science/technology component of endotoxin, a very potent immunostimulant. Raetz's group has identified the 10 en zymes that are required to make the sim plest form of lipid A that supports bacte rial cell growth and have determined the structures of some of the intermediates along the biosynthetic pathway. Most of the enzymes of lipid A biosynthesis are now available in recombinant form, and some have been used for the synthesis of lipid A analogs. Several are targets for the development of new antibacterial agents—including the first enzyme in the pathway, UDP-GlcNAc O-acyltransferase, which puts a fatty acid onto the sugar nucleotide UDP-GlcNAc (uridine-5/diphosphate-Af-acetylglucosamine). Last year, Raetz and biochemistry professor Steven L. Roderick of Albert Einstein College of Medicine, Bronx, N.Y., determined the structure of the acyltransferase [Science, 270, 997 (1995)]. The enzyme consists of three identical subunits, each of which contains a novel type of Glycoform of ribonuciease prepared enzymaticaiiy by Wong protein secondary struc and coworkers. The researchers iinked the tetrasaccharide ture called a left-handed sialyl Lewis X (bottom right) to the fi-amide nitrogen of the parallel β-helix. enzyme's asparagine-34 residue. Ribonuciease is shown in Raetz and coworkers, white and yellow, with pink representing disulfide bonds in collaboration with re and disulfide-bonded residues, and green asparagine-34. In searchers at Merck & sialyl Lewis X, green stands for carbon, red for oxygen, and blue for nitrogen. Co., Rahway, N.J., have also identified synthetic can biosynthetic pathway. Peptidogly- antibacterial agents that inhibit the sec cans are covalently linked networks of ond enzyme in the lipid A biosynthetic polysaccharide and polypeptide chains pathway, an acyl-UDP-GlcNAc deacetylase. The inhibitors are chiral hydroxamthat form the cell walls of bacteria. A better understanding of peptidogly- ic acids that bind to a metal in the active can biosynthetic enzymes is important site of the enzyme. The most potent was for analysis of the mechanism of action capable of curing mice of an E. colt infec of existing antibiotics and the identifica tion that was inevitably fatal in the ab tion of new antibacterial drugs. Penicil sence of the treatment. lin, for example, has long been known to At the symposium, biochemistry pro bind to and inactivate the enzymes that fessor Yuan C. Lee of Johns Hopkins Uni cross-link peptidoglycans. versity discussed some unusual oligosac Also focusing on the enzymes of bac charides in plant and insect glycopro terial cell wall biosynthesis is the group teins. According to Lee, mammalian of biochemistry professor Christian R. H. glycoproteins have been intensively stud Raetz of Duke University. Raetz and co ied, but information on carbohydrate workers are studying the biosynthesis of structures, processing pathways, and en the lipid A moiety, an acylated and phos- zyme systems in plant and insect cells is phorylated disaccharide of glucosamine much more fragmentary. found in the outer membrane of Es Lee, chemical engineering professor cherichia colt. Lipid A is required for the Michael J. Betenbaugh of Johns Hopkins, growth of bacteria and is the active and coworkers recently found that the
CARBOHYDRATES' COMPLEXITIES
Glycobiochemists attain a more detailed understanding of the involvement of carbohydrates in intricate life processes Stu Borman C&EN Washington
B
iochemists have come relatively late to the detailed study of biolog ical carbohydrates and glycoconjugates. The tremendous structural variabil ity of such compounds, the great difficul ty of synthesizing them, and the baffling complexity of the life processes in which they are involved have all tended to bog down the process. But if the presentations at a sympo sium on "Glycobiochemistry" at last month's American Chemical Society na tional meeting in Orlando are any indica tion, considerable progress now is being made in attaining a better understanding of the chemistry and biology of carbohy drate interactions. The symposium was organized by chemistry professor Hungwen Liu of the University of Minnesota, Minneapolis, and Chi-Huey Wong of the department of chemistry and Skaggs In stitute of Chemical Biology at Scripps Re search Institute, La Jolla, Calif. Carbohydrates and their derivatives play an essential role in biological process es, including "infection, cell adhesion, me tastasis, cell development, differentiation, regulation, and many signaling events," says Wong. "However, the mechanisms of these processes are not well understood, and currently there are no effective means used clinically to control carbohydratebased recognition. It is hoped that the meeting will stimulate further research in this field so that effective new strategies will be developed to combat carbohy drate-mediated diseases." There was a strong emphasis on biosynthetic enzymes at the symposium. For example, Christopher T. Walsh of the de partment of biological chemistry and mo lecular pharmacology of Harvard Medical School described detailed mechanistic and structural studies by his group on many of the enzymes in the peptidogly36 SEPTEMBER 30, 1996 C&EN
mon core) and the complex type (which Lipid A biosynthetic pathway is are much more struc 10-step process turally varied). But the secreted IgG glyco UDP-GlcNAc + fl-3-Hydroxymyristoyl-ACP forms are primarily of the complex type, O-Acyltransferase with no high-manHO^ nose types present. HO^U-0 Acetate In the area of ^L „ ΟΛ^Μ plant glycobiochemNH 2 1 < NH ^ ο ρ *>***>*» 0=( istry, Lee, Takahashi, UDP chief investigator HO' HO Song-Nan Su of the Veterans General Hospital, Taipei, Tai wan, and coworkers have found some unusual oligosaccha rides in BG60, an im portant allergen of Bermuda grass pol len, which causes al lergic responses in humans. The two most abundant oligo saccharides found by the group in their analysis of BG60 are -QT the first such struc tures ever found in plant glycoproteins in that they possess an a-(l,3)-linked fucose but no xylose. Oligosaccharides from plant glycopro teins normally con tain both fucose and xylose or, alternative ly, only xylose. This unique structure could Lipid A moiety have some bearing on the allergic reactions caused by BG60 and UDP-GlcNAc = uridine-S'-diphosphate-ZV-acetylglucosamine other glycoproteins in ACP = acyl carrier protein pollen. Professor Barbara production in insect cells of a mouse im Imperiali's group in the Division of munoglobulin (IgG)—a glycoprotein— Chemistry & Chemical Engineering at greatly increases when the chaperone California Institute of Technology is BiP (immunoglobulin binding protein) is studying the mechanism of protein glycoexpressed with it. cosylation, the biosynthetic process in And in collaboration with Noriko Ta- which carbohydrate groups are added to kahashi, director of the GlycoLab at Na- proteins. N-linked glycosylation, in kano Vinegar Co., Handa City, Japan, Lee which a carbohydrate is linked to an asand coworkers found that the varying ol paragine residue, is the predominant pro igosaccharide structures (glycoforms) of tein-carbohydrate modification in eukaryIgGs differ depending on whether they otic cells. Imperiali and coworkers have are retained intracellularly or secreted. made major progress in understanding The intracellular IgG glycoforms are of the mechanism, sequence specificity, both the high-mannose type (having only and structural consequences of the Noc-mannosyl residues attached to a com linked glycosylation process.
/A,
N-linked glycosylation is catalyzed by oligosaccharyl transferase. The enzyme catalyzes the formation of an N-C bond between the amide nitrogen atom of an asparagine residue and a carbon atom in GlcNAc from a lipid-linked oligosaccha ride. The reaction is cotranslational, in that it occurs while a nascent protein is still attached to the ribosome on which it is being synthesized. In the reaction, the lipid (dolichol pyrophosphate) is dis placed from the lipid-linked oligosaccha ride by the asparagine nitrogen, which acts as a nucleophile. The nitrogen atom of the asparagine amide group is not an inherently reactive nucleophilic group. But Imperiali and co workers have proposed a mechanism that rationalizes its reactivity and also helps explain the conformational selec tivity of the N-glycosylation process. The mechanism suggests that a unique hydro gen-bonding array set up by an "Asx-turn" conformation in the protein's enzymerecognition site facilitates deprotonation of the asparagine nitrogen by a basic res idue in the active site of the enzyme. This leads to a tautomerization that changes the amide into a highly reactive nucleophile capable of displacing doli chol pyrophosphate from the dolichololigosaccharide substrate. Based in part on their understanding of this mechanism, Imperiali and co workers have now designed and tested the first synthetic compounds that inhib it oligosaccharyl-transferase-catalyzed gly cosylation [/. Am. Chem. Soc, 118, 7636 (1996)]. The peptidyl compounds, which were patterned after the enzyme-rec ognition site, inhibit glycosylation at nanomolar concentrations. Such inhibi tors have potential diagnostic, therapeu tic, and synthetic applications. Studies on the kinetics and mecha nism of oligosaccharyl transferase-catalyzed N-linked glycosylation are also be ing carried out by James K. Coward and coworkers in the College of Pharmacy and department of chemistry of the Uni versity of Michigan, Ann Arbor. Coward's group has used synthetic peptide substrates containing stable iso topes to study the detailed catalytic action of the enzyme. Some researchers have proposed that the mechanism of oligosac charyl transferase involves the formation of reactive tetrahedral intermediates de rived from the asparagine residue of the substrate. These proposals have been con ceived by analogy with well-established mechanisms for related glutaminedependent amidotransferase reactions. SEPTEMBER 30, 1996 C&EN 37
science/technology
w^.
from the nucleotide substrate. "In the E. colt and mammalian enzymes, the eviProposed activation of oligosaccharyl transferase dence that a thiyl radical is involved is, I think, quite strong but indirect," says ^r Peptide or protein Stubbe. But her group has been able to O^N, H obtain direct evidence for the existence + Enzyme-base-H of the thiyl radical in another bacterial system. "Finding a sulfur radical and finding nucleotide radical intermediates has been incredibly exciting for me," she says, "because this mechanism was very Base-enzyme unprecedented but is now turning out to Dolichol-PP-GlcNAc-oligosaccharide be okay." Base-enzyme = basic residue in active site of oligosaccharyl transferase. PP = pyrophosphate. Stubbe's group, in collaboration with R r side chain of any amino acid (except proline). James R. McCarthy and coworkers at Source: Barbara Imperial! and coworkers Marion Merrell Dow Research Institute (now Hoechst Marion Roussel), CincinHowever, Coward and coworkers nism her group has proposed for ribonu- nati, Ohio, have also conducted detailed have found evidence indicating that such cleotide reductases, a family of enzymes studies on ribonucleotide reductase in35, 8381 a mechanism for oligosaccharyl trans- that catalyze the conversion of nucleo- hibitors [Biochemistry, ferases is unlikely. In addition, they have tides to deoxynucleotides as part of the (1996)]. The inhibitors, which were deconducted the first complete steady-state DNA biosynthesis process in all organ- signed by the Marion Merrell Dow group kinetic analysis of the oligosaccharyl isms. All the chemistry of this process on the basis of the thiyl-radical mechatransferase reaction, enabling them to takes place on the ribose sugar group of nism, are considered potential antitumor and antiviral agents. evaluate the specificity of the enzyme for the nucleotide. particular peptide and lipid-linked oligoThe University of Minnesota team led Stubbe and coworkers are using saccharide substrates. techniques such as stopped-flow ultra- by symposium co-organizer Liu has also In collaboration with biochemist violet-visible spectroscopy and rapid- come up with a radical mechanism for Frank Maley of the New York State De- freeze-quench electron paramagnetic the enzymatic biosynthesis of deoxy sugpartment of Health, Albany, Coward and resonance spectroscopy to detect the ar nucleotides. In this case, the reaction coworkers have also analyzed the in vitro nucleotide radical intermediates formed is a deoxygenation that is the second-tointeractions of oligosaccharyl transferase in the course of these very complex re- last step in the biosynthesis of cytidine with peptides of varied length containing actions. Confirmatory evidence for the diphosphate-ascarylose, a dideoxyhexose glycosylation sequons (short recognition radical mechanism they've proposed found in a bacterial antigen. sequences). Based on these studies, they has been hard to obtain, but their efThe reaction is catalyzed by two enwere able to achieve the in vivo glycosy- forts lately have been paying off. Stubbe zymes, a dehydrase and a reductase. Delation of the enzyme invertase. "The im- says her group has recently observed tailed chemical and spectroscopic invesportant result of this research," says radicals "that no one has ever seen be- tigations led Liu and coworkers to proCoward, "is now one can think about fore in reactions catalyzed by these en- pose that it is a redox process involving designing sites into peptides and pro- zymes. It's the first evidence for nucleo- a unique radical form of pyridoxamine-5'teins and making reasonable predictions tide radical intermediates along the re- phosphate, an enzyme cofactor derived as to whether or not a particular sequon action pathway." from vitamin B6 (pyridoxine). This reprewill be glycosylated." The key point of the proposed catalyt- sents the first reported example of parJoAnne Stubbe of the departments of ic mechanism of ribonucleotide reduc- ticipation of a pyridoxamine-5,-phoschemistry and biology at Massachusetts tases is that a transient thiyl (sulfur) radi- phate radical in a redox reaction. Currently, Liu and coworkers are exInstitute of Technology discussed at the cal initiates the nucleotide reduction promeeting an unusual radical-based mecha- cess by abstracting a hydrogen atom amining the biosynthetic pathways of
Ascarylose biosynthesis may proceed by radical mechanism H,Q Dehydrase and reductase
2
CDP-6-deoxyL-fA?reo-D-glycero4-hexulose CDP = cytidine diphosphate Source: Hung-wen Liu and coworkers.
38
SEPTEMBER 30, 1996 C&EN
-O 3 PC/ Pyridoxamine-5-phosphate radical intermediate
OCDP OH CDP-3,6-dideoxyD-glycero-D-glycero4-hexulose
[OCDP OH CDP-ascarylose
other deoxyhexoses, some of which may use similar radical mechanisms. Liu says these further studies could conceivably "provide the basis for defining a new mechanistic class of dehydrases and lead to the development of novel antibacteri al agents." Perry A. Frey of the Institute for En zyme Research at the University of Wis consin, Madison, spoke at the sympo sium on the structures and mechanisms of action of two E. coli enzymes required for galactose metabolism. One of these is galactose-1-phosphate uridylyltransferase, the human version of which is de fective in the inborn disease galac tosemia, in which infants are unable to metabolize galactose, a constituent of the lactose in milk. The enzyme's structure and mecha nism—determined by Frey, graduate stu dent Joseph E. Wedekind, and crystallographer Ivan Rayment—suggest a chemi cal basis for the inactivity of the principal galactosemic variant of the enzyme. Al though the researchers reported the structure of the E. coli version of the en zyme, the human enzyme is genetically homologous and displays extensive amino acid sequence identity with the E. coli enzyme, making the findings poten tially relevant to humans. The enzymatic reaction proceeds in two steps. In the first step, UDP-glucose reacts with histidine-166 in the E. coli en zyme to form a covalent intermediate in which histidine is linked to uridine monophosphate (IMP), in the second step, the intermediate reacts with galactose-1-phosphate to form UDP-galactose, with release of the free enzyme. The detailed structure of the cova lent intermediate in the E. coli enzyme was reported by Frey and coworkers earlier this month [Biochemistry, 35, 11560 (1996)]. It shows the phosphate group of UMP hydrogen-bonded to the side chain of the enzyme's glutamine168 residue. The normal human enzyme also has glutamine at the corresponding position in its sequence, but the principal human galactosemic variant has arginine at this position. The researchers believe that ar ginine overstabilizes the intermediate, thus preventing it from reacting with galactose-1-phosphate in the second step. In a development with potential for commercialization, Stephen G. Withers and coworkers of the Protein Engineer ing Network of Centres of Excellence and the departments of chemistry and microbiology at the University of British
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science/technology Columbia, Vancouver, have engineered thesis of oligosaccharides that mediate SLex is currently undergoing clinical glycosidases that synthesize but do not specific recognition and signaling pro trials as an anti-inflammatory drug, but degrade oligosaccharides, increasing the cesses. A recent tour de force by Wong, new and better anti-inflammatory agents enzymes' efficiency at catalyzing oligo graduate student Krista Witte, and re are needed to inhibit selectin-carbohysaccharide synthesis. search associate Pamela Sears was their drate interactions. Wong and coworkers Normally, glycosidases catalyze the use of subtilisin and glycosyltransferases are using chemoenzymatic methods to hydrolysis of glycosidic bonds, the bonds to synthesize glycoforms of the protein make compounds that mimic the active between sugar units in oligosaccharides. ribonuclease Β via glycopeptide conden conformation of SLex but are simpler, The natural enzymes potentially can be sation and sequential glycosylation. The more stable, more potent, and perhaps used to catalyze the reverse reaction to ability to synthesize glycoproteins in this orally active as well (SLex can't be admin synthesize oligosaccharides. But the oli manner "provides a new way to study istered orally). gosaccharide product formed is a sub the structure and function of glycopro Wong's group recently synthesized fustrate for hydrolysis by the enzyme, mak teins," says Wong. cosylated peptides that in some cases are ing it difficult to obtain high yields of oli Wong's group also has focused on the five to 10 times more active than SLex at gosaccharides using glycosidases. synthesis of sialyl Lewis X (SLex) mimet- inhibiting E-selectin interactions \J. Am. Withers and coworkers now have pro ics. SLex, a tetrasaccharide found on Chem. Soc, 118, 6826 (1996)]. And at the duced mutant glycosidases in which one blood cell surfaces, binds to selectins on glycobiochemistry symposium, Wong dis cussed some new mimetics that or two key amino acids have are 20 times more active than been changed in the active site. SLex in E-selectin inhibition and In one type of enzyme called a re more than 100 times more active taining glycosidase (because it hyin P-selectin inhibition. Another drolyzes glycosidic bonds with re approach Wong's group is taking tention of configuration), they is to block the biosynthesis of have changed a normally nucleoSLex by inhibiting the crucial en philic amino acid in the active site zyme fucosyltransferase with ei from glutamate to alanine. This ther a fluorinated sugar nucleo makes it impossible for the mu tide or a synergistic inhibitor tant enzyme to form adducts with complex of an azatrisaccharide the substrate—a key step in the and guanosine diphosphate \J. mechanism of action of the natu Am. Chem. Soc, 118, 7653 ral enzyme. (1996)]. Hence, the mutant enzyme Samuel J. Danishefsky, director cannot hydrolyze glycosidic of the Laboratory for Bioorganic bonds. But it can still catalyze Chemistry at Memorial Sloanthe formation of glycosidic Kettering Cancer Center, New bonds to form oligosaccharides, York City, and professor of chem when used in conjunction with istry at Columbia University, con glycosyl fluorides of opposite cluded the session with a presen anomeric configuration to that tation on a nonenzymatic synthet of the natural substrate. Such Frey and coworkers find that uridine monophosphate ic strategy involving glycals— glycosyl fluorides do not func (yellow bonds) is hydrogen-bonded to glutamine-168 in carbohydrates with a double tion with the wild-type enzyme the covalent intermediate formed in the reaction bond in the ring. Danishefsky and but are good glycosyl donors catalyzed by E. coli galactose-1-phosphate coworkers have championed the with the mutants because they uridylyltransferase. His = histidine, Pro = proline, Gly = glycine, Gin = glutamine. Gray = carbon, red = oxygen, concept of using glycals as build mimic the structure of enzymeblue = nitrogen, dark pink = phosphorus. ing blocks for the construction of substrate adducts formed by the biologically important oligosac natural enzyme. Withers and coworkers have not yet the surfaces of the endothelial cells that charides and glycoconjugates [Angew. published in the scientific literature on line blood capillaries. Selectins, which in Chem. Int. Ed. Engl, 35, 1381 (1996)]. Their recent achievements have includ these enzymatic syntheses, but they have clude E-selectin and P-selectin, are carbo filed for U.S. and Canadian patents on hydrate-binding proteins. SLex-selectin ed the solid-phase convergent synthesis of the technology. According to Withers, binding is an important step in the in a trisaccharide tripeptide and a trisaccha"This approach has, even at this early flammatory response, a normal body pro ride pentapeptide. In unpublished work, stage, been carried out on a gram scale. cess for tissue repair following infection they have also used glycals to synthesize the chitobiose core structure common to There is no reason this should not be fea or injury. But if the inflammatory response all N-linked glycopeptides. sible on the kilogram scale and greater. It One of the group's ultimate goals is to should also be possible to use immobi goes overboard, normal tissue is de lized enzyme systems." Oligosaccharide stroyed instead of being repaired. Selec- use glycal techniques to synthesize a yields of up to 92% have been obtained tin-carbohydrate interactions can also high-mannose antennary structure termi occur at a very early stage of cancer nating with human blood group anti with the mutant enzymes. Co-organizer Wong and coworkers at metastasis. Several groups are trying to gens—exemplifying the increasingly dif Scripps Research Institute specialize in design carbohydrate-based drugs to ficult challenges that glycobiochemists will face in the future.^ the enzymatic and chemoenzymatic syn control these interactions. 40 SEPTEMBER 30, 1996 C&EN
