paper alert

471

A selection of interesting papers that were published inthe two months before our press date in major journalsmost likely to report significant results in chemical biology.

• of special interest•• of outstanding interest

Current Opinion in Chemical Biology 2001, 5:471–479

Selected by Lisa MatthewsDana-Farber Cancer Institute, Boston, USA

e-mail: [email protected]

• A literature network of human genes for high-throughputanalysis of gene expression. Jenssen TK, Laegreid A,Hovig E: Nat Genet 2001, 28:21-28.Significance: This paper describes a novel method for gener-ating annotated gene networks that can be used to analysegene expression on a genome-wide scale.Findings: A network of over 13,700 genes was generated byassigning ‘links’ between genes that are co-cited in MEDLINE

records. The genes in these networks were annotated using theMeSH and Gene Ontology terms. The authors demonstrate theaccuracy of this approach by manually examining the biologicalrelevance of 1000 predicted associations. Between 60% and72% of predicted links were correct. In addition, the networkscorrelated well with gene clusters observed in publishedmicroarray analyses.

• Identification of novel small RNAs using comparativegenomics and microarrays. Wasserman KM, Repoila F,Rosenow C, Storz G, Gottesman S: Genes Dev 2001,15:1637-1651.Significance: This paper describes an approach that combinesboth comparative and functional genomics to identify novelsmall RNAs.Findings: sRNAs are difficult to identify. Although previouslycharacterized sRNA sequences, found in intergenic regions,are highly conserved between Escherichia coli andSalmonella, they cannot be identified on the basis ofsequence determinants. The authors apply both BLAST

analyses of intergenic regions of E. coli, Salmonella andKlebsiella pneumonia and high-density oligonucleotide

probe arrays to identify 17 new sRNAs, more than doublingthe number previously known.

•• Global analysis of protein activities using proteomechips. Zhu H, Bilgin M, Bangham R, Hall D, Casamayor A,Bertone P, Lan N, Jansen R, Bidlingmaier S, Houfek T et al.:Sciencexpress 2001:1-10.Significance: This paper describes the construction of the firstnear complete proteome microarray chip and its use in proteome-wide screens for protein interactions.Findings: The authors describe the productions of a proteinmicroarray containing 80% of yeast proteins and the use of thisarray in identifying proteins that bind calmodulin and phospho-tidylinositid on a proteome-wide scale. The authors predict that80% of the arrayed proteins are full length and are present in suf-ficient quantity for screening and that similar approaches could beapplied for comparable analyses in humans and other eukaryotes.

Selected by Nicola PohlIowa State University, Ames, USA


•• Generality of peptide cyclization catalyzed by isolatedthioesterase domains of nonribosomal peptide syn-thetases. Kohli RM, Trauger JW, Schwarzer D, Marahiel MA,Walsh CT: Biochemistry 2001, 40:7099-7108.Significance: Peptide and polyketide natural products areoften isolated as constrained macrocyclic structures that aredifficult to achieve by chemical cyclization methods. The termi-nal section (a thioesterase) of the large biosynthetic proteinsresponsible for these macrocycles facilitate cyclization andconcomitant cleavage from the protein of the growing chain.Surprisingly, these thioesterase domains are also functional asmacrocyclization catalysts when cloned and expressed as separate proteins and can serve as reagents for the in vitrosynthesis of novel macrocycles.Findings: The thioesterase domains of the three different synthases that produce tyrocidine, gramicidin S, and cyclic lipoheptapeptide surfactin A were expressed as individual proteins and used to make macrocycles from various syntheticpeptides. The tyrocidine thioesterase could generate an array ofmacrocycles from N-acetyl cysteamine thioesters of 6–14 aminoacid peptides with comparable kinetic efficiencies. A solid-phasesynthesis strategy for these peptide analogs is also reported.

•• Assessing the balance between protein–protein interac-tions and enzyme–substrate interactions in the channelingof intermediates between polyketide synthase modules.Wu N, Tsuji SY, Cane DE, Khosla C: J Am Chem Soc 2001,123:6465-6474.Significance: Modular polyketide synthases (PKSs) responsi-ble for the biosynthesis of erythronolide are made of modulesthat contain several enzymatic domains that carry out a seriesof reactions to build a polyketide chain from a substrate that iscovalently linked to the acyl carrier protein (ACP) domain. One

Chemical biologyPaper alert

Contents (chosen by)

471 Proteomics and genomics (Matthews)471 Biocatalysis and biotransformation (Pohl)472 Bio-inorganic chemistry (Cammack)473 Combinatorial chemistry (Hall)474 Next generation therapeutics (Projan)475 Analytical techniques (Cass)476 Mechanisms (Stewart)477 Model systems (Roberts and Sanders)477 Biopolymers (Flitsch, Lowden and Newman)

Proteomics and genomics

Biocatalysis and biotransformation

strategy to expand the use of these synthases for the combina-torial biosynthesis of new polyketides relies on the fusion ofvarious intact modules; however, more information is neededabout the molecular recognition features of each module aswell as the mechanism of chain transfer between modules. Inthis paper, the channeling of covalently bound intermediates isshown to convey a significant kinetic advantage in chain processing, especially for poor substrates, thereby allowing theproteins incredible synthetic flexibility.Findings: The four diastereomers of 2-methyl-3-hydroxy-pen-tanoic acid were individually linked to form the thioester ofeither an ACP with a protein linker or of N-acetylcysteamine(NAC). These substrates were then assayed with recombi-nant modules 2, 5, and 6 of the erythronolide PKS, eachcontaining a protein linker that binds to the linker of the ACPon one end and to a thioesterase domain at the opposite end.The kcat and kcat/Km values were measured for the reactionsof each combination. The modules showed an intrinsic prefer-ence for certain diastereomers as previously shown, but werestill able to process the poor substrates into products whenpresented as ACP adducts, although no product wasdetected when the substrates were presented as NACadducts. The kcat for the reactions increased 10-fold to morethan a 100-fold when substrates were channeled by the ACPrather than introduced by diffusion. In addition, the transferstep from the donor ACP to the next ketosynthase domainwas shown to be a reversible reaction.

• Production of polyunsaturated fatty acids by polyketidesynthases in both prokaryotes and eukaryotes. Metz JG,Roessler P, Facciotti D, Levering C, Dittrich F, Lassner M,Valentine R, Lardizabal K, Domergue F, Yamada A et al.:Science 2001, 293:290-293.Significance: The biosynthesis of polyketide natural productsand long-chain fatty acids share some of the same reactions butthe enzymes involved in each pathway are unique. Crossover ofany of these enzymes into the other biosynthetic pathway couldpotentially expand greatly the diversity of compounds that couldbe produced using combinatorial biosynthesis.Findings: Genes encoding the biosynthetic pathway of the verylong chain polyunsaturated fatty acid eicosapentanoic acidwere sequenced from marine prokaryotes and eukaryotes andsome of the biosynthetic genes were surprisingly found to bemore similar to polyketide synthases than to fatty acid synthases. These newly discovered polyketide synthases areprobably unique in structure and mechanism to their previouslyidentified counterparts. Other enzymes that further functionalizethis class of polyunsaturated fatty acids were also discovered.

• Construction of desosamine containing polyketidelibraries using a glycosyltransferase with broad substratespecificity. Tang L, McDaniel R: Chem Biol 2001, 8:547-555.Significance: The glycoside components of polyketide naturalproducts are critical in the biological activities of this importantclass of therapeutic, yet are difficult to synthesize and attach tothe polyketide. A combinatorial biosynthetic approach thatwould allow the incorporation of different glycoside analogswould greatly increase the size and diversity of polyketidelibraries for screening purposes.Findings: The glycosyl transferase that transfers the deoxy-sugar desosamine to picromycin was found to have broadsubstrate tolerance for various polyketide acceptors. The geneencoding this desosaminyl transferase was inserted into the

chromosome of a heterologous bacterial host along with othernecessary genes for desosamine biosynthesis. Plasmidsencoding the biosynthesis of genetically modified polyketideswere then screened in this host and a library of different desosaminyalted polyketides was obtained. The addition of thedesosamine to the members of this library resulted in somecompounds that showed antibiotic activity.

• Nativelike enzyme properties are important for optimumactivity in neat organic solvents. Griebenow K, Vidal M, Baéz C,Santos AM, Barletta G: J Am Chem Soc 2001, 123:5380-5381.Significance: The optimization of enzymatic reactions in pureorganic solvents is extremely difficult, but the rewards caninclude an increased substrate range and novel biocatalytictransformations. A general procedure to quickly analyze theeffect of solvents on potential enzyme activity would greatly aidin adapting enzymatic catalysis to organic solvents.Findings: Reasoning from evolutionary adaptation argumentsthat optimal catalysis in organic solvents will only result whenboth the structure of the enzyme and its conformational mobil-ity most closely match those of the enzyme in aqueous solution,the authors measured the change in the conformational mobil-ity and secondary structure of subtilisin Carlsberg with variousadditives in dioxane by thermal denaturation. Conditions thatcaused the secondary structure and conformational mobility ofthe enzyme to be most like the optimal structure and mobility inwater also resulted in the highest activity in organic solvent.

Selected by Richard CammackKing’s College London, London, UKe-mail: [email protected]

•• Femtomolar sensitivity of metalloregulatory proteinscontrolling zinc homeostasis. Outten CE, O’Halloran TV:Science 2001, 292:2488-2492.Significance: The free or loosely bound Zn2+ ions in the cellare at extremely low concentrations, rather than, as previouslybelieved, available as a cytosolic pool. The cellular distributionof zinc, as for iron and copper, is tightly controlled.Findings: Escherichia coli cells growing on metal-depletedmedium, accumulated significant amounts of zinc, correspond-ing to a total concentration of 0.2 mM. However, theconcentration of free Zn2+ was estimated, from the activity of the Zur and ZntR DNA-binding proteins, to be six orders ofmagnitude less than one atom of free Zn2+ per cell. These twometalloregulatory proteins control the uptake and efflux of zinc,so that the intracellular concentration of zinc is maintainedwithin a narrow range around 0.2 fM.

• Fast deuterium access to the buried magnesium/man-ganese site in cytochrome c oxidase. Florens L, Schmidt B,McCracken J, Ferguson-Miller S: Biochemistry 2001,40:7491-7497.Significance: In order to preserve electrostatic neutrality, electron-transfer proteins have specific pathways for transfer ofprotons, as well as electron-transfer chains. For cytochrome coxidase, this contributes to the mechanism of proton pumpingacross the membrane. It is shown that a Mg2+ or Mn2+ ion at aconserved site in cytochrome c oxidase is kinetically competentto be part of the proton-transfer pathway.

472 Paper alert

Bio-inorganic chemistry

Findings: The ion-binding site lies in a predicted water channel,leading from the surface to the CuA and heme a in the crystalstructure of the protein. After mixing with D2O and rapid freezing,the presence of deuterium coordinated to the Mn2+ ion wasdetected by electron spin-echo spectroscopy. All three water mol-ecules bound to the ion were exchanged within 11 ms, indicatingan apparent proton transfer rate constant higher than 3000 s–1,which is fast enough to participate in the enzyme reaction.

• The giant protein AHNAK is a specific target for the cal-cium- and zinc-binding S100B protein — potentialimplications for Ca2+ homeostasis regulation by S100B.Gentil BJ, Delphin C, Mbele GO, Deloulme JC , Ferro M,Garin J, BaudierJ: J Biol Chem 2001, 276:23253-23261.Significance: S100 proteins are small calcium-binding proteins that are expressed tissue-specifically. S100B is abundant in brain, where it is overexpressed in Alzheimer’s disease and Down’s syndrome. The target for binding ofS100B is desmoyokin, the product of the human gene AHNAK.This high-mass phosphoprotein (Mr 700,000) appears to beinvolved in calcium homeostasis.Findings: Cloned S100B-binding fragments of AHNAK, identified by their affinity to S100 on a column, were found torepresent a repeated motif. Binding was found to be dependenton Ca2+ and further enhanced by Zn2+. Calmodulin was notbound at all. AHNAK has been found associated with L-typecalcium channels, and also phospholipase C-γ, which by releas-ing inositol 1,4,5-triphosphate, assists the release of calciumfrom intracellular stores.

• Tributyltin interacts with mitochondria and inducescytochrome c release. Nishikim A, Kira Y, Kasahara E, Sato EF,Kanno T, Utsumi K, Inoue M: Biochem J 2001, 356:621-626.Significance: Alkyl-tin complexes, used to protect ships fromfouling by invertebrates, are known to have a number of toxiceffects on mitochondria. Here it is shown that tributyltin, apotent inducer of apoptosis, causes release of cytochrome c.This effect is independent of the previously known effects oforganotin compounds, namely induction of mitochondrialswelling and the inhibition of ATP synthase.Findings: At concentrations above 5 nmol/mg protein, tributyltininduced release of cytochrome c from rat-liver mitochondria.Similar concentrations of tributyltin were shown to prevent thebinding of the adenine nucleotide translocator to phenylarsineoxide agarose, showing that it bound to vicinal thiols. The pro-posed mechanism is that tributyltin inhibits the translocator,leading to opening of the permeability transition pore, decreas-ing the membrane potential and releasing cytochrome c.

Selected by Dennis HallUniversity of Alberta, Edmonton, Alberta, Canada


• Dynamic deconvolution of a pre-equilibrated dynamiccombinatorial library of acetylcholinesterase inhibitors.Bunyapaiboonsri T, Ramstrôm O, Lohmann S, Lehn J-M,Peng L, Goeldner M: ChemBioChem 2001, 2:438-444.Significance: For dynamic combinatorial chemistry to be usefulin enzyme screening applications, efficient assays are requiredin order to quickly identify active library members that are

self-amplified upon binding to the enzyme. To this end, this workpresents the first application of a deconvolution approach tothe case of dynamic libraries.Findings: An equilibrating library of bis-cationic acylhydrazonecompounds was made based on the combination of fourhydrazides with a set of four monoaldehydes and five dialdehy-des in aqueous media. This library was screened againstacetylcholinesterase enzyme (from the electric ray Torpedo)based on the knowledge that some diammonium compoundsspaced with a long interammonium spacer have the ability toinhibit the active site of such enzymes. In this case, because ofthe relative instability of the targeted enzyme under the pH 4.0conditions required for equilibration of the library from its com-ponents, the library was pre-equilibrated before addition of theenzyme. A dynamic deconvolution strategy was applied inwhich each library building block is sequentially omitted in aseries of assays. The nature of the most important buildingblocks can then be deduced from the biological activity of eachsubmixtures. This allowed the identification of a potent bis-pyri-dinium inhibitor (inhibition constant = 1.09 nanomolar).

• Precipitons-functional protecting groups to facilitateproduct separation: applications in isoxazoline synthesis.Bosanac T, Yang J, Wilcox CS: Angew Chem Int Ed Engl2001, 40:1875-1879.Significance: There is a need for new methods for productisolation that combine the advantages of solution- and solid-phase synthesis and thus can help facilitate and acceleratelibrary chemistry. The controlled precipitation technique presented in this paper provides a new alternative to otherphase transfer techniques.Findings: A stilbene-containing protecting group containing analcohol anchor was synthesized. Whereas the cis isomer is soluble in several solvents, the trans isomer, accessible bychemical or photochemical isomerization, was found insolublein ether, hexanes, and methanol. Such a group of atoms thatcan be attached on purpose to a substrate then isomerized tofacilitate product isolation by precipitation is termed a ‘precipi-ton’. Therein, several alkenoates were coupled with theprecipiton and were reacted with several types of nitrile oxidesin ether. After completion of the reaction, the ether was evaporated and the resulting crude isoxazoline products weredissolved in THF. Addition of diphenyl disulfide followed byheating the mixture to reflux caused the desired cis-transisomerization of the stilbene group and consequent precipita-tion of the trans-stilbene conjugated isoxazoline products.Soluble by-products such as the furoxan, formed from theexcess nitrile oxide, were eliminated easily through rinsing the precipitate, thereby providing a pure solid.

• Use of diversity-oriented synthesis to discover galan-thamine-like molecules with biological properties beyondthose of the natural product. Pelish HE, Westwood NJ,Feng Y, Kirchhausen T, Shair MD: J Am Chem Soc 2001,123:6740-6741.Significance: Small-molecule libraries based on natural prod-uct scaffolds are usually made with the goal of improving theknown biological properties of the parent natural product. Thiswork shows that natural-product-based libraries can also beused successfully to discover molecules with novel, unsus-pected biological properties.Findings: A 2527-membered library of compounds was syn-thesized based on the postulated biosynthesis of galanthamine,

Paper alert 473

Combinatorial chemistry

a potent acetylcholinesterase inhibitor. The initial precursor wascoupled to a siloxy linker onto macrobeads (500–600 µm diam-eter). Construction of the galanthamine scaffold was effectedby the biomimetic oxidation of a diphenol precursor. Then, diver-sity-generating operations included aryl ether formation via aMitsunobu reaction, conjugate addition onto an enone, sec-ondary amine acylation (or alkylation) and imine formation on acyclohexanone ring. Each bead was arrayed into single wells of384-well plates and the compounds were liberated by treat-ment with hydrofluoric acid. The library was eventually screenedfor the discovery of compounds that interfere with the secretorypathway in a cell-based phenotypic assay using a fluorescentfusion protein. One compound, thereafter called secramine,was identified as a potent inhibitor of protein trafficking from theGolgi apparatus to the plasma membrane down to 2 micromo-lar concentration. Galanthamine itself had no such activity,thereby confirming the potential of natural-product-basedlibraries to turn up compounds with novel biological properties.

• Parallel synthesis and biocatalytic amplification of across-conjugated cyclopentenone library. Jang WB, Hu H,Lieberman MM, Morgan JA, Stergiades IA, Clark DS, Tius MA:J Comb Chem 2001, 3:346-353.Significance: There is a need for new methods and strategiesthat can optimize the number of analogues around a given scaffold in diversity-oriented synthesis. This work shows thatbiocatalytic reactions can help amplify the functional diversityand number of analogues from small-molecule libraries firstassembled using chemical methods.Findings: A small model parallel library of 28 cross-conjugatedcyclopentenones was made in solution phase from the cyclocon-densation of three allenyl ethers and 10 α-β-unsaturated amides.The size and diversity of the library were then expanded using bio-catalytic transformations performed on selected library memberssuch as halohydratation by soybean peroxidase, ketone reductionwith baker’s yeast, acylation by protease/lipase mixtures, and oxidations of primary alcohols with soybean peroxidase. The complete library was tested in different screens for anticancer,antifungal, antibacterial, and antimycobacterial activities. Activecompounds were found in all screens, providing promising leadsfor eventually designing second-generation libraries.

• Sequence-selective peptide detection by small syntheticchemosensors selected from an encoded combinatorialchemosensor library. Iorio EJ, Shao Y, Chen C-T, Wagner H,Still WC: Bioorg Med Chem Lett 2001, 11:1635-1638.Significance: The development of selective chemosensors forsmall biomolecules is an important but rather difficult task. Asshown in this communication, combinatorial approaches can help accelerate the discovery of novel chemosensors thatcan distinguish between random peptides.Findings: A small 448-membered tag-encoded library of two-armed chemosensors was made according to a modular designreported previously in the same laboratory. These arms areassembled from a diazo dye unit with aromatic acids and cyclicdiamines as diversity components. The resulting receptors arealso functionalized with a dansyl unit and are thus based on afluorescence energy transfer mechanism for detection.Screening for binding against two model tripeptides, throughlooking for fluorescence enhancement in chloroform, revealedtwo distinct chemosensors. Further studies and binding con-stant measurements in solution allowed the identification of themost important binding elements in the receptors. This work

constitutes a successful proof of concept, demonstrating thesuitability of a combinatorial approach towards the eventual discovery of specific chemosensors for any small peptide.

• Combinatorial synthesis of cholesterol ester transfer protein–mRNA ligands and screening by nondenaturinggel-electrophoresis. Baumann M, Bischoff H, Schmidt D,Griesinger C: J Med Chem 2001, 44:2172-2177.Significance: Because of its high structural and functionaldiversity, RNA remains a difficult target for rational drug design.Provided that relatively high-throughput assays such as the onedescribed in this article are used, combinatorial approachescan be of significant help in identifying promising ligands forlead development.Findings: A 625-membered library of heptapeptides ofsequence Lys-XXXX-Lys-Cys-NH2 was assembled bysplit-pool solid-phase synthesis. The library was randomizedat four positions (X) either with cationic residues (lysine andarginine) to enable high-affinity to polyanionic RNA, or withhydrophobic residues (tyrosine, leucine and isoleucine) toenhance binding selectivity. The library was targeted againstthe 23-nucleotide RNA from the 5′-untranslated region of thecholesterol ester transfer protein (CETP). After cleavage fromthe resin, the peptides were conjugated via the cysteine to apolyethylene-glycol linker in order to obtain a larger retarda-tion effect in the gel-electrophoresis affinity assay. Librarymembers were screened with the target RNA in 25 mixturescontaining 25 different compounds in each lane of a non-denaturing polyacrylamide gel. Then, in a significant effortrequired to avoid sophisticated decoding techniques, all compounds from mixtures causing the largest gel-shift werere-synthesized and ran individually in the gel-shift assay touncover the most active sequences from the original mixture.The 27-nucleotide (HIV-1) TAR RNA was used to controlbinding specificity for the tightest binding peptides. Thenature of the interactions in the complex between the mostpromising ligands and the RNA target were further investi-gated by circular dichroism, ultraviolet measurements, andnuclear magnetic resonance. In particular, one peptide(Lys-Tyr-Lys-Leu-Tyr-Lys-Cys-NH2) showed micromolar affinityto the CETP mRNA.

Selected by Steven ProjanWyeth-Ayerst Research, Pearl River, New York, USA


• Identification of a plasmid encoding SHV-12, TEM-1, anda variant of IMP-2 metallo-ββ-lactamase, IMP-8, from a clinical isolate of Klebsiella pneumoniae. Yan J-J, Ko W-C,Wu J-J: Antimicrob Agent Chemother 2001, 45:2368-2371.Significance: As multidrug resistance among Gram-positivebacteria has resulted in virtually ‘pan-resistant’ strains of staphy-lococci and enterococci, there has been limited attention to asimilar phenomenon taking place among Gram-negative bacte-ria, especially the respiratory pathogens Acinetobacter baumaniiand Klebsiella pneumoniae. Increasingly, nosocomial isolates ofthese pathogens have demonstrated broad antibiotic resis-tance. This publication documents a strain of K. pneumoniaethat carries three different β-lactamase genes (including oneencoding a metallo-β-lactamase), rendering the strain resistant

474 Paper alert

Next generation therapeutics

to virtually all β-lactam antibiotics. This report is ostensibly similar to one published earlier this year (Yigit et al.,Antimicrob Agent Chemother 45:1151); however, in thisinstance the authors identified a metallo-β-lactamase gene thatcontributed towards the carbapenem resistance of their clinicalisolate, a major difference from the previous publication.Findings: The authors were able to transform a susceptible donorstrain of Escherichia coli, by trasnconjugation, to β-lactam resistance. A single plasmid, pEKO787D1, apparently carried thegenes coding for three different β-lactamases. This was confirmedby isoelectric focusing and antimicrobial susceptibility testing.Both the donor strain and the transconjugant had the same β-lac-tam resistance profile with good susceptibility demonstrated onlywith a combination of aztreonam and clavunlanic acid (a combi-nation not approved in the USA). The authors proceeded tosequence the metallo-β-lactamase gene and found that it differedfrom the imp-2 gene by four nucleotides (which would result intwo amino acid differences). This novel metallo-β-lactamase gene,found on an integron, was designated imp-8.

• Efficacy of linezolid in treatment of experimental endocardi-tis caused by methicillin-resistant Staphylococcus aureus.Dailey CF, Dileto-Fang CL, Buchanan LV, Oramas-Shirey MP,Batts DH, Ford CW, Gibson JK: Antimicrob Agent Chemother2001, 45:2304-2308.Significance: It has been a widely held dogma that the treatmentof deep-seated bacterial infections requires the use of bacterici-dal agents. However, in this publication, the authors demonstratethat, for just such an infection (infective endocarditis), linezolid, astrictly bacteriostatic agent can provide a significant reduction inviability of methicillin-resistant Staphylococcus aureus (MRSA).Findings: The authors tested their novel, Gram-positive, antibac-terial agent linezolid (which is a synthetic, bacteriostatic proteinsynthesis inhibitor) in a rabbit model of infective endocarditis. Inthis model, a catheter is inserted into the right carotid artery andthrough the left ventricle, and bacteria (in this case MRSA) areintroduced a day later distally (through an ear vein) and varioustherapies are instituted some 18–24 hours later. The catheter uniformly becomes the focus of a bacterial vegetation and thenumber of viable bacteria are determined at some point after theintroduction of the bacteria (here, five days after initiation of therapy). The authors demonstrated a dose-dependent effect of linezolid, achieving a six-log reduction in viable bacteria at the highest dose tested compared with in untreated controls. Thesereductions were also reflected in enhanced survival of the infectedrabbits and parallel reductions in viable counts recovered from therabbits’ kidneys. Although comparable levels of reduction in viabil-ity required amounts of linezolid that were threefold higher than forvancomycin (75 versus 25 mg/kg). This difference mirrors the relative level of in vitro potency of the two agents. Perhaps moreremarkable is that the linezolid was administered orally whereasthe vancomycin was administered intravenously.

Selected by Tony CassImperial College of Science, Technology and Medicine, London, UK


•• Quantum-dot-tagged microbeads for multiplexed opticalcoding of biomolecules. Han M, Gao X, Su JZ, Nie S:Nat Biotechnol 2001, 19:631-635.

Significance: High-throughput, multiplex methods of analysiscome in many different formats; for example, microarrays usespatial encoding to relate molecular identity and location.Where multiplex assays are carried out with bead-supportedreagents, an encoding signal needs to be used to relate molecular identity and bead identity. Fluorescence has beenused for this encoding process; however, the number of codesis highly dependent on the intensity and spectral width of theencoding species. Moreover, the emitters should all be capableof excitation at the same wavelength. In this paper, quantumdots (QDs) embedded in microbeads are presented as efficientencoding materials.Findings: QDs made from cadmium sulfide capped with zincsulfide and of varying size were embedded in poly(styrene)beads. It was shown by spectral imaging that the emissioncharacteristics were largely unaffected by this process and typical spectral half widths were around 20 nm. Encoding useda combination of wavelength and intensity and it was demon-strated that these beads could be used in fluorescence-basedDNA hybridisation schemes, in which the fluorescence on the DNA had emission characteristics well separated from theencoding emission.

•• Determination of protease cleavage site motifs usingmixture-based oriented peptide libraries. Turk BE, Huang LL,Piro ET, Cantley LC: Nat Biotechnol 2001, 19:661-667.Significance: The completion of the first draft of the humangenome and the development of powerful bioinformatics toolshas enabled sequence-based assignment of function to manypreviously unidentified proteins. Although such approaches canassign a gene sequence to a particular class of protein, it is stillnecessary to develop high-throughput methods to rapidlydetermine exact substrate specificities of a putative enzyme.This paper describes a rapid method to ascertain the cleavagesite specificity of proteases using mixed peptide librariesFindings: A completely random mixture of 12-mer peptides,acetylated at the amino terminus was treated with variousmatrix metalloproteases and the reaction products then subjected to amino-terminal sequencing. Because of theblocked amino terminus in the original peptides, the sequencesare derived from the carboxy-terminal fragment peptides. The relative amount of each amino acid released during an Edmandegradation cycle reflects the relative specificity of the protease. A second library, unblocked on the amino terminusand carrying a carboxy-terminal biotin, was used to determinethe amino-terminal cleavage motif. Using the motifs determinedin this fashion, putative substrates could be identified throughsequence database searches.

• Image metrics in the statistical analysis of DNAmicroarray data. Brown CS, Goodwin PC, Sorger PK:Proc Natl Acad Sci USA 2001, 98:8944-8949.Significance: Spotted DNA microarrays are amongst the com-monest approach to ‘global’ expression profiling. Typically, thespotted cDNA probes are hybridised to fluorescently labelledtarget DNA and the resulting fluorescent array imaged andanalysed. Increasing use of these microarrays have shown that,despite their undoubted power, they are prone to various artefacts that can confound interpretation.Findings: A variety of artefacts and sources of error were iden-tified in a 6200 element spotted array. An important source oferror arose from the estimation of background fluorescenceintensities; using the local background was not wholly

Paper alert 475

Analytical techniques

satisfactory as a correction method as the non-specific bindingof target appeared different between non-complementary DNAand plain glass surfaces. Other artefacts included clumping,scratches and dye separation. A statistical metric, the spot ratiovariability, was chosen to characterise the irregularity of thespots and used to assign significance to estimates of individualexpression ratios.

•• Fluorescent sensors for Zn2+ based on a fluoresceinplatform: synthesis, properties and intracellular distribu-tion. Burdette SC, Walkup GK, Spingler B, Tsien RY,Lippard SJ: J Am Chem Soc 2001, 123:7831-7841.Significance: The production of sensing molecules for intracellular imaging of important metabolites is a key area incell biology. Although probes for ions such as protons and calcium have been available for a number of years, numerousother species would benefit from the availability of similarreagents. Such fluorosensors should exhibit high affinity, highquantum yield and significant changes in emission wavelengthand/or intensity upon analyte binding. In this regard, zinc is animportant target analyte for neurobiology and this paperdescribes new zinc fluorosensors.Findings: Two fluorescein-based zinc sensors are described andcharacterised. They fulfil the criteria described above with affinitiesin the sub-nanomolar region, quantum yields of 0.9 and 3–5-foldchanges in fluorescence when bound to zinc and, in addition, arewater-soluble. The reagents were shown to be suitable forlabelling COS-7 cells ad subsequent fluorescence imaging.

• Guest-induced diminishment in fluorescence quenchingand molecule sensing ability of a novel cyclodextrin–pep-tide conjugate. Hossain MK, Hamasaki K, Takahashi K,Mihura H, Ueno A: J Am Chem Soc 2001, 123:7435-7436.Significance: Cyclodextrin (CD) molecules have long beenknown to acts as hosts for a variety of small-molecule guests.To use CDs as sensing elements, the basic CD structure needsto be elaborated such that the guest occupancy generates areadily measurable signal. To this end, helical peptide conju-gates of CD were synthesised that also carried pyrene andnitrobenzene groups on the peptide backboneFindings: The helical peptide had its pyrene fluorescencequenched because of the close proximity of the nitrobenzenegroup that was bound in the CD cavity. Displacement of thenitrobenzene from the cavity by a guest molecule such as lithocholic acid resulted in a relief of quenching.

Selected by Jon D StewartUniversity of Florida, Gainesville, Florida, USA


• Ethylbenzene dehydrogenase, a novel hydrocarbon-oxidiz-ing molybdenum/iron-sulfur/heme enzyme. Kniemeyer O,Heider J: J Biol Chem 2001, 276:21381-21386.Significance: This is the first example of an enzyme-mediated oxidation of a non-activated hydrocarbon that does not requireoxygen. The ability to couple stereoselective hydrocarbon oxidation with an inorganic acceptor (ferricenium hexafluorophos-phate) provides a new, potentially useful tool for biocatalyticasymmetric organic synthesis that avoids problems associatedwith nicotinamide cofactor recycling.

Findings: Cells of denitrifying bacteria Azoarcus sp. EbN1grown with ethylbenzene as the source of carbon and energyunder anaerobic conditions produced relatively high levels of eth-ylbenzene dehydrogenase activity (22 nmol/min/mg protein). Theoxidation was highly stereoselective and (S)-1-phenethyl alcoholwas the only observed product. Ethylbenzene dehydrogenase,found exclusively in the soluble fraction (100,000 g supernatant),was purified by conventional chromatographic methods underanoxic conditions. The 155 ± 15 kDa enzyme was determined tobe an αβγ trimer containing one molybdenum atom, four Fe4S4clusters and one heme moiety. Ethylbenzene was bound verytightly, and the KM value was estimated to be <2 µM. Apart fromn-propylbenzene, other alkylated aromatics were not oxidized.

•• Molecular structure of dihydroorotase: a paradigm forcatalysis through the use of a binuclear metal center.Thoden JB, Phillips GN Jr, Neal TN, Raushel FM, Holden HM:Biochemistry 2001, 40:6989-6997.Significance: The crystal structure of dihydroorotase withbound substrate and product revealed that each active siteunexpectedly contained two bound Zn(II) ions bridged by a car-bamoylated lysine sidechain, analogous to the arrangementfound in urease. These discoveries have significantly clarifiedthe catalytic mechanism of a critical enzyme in de novo pyrimi-dine biosynthesis, and sequence alignments suggest that thelessons may be broadly applicable.Findings: Dihydroorotase from Escherichia coli was co-crystal-lized with substrate (N-carbamoylaspartate) and the structurewas determined to 1.7 Å resolution. The dimeric protein con-tains two active sites: one contained bound substrate and theother product. Although prior work had suggested a mononu-clear Zn(II) site per subunit, clear evidence was found for twoZn(II) ions bridged by a carbamoylated lysine sidechain. Bothβ-carboxylate oxygens of bound substrate interacted with one ofthe two Zn(II) ions. This interaction might convert one oxygen toa sufficiently good leaving group for the intramolecular acylation,which is known to proceed without additional energy input.

• Discovery of a novel enzyme, isonitrile hydratase, involvedin nitrogen-carbon triple bond cleavage. Goda M, Hashimoto Y,Shimizu S, Kobayashi M: J Biol Chem 2001, 276:23480-23485.Significance: This is the first time that an enzymatic conversion ofthe isonitrile functional group has been observed. Some naturalproducts that contain this moiety are lethal poisons, and the bacterial enzyme characterized here may be a detoxification route.Findings: Soil microorganisms were cultured for two monthswith glycerol as the carbon source in the presence of 0.01%cyclohexyl isocyanide. This level of isonitrile is toxic to virtuallyall microbes; however, a Pseudomonas putida strain was eventually isolated that was resistant to these conditions.Resistance involved conversion to N-cyclohexylformamide by a59 kDa homodimeric protein that appeared to contain noexogenous cofactors. Sulfhydryl-modifying reagents signifi-cantly diminished the catalytic activity. Interestingly, theorganism was unable to grow on N-cyclohexylformamide, suggesting that isonitrile hydration was simply for detoxification,rather than the first step in catabolism.

•• Biosynthesis of the thiazole moiety of thiamin inEscherichia coli: identification of an acyldisulfide-linked pro-tein–protein conjugate that is functionally analogous to theubiquitin/E1 complex. Xi J, Ge Y, Kinsland C, McLafferty FW,Begley TP: Proc Natl Acad Sci USA 2001, 98:8513-8518.

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Significance: This study provides the first example of an acyldisulfide biosynthetic intermediate. In addition, the data stronglysupport previously postulated parallels between thiaminpyrophosphate biosynthesis and the ubiquitin pathway ofhigher organisms. Indeed, the former is suggested to be theevolutionary ancestor of the latter.Findings: The Escherichia coli ThiF protein was known to catalyze the formation of the carboxy-terminal acyl-AMP derivedfrom the ThiS protein and ATP. Given the similarity betweenproteins involved in thiamin biosynthesis and the eukaryoticubiquitin pathways, it was suspected that the ThiS-acyl-AMPmight subsequently form a covalent intermediate with ThiF. Thispossibility was probed by analyzing the reaction mixture bymass spectrometry, which suggested an acyl disulfide linkagebetween ThiS and ThiF (ThiS-CO-S-S-ThiF). Iodoacetatelabelling followed by mass spectral analysis indicated thatCys182 of ThiF was involved in this disulfide linkage. This sup-position was confirmed by the behavior of the ThiF Cys182Sermutant, which formed the ThiS-acyl-AMP but was unable toform the covalent acyl disulfide intermediate. Based on thesedata, a mechanism for thiamin biosynthesis was proposed.

Selected by Sarah L Roberts and Jeremy KM SandersUniversity of Cambridge, Cambridge, UK


•• Cystine-based oligoureas: a new class of hydrogen-bonding electroneutral anion receptors. Ranganathan D,Lakshmi C: Chem Commun 2001:1250-1251.•• A cyclic hexapeptide containing L-proline and 6-aminopi-colinic acid subunits binds anions in water. Kubik S,Goddard R, Kirchner R, Nolting D, Seidel J: Angew Chem IntEd Engl 2001, 40:2648-2650.Significance: In spite of the vast range of supramolecular recep-tors that have been reported over the past two decades, thereare only a handful of designs for anion receptors. Of thosedescribed, most are based on protonated macro, mono or poly-cyclic amines and employ coulombic interactions for recognition.In nature, however, it is neutral anion-binding proteins that regu-late the transport of anions, largely through the use of hydrogenbonds. In both of these papers, the authors introduce amino-acid-based macrocycles, neutral receptors that mimic natural systemsand represent a new class of electroneutral anion receptors. Findings: Ranganathan and Lakshmi have designed and synthesized L-cystine-based cyclic oligourea macrocycles, theresulting receptors containing multiple urea groups distrib-uted symmetrically all over the ring framework. The anionrecognition properties of the receptors were studied and itwas found that they show remarkable affinity and selectivity,according to size complementarity, for planar polyoxyanions.In the second example, Kubik et al. have utilised a cyclic hexapeptide containing L-proline and 6-aminopicolinic acid.This macrocycle is shown to bind anions such as halides andsulfates in a cavity formed by the aggregation of twocyclopeptide molecules.

• Crystal structure of a synthetic cyclodecapeptide for tem-plate-assembled synthetic protein design. Peluso, S,Rückle T, Lehmann C, Mutter M, Peggion C, Crisma M:ChemBioChem 2001, 2:432-437.

Significance: Topological templates are synthetic scaffoldsthat direct functional groups or structural elements in well-defined spatial arrangements. Ideally, such scaffolds mimicstructural and functional features of peptide ligands and pro-teins surfaces and they find widespread applications in proteinde novo design and peptide mimicry for drug design. In thispaper, the authors describe a new generation of scaffolds,aimed at effectively restricting the template conformation to adouble β-II′ hairpin similar to that found in gramicidin S in thesolid state.Findings: A de novo designed cyclodecapeptide was synthe-sised and its postulated three-dimensional structure in the solidstate and in solution was proven. The template was crystallizedand its structure determined by X-ray diffraction, showing anantiparallel β-sheet backbone conformation connected by twotype II′ β turn hairpins. The three-dimensional structure of theartificial template was then studied in solution by NMR andagain shown to be compatible with a β-sheet plane.

Selected by Sabine FlitschEdinburgh University, Edinburgh, UK


• Structure, mechanism and engineering of a nucleotidylyl-transferase as a first step toward glycorandomisation.Barton WA, Lesniak J, Biggins JB, Jeffrey PD, Jiang JQ,Rajashankar KR, Thorson JS, Nikolov DB: Nat Struct Biol2001, 8:545-551.Significance: Nucleotidyldiphosphate sugars are important co-factors for the in vivo and in vitro biosynthesis of glycocon-jugates. This report shows for the first time how a range ofnatural and unnatural sugar cofactors can be synthesised fromthe triphosphate and sugar phosphate using a rationally re-designed transferase as a catalyst. In combination with glycosyltransferases, these activated sugar cofactors should find applications in the enzymatic synthesis of glycoconjugate libraries.Findings: The structure of α-D-glucopyranosyl phosphatethymidylyltransferase in complex with substrate and productwas determined to 2.1 Å resolution. Structure-based engineer-ing of the transferase produced enzymes with altered selectivitytowards sugar phosphates.

• Parallel synthesis of oligosaccharide conjugatedenediynes onto silyl-linked solid support. Matsuda A, Doi T,Tanaka H, Takahashi T: Synlett 2001, 7:1101-1104.Significance: This paper shows how the synthesis of highlycomplex and chemically fragile glycoconjugates can beachieved on solid support.Findings: A 14-member library of enediyne glycoconjugateDNA cleaving agents with mono-, di- and trisaccharides wassynthesised on solid support (crowns) using trialkylsilane linkers.

• Carbohydrate self-recognition mediates marine sponge cellular adhesion. Haseley SR, Vermeer HJ, Kamerling JP,Vliegenthart FG: Proc Natl Acad Sci USA, 2001, 98:9419-9424.Significance: Carbohydrate–carbohydrate interactions on cellsurfaces are difficult to measure and few examples have beenconvincingly described so far. The present paper describes surface plasmon resonance studies on the self-recognition of a

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Biopolymers

defined carbohydrate epitope form a sponge showing that thisrecognition process is calcium-dependent.Findings: A known sulfated disaccharide epitope fromMicrociona prolifers was conjugated to bovine serum albuminand its self-association studied using surface plasmon reso-nance. The interaction was found to be weak but highly specificin the presence of calcium ion, but not magnesium or man-ganese ions. Furthermore, control studies with similar negativelycharged sugars showed that this interaction was not just elec-trostatic, but highly dependent on the carbohydrate structure.

• Bt toxin resistance from loss of a putative carbohydratemodifying enzyme. Griffitts JS, Whitacre JL, Stevens DE,Aroian RV: Science 2001, 293:860-864.Significance: The Caenorhabditis elegans gene responsiblefor insect resistance against insect toxins used in transgeniccrops has been identified and an interesting molecular mecha-nism for resistance based on loss of carbohydrate modificationhas been proposed.Findings: A Bacillus thuringiensis toxin resistance gene(bre-5) from C. elegans is predicted by BLAST and proteindomain searches and by biochemical data to encode for aβ-1,3-galactosyltransferase. It is proposed that this transferaseforms a carbohydrate structure on proteins and lipids at thegut surface that is necessary for toxin binding. In the absenceof these carbohydrate structures, the toxin cannot bind, result-ing in resistance. Such a mechanism would explain previousresults which showed that a single toxin can bind to receptorsthat are unrelated in protein sequence, but might carry thesame carbohydrate-recognition sequence.

• Galactan biosynthesis in Mycobacterium tuberculosis.Kremer L, Dover LG, Morehouse C, Hitchin P, Everett M,Morris HR, Dell A, Brennan PJ, McNeil MR, Flaherty C et al.:J Biol Chem 2001, 276:26430-26440.Significance: A novel glycosyltransferase involved in thebiosynthesis of some unique cell wall peptidoglycans inMycobacterium tuberculosis has been identified. The trans-ferase is unusual in that it catalyses two different chemicalsteps. It also has promise as a novel molecular target to combat mycobacterial infections.Findings: The polymerisation of the galactan region of themycolyl–arabinoglycan complex was defined at a biochemi-cal and genetic level to consist of alternating 1,5- and1,6-linked galactofuranosyl units. Interestingly, formation ofboth linkages was catalysed by one enzyme, which is anexception to the ‘one enzyme, one linkage’ rule generallyapplicable for glycosyltransferases.

Selected by Philip AS LowdenUniversity of Exeter, Exeter, UKe-mail: [email protected]

•• Direct observation of hole transfer through DNA by hopping between adenine bases and by tunnelling.Giese B, Amaudrat J, Köhler A-K, Spormann M, Wessely S:Nature 2001, 412:318-320.Significance: This paper is a significant advance in understand-ing the phenomenon of DNA-mediated electron transfer. As wellas presenting an exciting challenge to experiment and theory, anexplanation of this process will aid in our understanding of oxida-tive stress and in the construction of DNA-based electronicdevices. The results presented should help to resolve some of thecontradictory observations that have previously been reported.

Findings: The authors measured the efficiency of charge transfer through DNA duplexes, initiated by photolytic decomposition of a 4′-acyl nucleotide to a radical cation, andterminated at an easily oxidised GGG sequence. They foundthat the rate of electron transfer between guanosine residuesdecreases with increasing distance only up to a separation ofthree AT base pairs. They attribute this to a change in mecha-nism from a direct tunnelling interaction between guanosines atshort distances to a thermally induced hopping of chargesbetween adenosines at longer distances.

•• RNA-catalysed amino acid activation. Kumar RK, Yarus M:Biochemistry 2001, 40:6998-7004.Significance: This is the first report that RNA can catalyse theactivation of amino acids as acyl phosphates. A requirement ofthe ‘RNA world’ theory of the origin of life is that RNA catalystsshould have been able to synthesise the first proteins. Withthese results, all the necessary steps for coded protein synthe-sis by RNA have now been demonstrated – amino acidactivation, amino acyl-RNA formation, peptide bond formation,and association of codons with individual amino acids. Findings: In vitro selection was used to discover a set of RNAsequences that catalyse formation of aminoacyl phosphates fromamino acids and a 5′-triphosphate on the RNA. The selectionwas performed using the easily trapped 3-mercaptopropionicacid as substrate but amino acids were found to be good sub-strates also. The reaction was shown to be specific for thecarboxylate group and the reaction product was confirmed to bethe amino acyl-monophosphate at the 5′-terminal guanosine bynuclease digestion and comparison with synthetic standards.The reaction was shown to have a pH optimum of 4–4.5 and to be dependent on Ca2+. Rate data could be fitted toMichaelis–Menten kinetics.

•• A crystallographic map of the transition from B-DNA toA-DNA. Vargason JM, Henderson K, Ho PS: Proc Natl AcadSci USA 2001, 98:7265-7270.Significance: This paper reports the most complete descriptionto date of the pathway from B-DNA to A-DNA. These data willprove useful in understanding the dynamic properties of DNAand its deformation by proteins such as the TATA-binding protein.Findings: The authors have determined a series of single-crys-tal structures for the duplex d(GGCGCC)2 and variantscontaining brominated or methylated cytosines. They observe13 unique conformations that can be linked in a smooth progression from B-form to A-form. The most important intermediate structure shows half of each strand converted intoA-form with A- and B-form nucleotides opposite each other.

Selected by Richard NewmanEuropean Bioinformatics Institute, Cambridge, UK


•• Three-dimensional structure of cyanobacterial photosys-tem I at 2.5 Å resolution. Jordan P, Fromme P, Witt HT,Klukas O, Saenger W, Krauss N: Nature 2001, 411:909-917.Significance: Life on Earth depends on oxygenic photosynthe-sis, the conversion of light energy from the Sun to chemicalenergy. In plants, green algae and cyanobacteria this process isdriven by the cooperation of two large protein–cofactor complexes, photosystems I and II located in the thylakoid photosynthetic membranes.Together they absorb light and convert it into NADPH and a trans-membrenane electrochemical potential gradient of protons. The

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NADPH and ATP molecules are used to power the biochemicalreactions that convert atmospheric CO2 to organic molecules.Understanding the catalysis involved in the fundamental reactionof photosynthesis has been brought one step closer.Findings: The crystal form of PSI, isolated from the thermophiliccyanobacterium Synechococcus elongates is a trimer with a totalmass of ~1 MDa. The high-resolution structure reveals new details,such as the location of bound lipid molecules (PSI contains fourspecifically bound lipid molecules, one of which provides a ligandfor a chlorophyll molecule), as well as the position and orientations

of the cofactors involved in primary charge separation and electrontransfer. In particular, the new data provide detailed informationabout the coordination sites for binding chlorophylls (in somecases the chlorophylls are positioned in such a way as to interactelectronically with their nearest neighbours and thus aid the trans-fer of energy from the light harvesting chlorophylls to the P700cofactor), carotenoids, lipids and quinines to proteins. This struc-tural information on the proteins and cofactors and theirinteractions provides a basis for understanding how the high efficiency of PSI in light capturing and electron transfer is achieved.

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