystic fibrosis transmembrane conductance regulator (CFTR) were bound to beads via a biotin–streptavidin link and the beads were encoded with different combinations of fluorophores such that each probe’s sequence corresponded to a particular encoding scheme. Beads were then randomly dispersed into 3µm wells at the end of an optical-fibre bundle such that each bead was individually addressable. Using this array, the different CFTR targets could be identified with detection limits in the nM region and hybridisation times of around five hours. Anchored multiplex amplification on a microelectronic chip array. Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M: Nat Biotechnol 2000, 18:199-204. • Significance: Two problems with multiplex PCR of several genes in a mixture are the occurrence of primer–primer interactions and the different optimal conditions for the amplification of different sequences. One way to circumvent these problems is to spatially localise probes and target DNA in different regions on an electronic chip. Electrophoretic migration also accelerates the subsequent hybridisation reaction. Findings: Isothermal amplification of target DNA using the strand displacement assay (SDA) was found to be compatible with the electronic chip structures. To demonstrate this approach, a 10-plex (i.e. 10 different genes) amplification of a mixture of human and bacterial targets was performed. The typical detection limit was around 10,000 molecules of target with low cross reactivity between the different targets. Intracellular measurements in mammary carcinoma cells using fiber-optic nanosensors. Cullum BM, Griffin GD, Miller GH, Vo-Dinh T: Anal Biochem 2000, 277:25-32. • Significance: The biochemical analysis of single cells is attracting considerable interest, a variety of approaches have been used including mass spectrometry and microelectrode methods. Optical sensing techniques have been extended into this domain with the use of optical–fibre nanosensors that can be inserted into single cells for fluorescence measurements. Findings: Optical fibres with a diameter of 40 nm were coated on their tips with an antibody to benzo[a]pyrene tetrol (BPT). The fibres were then inserted into individual rat liver epithelial or mammary carcinoma cells. BPT uptake into cells immersed in a solution of BPT could be measured by fluorescence using the optical fibres; a detection limit of around 6 pM was determined. Rapid nanopore discrimination between single polynucleotide molecules. Meller A, Nivon L, Brandin E, Golovchenko J, Branton D: Proc Natl Acad Sci USA 2000, 97:1079-1084. • Significance: High-throughput analysis of DNA at low copy number has important applications in human genotyping. Ideally, the analysis be rapid and should not require labelling of the DNA. Findings: Diffusion of single DNA molecules through an α-hemolysin pore was found to produce a transient ion blockade that could be measured by patch-clamp methods. In a mixture of polynucleotides of different size and base composition, the components showed distinct behaviour in terms of the

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transit time through the pore and the magnitude of the ion blockade current. Even where the size and composition were the same, different sequences could be distinguished. An analysis of the distributions of transit time and current could be used to identify the different nucleotides present. Activity-based protein profiling: the serine hydrolases. Liu Y, Patricelli MP, Cravatt BF: Proc Natl Acad Sci USA 1999, 96:14694-14699. •• Significance: The parallel determination of multiple enzyme activities in cell extracts is a valuable complement to the measurement of gene expression and protein levels. Variations in the profiles of enzymatic activity between tissues or between diseased and healthy cells could guide the discovery of new drugs. Findings: A fluorophosphate (FP)–biotin derivative was synthesised and used to irreversibly modify serine proteases in tissue extracts. As the compound only modifies catalytically active proteases, its degree of reaction is a measure of the amount of active enzyme present. Modified proteins are detected by avidin blotting following electrophoretic separation. Tissue-specific profiling of serine proteases was demonstrated with this method.

Mechanisms Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA. Gu X, Liu Y, Santi DV: Proc Natl Acad Sci USA 1999, 96:14270-14275. • Significance: The mechanistic course of an essential tRNA modification enzyme has been established along with its relationship to other enzyme-catalyzed reactions involving the pyrimidine ring of uracil. Findings: Incubation of pseudouridine synthase I with yeast tRNAPhe containing 5-fluorouracil at the site modified by this enzyme (position 39) led to formation of a covalent intermediate that was stable to detergent and urea but heat-labile. By incorporating radioactive isotopes at various positions in the 5-fluorouracil-modified tRNA, it was established that the covalent linkage involved the pyrimidine of 5-fluorouracil. All of the data were consistent with an intermediate formed by conjugate addition of the sidechain carboxylate of Asp60 to the 6-position of the fluorouracil ring with protonation occurring on the opposite face to yield the cis-5S,6R stereochemistry. This intermediate is believed to be analogous to that formed during the normal catalytic cycle in which the pyrimidine of U39 is isomerized to form ψ39 and it provides a logical chemical mechanism for this conversion. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain. Jaeger L, Wright MC, Joyce GF: Proc Natl Acad Sci USA 1999, 96:14712-14717. •• Significance: This paper reports a strategy for exploring the sequence space associated with the randomized portion of a ribozyme more completely, while still affording a high probability of maintaining a three-dimensional structure conducive to catalysis. Findings: The hypothesis that a pre-existing RNA domain derived from the Tetrahymena ribozyme could serve as a ‘scaffold’ for evolving new ribozyme catalysts was tested by appending three loops whose sequences were completely randomized (total of 85 positions). A library of 1016 different sequences was screened for the ability to undergo self-ligation; and ten rounds of selection yielded catalysts with rate constants

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of 0.26 min–1 (>106-fold greater than the background rate). A parallel experiment using a simple 85-nucleotide RNA in which all positions were varied failed to yield catalysts for the self-ligation reaction, perhaps because the total size of the individual ribozymes was insufficient for both substrate binding and catalysis for this sophisticated reaction. The major advantage of the strategy outlined in the paper is that it allows access high to sequence diversity whilst retaining a common core that provides a useful overall structure, similar to the situation in antibodies. One polypeptide with two aminoacyl-tRNA synthetase activities. Stathopoulos C, Li T, Longman R, Vothknecht UC, Becker HD, Ibba M, Söll D: Science 2000, 287:479-482. •• Significance: This is the first demonstration that a single tRNA synthetase can accept two different amino acids and two different tRNAs while forming only the cognate aminoacylated tRNAs in both cases. Findings: The genomes of the thermophilic methanogens Methanococcus jannaschii and Methanobacterium thermoautotrophicum both appear to lack homologs of cysteinyl-tRNA synthetase. A single protein was purified from M. jannaschii that catalyzed the aminoacylation of tRNACys; unexpectedly, the sequence of this protein suggested that it was a homolog of prolyl-tRNA synthetase. A variety of kinetic experiments established that this single protein catalyzed the aminoacylation of tRNACys and both tRNAPro molecules, but only with the correct amino acid (cysteine and proline, respectively). Apart from accepting two very different amino-acid substrates and tRNAs, the other properties of the M. jannaschii enzyme were very similar to those of other prolyl-tRNA synthetases. Phylogenetic analyses were not useful in detecting the expanded substrate specificity of the M. jannaschii synthetase and it is not clear how widespread this phenomenon may be. Assessment of protein-tyrosine phosphatase 1B substrate specificity using “inverse alanine scanning”. Vetter SW, Keng Y-F, Lawrence DS, Zhang Z-Y: J Biol Chem 2000, 275:2265-2268. • Significance: A simple and generally applicable approach for determining the specificity of enzymes that accept peptide substrates is described. The method provides information on individual sequences while requiring a relatively small number of library members. Findings: From the crystal structure of murine protein-tyrosine phosphatase 1B, it was predicted that Ac-Ala4Tyr(OPO3)–Ala4–CONH2 would be an acceptable substrate, and this was subsequently confirmed experimentally. 152 additional peptides were synthesized in which each alanine residue was replaced separately and sequentially with each of the 19 non-alanine residues. The kinetic parameters for each peptide were determined individually, which allowed the substrate preferences to be investigated at every position in a physiologically-relevant reaction. The results from the library screening reiterated known substrate preferences for protein-tyrosine phosphatase 1B, but also revealed several other unsuspected peptide sequences that were also excellent substrates. Conformational effects in biological catalysis: an antibodycatalyzed oxy-Cope rearrangement. Mundorff EC, Hanson MA, Varvak A, Ulrich H, Schultz PG, Stevens RC: Biochemistry 2000, 39:627-632. • Significance: The crystal structures reported in this clearly illustrate how selection for tighter binding to a transition-state

analog by the immune system can actually compromise catalytic activity for a reaction involving a flexible substrate. Findings: A catalytic antibody was raised against a flexible antigen that mimicked the transition state structure for an oxy-Cope rearrangement, a sigmatropic reaction related to that catalyzed by chorismate mutase. Surprisingly, although the germ-line precursor antibody that gave rise to the mature catalytic antibody possessed 40-fold lower affinity for the transition-state analog, it catalyzed the reaction with 35-fold higher efficiency. X-ray crystal structures of the germ-line precursor and the mature antibody in the presence and absence of transition-state analog, were therefore obtained to probe the origin of this phenomenon. These data revealed that the mature antibody bound the flexible transition-state analog in a conformation that disfavored the sigmatropic rearrangement; by contrast, the binding site of the germ-line antibody was more flexible, which might allow the substrate to more easily reach the reactive conformation.

Model systems Selected by Graham RL Cousins and Jeremy KM Sanders University of Cambridge, Cambridge, UK

Models of nitric oxide synthase: iron (III) porphyrin-catalysed oxidation of fluorenone oxime to nitric oxide and fluorenone. Wang CC-Y, Ho DM, Groves JT: J Am Chem Soc 1999, 121:12094-12103. •• Significance: Nitric oxide synthase (NOS) is a heme-containing monoxygenase that catalyses the oxidation of L-arginine to L-citrulline and NO, an important biomessenger. An understanding of the NOS reaction mechanism is of academic interest, but more importantly might facilitate the rational design of NOS inhibitors with significant pharmaceutical implications. The article describes the first Iron(III)-porphyrin-catalysed oxidation of oximes by O2 to nitric oxide, a model reaction for the NOS catalysed oxidation of N-hydroxyarginine to L-citrulline and NO. On the basis of their results, the authors propose a probable mechanism for NOS oxidation of L-arginine. Findings: A hydroxoiron(III) porphyrin catalysed the oxidation of fluorenone oxime with O2 to nitro iron(II) porphyrin, fluorenone and O-(9-nitro-fluorenyl)fluorenone oxime. A mechanistic study of the reported reaction suggests that the NOS-catalysed formation of NO is initiated by an Fe–O bond homolysis of the ‘iron porphyrin—N-hydroxyarginine’ complex. It is believed that this generates an arginine derived iminoxy radical that reacts with O2 to give a peroxyFe(III) species which can decompose to citrulline, NO and an oxoFe(IV) heme. α-D-riboses as models 5-(β-cyclodextrinylamino)-5-deoxy-α for nuclease, ligase, phophatase, and phosphorylase. Han MJ, Yoo KS, Chang JY, Ha T-K: Angew Chem Int Ed 2000, 39:347-349. • Significance: Synthetic ribose-containing polymers have been reported that show activity for DNA cleavage, the study of which strongly suggests that vic-cis-diol groups of the ribose rings play a significant role in catalysis. It is proposed that ribose-containing biopolymers might have similar activities to their synthetic counterparts, although studies of such biopolymers are rare. The authors report the synthesis of ribose-appended cyclodextrins and a study of their catalytic mechanisms for nuclease, ligase, phosphatase and phosporylase reactions with a view to investigating the significance of vic-cis-diol groups. This article prompts interest and future research into catalysis by ribose-containing polymers and confirms the importance of vic-cis-diols in catalysis.

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Findings: Four cyclodextrin derivatives — to each appended α-Dribose with different hydroxyl protection patterns — were synthesised and analysed for catalytic activity towards the hydrolysis and esterification of phosphates, and the phosphorylation of alcohols. Only substitution patterns maintaining vic-cis-diols displayed catalytic activity. This and further experiments enabled the authors to postulate that the vic-cis-diol forms hydrogen bonds to two phosphoryl oxygens, which activate the phosphorus atom thus rendering it susceptible to nucleophilic attack. Cholesterol esterase activity by in vitro selection of RNA against a phosphate transition-state analogue. Chun S-M, Jeong S, Kim J-M, Chong B-O, Park H, Yu J: J Am Chem Soc 1999, 121:10844-10845. • Significance: RNA biopolymers have attracted great interest as potential catalysts because of their unique conformations, molecular diversity, ease of generation and, of course, their biological importance — especially in support of the ‘RNA world’ hypothesis. Many attempts at introducing catalytic sites in natural and synthetic polymers by deploying transition state analogues have been reported, but only a few successes have been reported for the selection of RNA catalysts using hydrophobic transition-state analogues. The authors believe their results represent the first selection of a ribozyme that catalyses a reaction which requires nucleophilic or general base-activated nucleophilic substitution at the carbonyl functionality. Also suggested are possible applications of RNA molecules as antibody substitutes against hydrophobic molecules such as cholesterol and other steroid hormones. Findings: A cholesterol phosphate molecule, chosen as the transition-state analogue and hapten, was immobilised on agarose. The RNA pool was enriched by six cycles of affinity chromatography and elution procedures; 11 clones were generated. Of these clones, one showed a high binding constant with the hapten, 250-fold greater than the original RNA. This binding is manifested in a 110-fold rate enhancement for hydrolysis of the p-nitrophenyl substrate over the control reaction. The catalysis displayed saturation kinetics and was inhibited strongly by the transition-state analogue.

Biopolymers Selected by Sabine Flitsch and Philip AS Lowden* Edinburgh University, Edinburgh, UK *University of Exeter, Exeter, UK

Function of glycosyltransferase genes involved in urdamycin A biosynthesis. Trefzer A, Hoffmeister D, Kunzel E, Stockert S, Weitnauer G, Westrich L, Rix U, Fuchser J, Bindseil KU, Rohr J, Bechthold A: Chem Bio 2000, 7:133-142. • Significance: The unambiguous characterisation of all glycosyltransferases involved in the biosynthesis of urdamycin A opens up the exciting possibility of re-engineering sugar sidechains of such polyketide antibiotics and hence generation of analogues with improved therapeutic profiles. Findings: The four genes for the glycosyltransferases involved in the biosynthesis of the sugar sidechains of the polyketide antibiotic urdamycin A have been unambiguously assigned and characterised using gene inactivation and expression experiments. Some of the glycosyltransferases display acceptor-substrate flexibility, which should make them useful catalysts for the combinatorial synthesis of novel polyketide analogues. Fmoc-based synthesis of peptide-alpha-thioesters: application to the total chemical synthesis of a glycoprotein by

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native chemical ligation. Shin S, Winans KA, Backes BJ, Kent SBH, Ellman JA, Bertozzi CR: J Am Chem Soc 1999, 121:11684-11689. •• Significance: This is the first example of the synthesis of a glycoprotein by total chemical synthesis using native chemical ligation. This methodology should find wide application in glycoprotein research. Findings: The synthesis of proteins by ligation of unprotected peptide segments is one of the most promising methods for the total synthesis of medium-size to large-size proteins. This paper describes the first application of this method to glycoproteins, with the small (82–aa) antimicrobial glycoprotein diptericin as the target. The novel use of a combination of the alkanesulfonamide ‘safety-catch’ linker and Fmoc protection group strategies was found to be highly effective for the synthesis of the appropriate glycopeptide-α-thioester fragment. This could then be coupled to a glycopeptide bearing an amino-terminal cysteine residue to yield the glycoprotein with two sites of glycosylation. Mechanism of action and identification of Asp242 as the β-D-glucatalytic nucleophile of Vibrio furnisii N-acetyl-β α-Lcosaminidase using 2-acetamido-2-deoxy-5-fluoro-α idopyranosyl fluoride. Vocadlo DJ, Mayer C, He S, Withers SG: Biochemistry 2000, 39:117-126. • Significance: This is the first example of a covalent glycosylenzyme intermediate found in a glycosidase acting on 2-acetamido sugars. Findings: 2-Acetamido-2-deoxy-5-fluoro-α-L-idopyranosyl fluoride was designed and prepared as a mechanism- based inhibitor of N-acetyl-β-D-glucosaminidase enzymes. It was hydrolysed slowly by the Vibrio furnisii enzyme and kinetic parameters were measured. A high steady-state concentration of a glycosyl enzyme intermediate was observed by mass spectrometry, and tandem mass spectrometric analysis of proteolytic digests revealed the catalytic nucleophile to be Asp242. This mechanism for a family 3 N-acetyl-β-D-glucosaminidase is in contrast to that of family 20 enzymes, which employ anchimeric assistance from the acetamido group. Glycosyl fluorides can function as substrates for nucleotide phosphosugar-dependent glycosyltranferases. Lougheed B, Ly HD, Wakarchuk WW, Withers SG: J Biol Chem 1999, 274:37717-37722. •• Significance: The use of glycosyl fluorides in place of nucleotide sugar donors could lead to a dramatic reduction in the expense and difficulty of oligosaccharide synthesis using glycosyltransferases. These results also lay the foundations for more detailed mechanistic studies of these fascinating enzymes. Findings: α-Galactosyl fluoride is demonstrated to be an effective substitute for uridine-5-diphosphogalactose (UDP-Gal) as a substrate for the α-galactosyltransferase from Neisseria meningitidis. Different glycosyl acceptors were selectively glycosylated and reactions could be scaled up to the multi-milligram level. Catalytic quantities of uridine-5-diphosphate were found to be essential for enzyme activity and UDP-Gal was synthesised in the absence of other acceptors. These results imply that glycosyltransferases may proceed via glycosyl–enzyme intermediates. DNA computing on surfaces. Liu Q, Wang L, Frutos AG, Condon AE, Corn RM, Smith LM: Nature 1999, 43:175-178. • Significance: This is the first published example of DNA computation performed on a solid support. These results provide a

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further demonstration of the potential power of DNA-based computational protocols and point the way towards automated DNA computers. Findings: An example of a particularly difficult problem in mathematical logic (3-SAT) was solved using DNA-based methods. DNA ‘words’, representing all sixteen possible solutions, were immobilised on a gold surface. Each round of computation involved hybridisation of complementary sequences satisfying each part of the problem, nuclease digestion of unhybridised sequences and denaturation of double-stranded DNA. Final readout of the solution was performed by PCR and hybridisation to an addressed array. Much more complicated problems could be tackled with relatively few extra steps. Single-base mismatch detection based on charge transduction through DNA. Kelley SO, Boon EM, Barton JK, Jackson NM, Hill MG: Nucleic Acids Res 1999, 27:4830-4837. •• Significance: This is an exciting new strategy for the detection of single base mismatches, with advantages over the traditional methods that measure the strength of hybridisation. The electrochemical response is insensitive to sequence context and mismatch identity, enabling detection of mismatches with comparable stability to Watson–Crick base pairs; additionally, probe molecules are non-covalently bound, simplifying sample preparation. Findings: Films of double-stranded DNA were prepared on the surface of gold electrodes. The electrochemical response of redox-active intercalators was found to be sensitive to the presence of a range of single base-pair mismatches in different sequence contexts. Similar results were obtained with films formed by reversible in situ hybridisation. The sensitivity could be enhanced by coupling the electrode-intercalator electron transfer to electrocatalytic processes in solution. Specific mutations in a viral RNA pseudoknot drastically change ribosomal frameshifting efficiency. Kim Y-G, Maas S, O’Neill A, Rich A: Proc Natl Acad Sci USA 1999, 96:14234-14239. • Significance: These studies are an important step in our understanding of how RNA three-dimensional structure can have a profound influence on its biological function and provides useful data for the development of antiviral drugs that target frameshifting. Findings: Ribosomal frameshifting is an essential process in the regulation of protein synthesis in many viruses. In most cases, RNA pseudoknots are required to stimulate frameshifting. On the basis of the previously determined crystal structure of the beet western yellow virus pseudoknot, a series of mutations were introduced to probe the contribution of different structural features to efficient frameshifting. Disruption of

nucleotides required for key tertiary interactions reduced frameshifting efficiency, while some other mutations caused an unexpected decrease or even increases in efficiency, demonstrating probable pseudoknot–ribosome interactions. Selected by Richard Newman Imperial Cancer Research Fund, London, UK

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-bisphosphate-specific PH domain. Hodgkin MN, Masson MR, Powner D, Saqib KM, Ponting CR, Wakelam JO: Curr Biol 2000, 10:43-46. •• Significance: The signalling pathway leading to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. The localisation and regulation of PLD is shown to be dependent upon a phosphatidylinositol-4,5-bisphosphate-specific pleckstrin homolgy (PH) domain. Findings: Sequence analysis indicating the presence of a PH domain was used to instigate studies, using surface plasmon resonance on supported lipid monolayers, of the binding of PLD1b to phosphatidylethanolamine, phosphatidylcholine and an activating lipid, phosphatidylinositol 4,5-bisphosphate. Results were obtained that suggest a key role for the PH domain in PLD function. Confocal microscopy results confirmed that human PLD1 containing a functional PH domain is critical in regulating the subcellular localisation of PLD1b, possibly by mediating its interaction with polyphosphoinositide-containing membranes, or possibly by inducing a conformational change in the enzyme, thereby regulating catalytic activity. Trienoic fatty acids and plant tolerance of high temperatures. Murakami Y, Tsuyama M, Kobayashi Y, Kodama H, Iba K: Science 2000, 287:476-479. •• Significance: Photosynthesis is inhibited by moderate to high temperatures but the causes of this inhibition are not clear. This study shows that the number of unsaturated lipids in the thylakoid membrane of chloroplasts — which contain the light absorbing system, electron import chain and ATP synthase — is important in determining a plant’s ability for growth and photosynthesis at temperatures of 35°C or more. This report may provide valuable information about the best approach to engineering plants that can carry out photosynthesis in the face of heat stress. Findings: Transgenic tobacco plants in which the gene encoding chloroplast omega-3 fatty acid desaturase, which synthesises trienoic fatty acids, was silenced, contained a lower level of trienoic fatty acids than wild-type plants and grew much better than controls at higher temperatures.