Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase.

The biosyntheses of oligosaccharides and glycoconjugates are conducted by glycosyltransferases. These extraordinarily diverse and widespread enzymes catalyze the formation of glycosidic bonds through the transfer of a monosaccharide from a donor molecule to an acceptor molecule, with the stereochemistry about the anomeric carbon being either inverted or retained. Human ABO(H) blood group A α-1,3-N-acetylgalactosaminyltransferase (GTA) generates the corresponding antigen by the transfer of N-acetylgalactosamine from UDP-GalNAc to the blood group H antigen. To understand better how specific active-site-residue protons and hydrogen-bonding patterns affect substrate recognition and catalysis, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection and time-of-flight neutron diffraction data were collected to 2.5 Šresolution at the PCS to ∼85% overall completeness, with complementary X-ray diffraction data collected from a crystal from the same drop and extending to 1.85 Šresolution. Here, the first successful neutron data collection from a glycosyltransferase is reported.

Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583.

AIMS: To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. METHODS AND RESULTS: The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C-terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4-nitrophenyl N,N'-diacetyl-beta-D-chitobioside with K(m) values of 1.6 and 2.1 mmol l(-1), respectively, and k(cat) values of 21.6 and 6.5 s(-1). The enzymes also showed activity towards 4-nitrophenyl beta-D-N, N', N''-triacetylchitotriose and carboxy-methyl-chitin-Remazol Brilliant Violet but not towards 4-nitrophenyl N-acetyl-beta-D-glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4-nitrophenyl beta-D-cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes. CONCLUSION: The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food-manufacturing plants.

Structure and energetics of the glucoamylase-isomaltose transition-state complex probed by using modeling and deoxygenated substrates coupled with site-directed mutagenesis.

Molecular recognition, site-directed mutagenesis, and molecular modeling are combined to describe hydrogen bonds important for formation and catalysis of the Aspergillus niger glucomylase-isomaltose complex. This analysis of the energetics of the transition-state complex identified OH-4', -6', and -4 as critical for isomaltose hydrolysis. Side-chains hydrogen bonded to isomaltose OH-4 (reducing end unit, i.e. at glucoamylase binding subsite 2) induced substrate conformation adjustment to optimize binding energy contributed by charged hydrogen bonds to OH-4' and -6' at the non-reducing unit (i.e. at subsite 1). These interactions were evident in the modeled complex of glucoamylase and isomaltose in the preferred trans-gauche conformation. Kinetic analysis demonstrated reductions in kcat of 10(3) to 10(5)-fold for the corresponding deoxy- and O-methyl analogs of isomaltose. Analysis of two mutants at the level of subsite 2, Glu 180-->Gln and Asp 309-->Glu, showed the binding energy for the enzyme-transition state complex, delta delta G, contributed by OH-3 and -4 to be 6-7 kJ mol-1 weaker than with wild-type enzyme. Unexpectedly, however, substitution of isomaltose OH-4' and -6' (at subsite 1) resulted in 10 to 12 kJ mol-1 lower delta delta G++ for both the mutants. Mutation at subsite 2, therefore, strongly perturbed distant transition-state stabilizing interactions. This was confirmed with 4'- and 6'-deoxy analogs of the conformationally biased methyl 6-R-C-methyl-alpha-isomaltoside, readily adopting trans-gauche conformation, that exhibit full delta delta G++ 18 to 20 kJ mol-1 for both mutants and wild-typ-. Glucoamylase, during catalysis, thus seems to induce a change from the predominant solution gauche-gauche conformer to trans-gauche isomaltose. This leads to enhanced binding at subsite 1 in the enzyme transition-state complex.

Biocatalytic synthesis of oligosaccharides.

Tremendous advances in biocatalytic approaches to oligosaccharide synthesis have taken place in the past two years. The use of isolated enzymes, both glycosyltransferases and glycosidases, or engineered whole cells allows the preparation of natural oligosaccharides and analogs required for glycobiology research.

Absorption spectra of cytochrome P450CAM in the reaction with peroxy acids.

The reaction of Fe(III) cytochrome P450CAM with m-chloroperbenzoic acid was studied by rapid scanning absorption spectroscopy. Native low-spin enzyme produced spectra characteristic of two reaction phases that were marked by time intervals with isosbestic positions. The high-spin enzyme substrate complex yielded a series of Soret-region spectra whose properties were dependent on peracid concentration. The simplest model describing the results was a sequence of at least two spectral intermediates, that were not entirely homologous with data measured in reactions with microsomal P450LM2. Comparisons with related heme protein states indicate higher Fe(IV) oxidation levels provide a plausible interpretation of the P450CAM spectra.

Stereochemistry and cofactor identity status of semicarbazide-sensitive amine oxidases.

The relationship between the soluble copper topaquinone amine oxidases, the membrane bound semicarbazide-sensitive amine oxidases and lysyl oxidase remains unclear. The stereochemical course of substrate oxidation has been determined for each enzyme type and these studies suggest that SSAO and lysyl oxidase are closely related mechanistically, and that they are distinct from the copper amine oxidases. Both lysyl oxidase and SSAO catalyze the oxidation of tyramine with removal of the pro-S hydrogen from C-1 of this substrate. The copper amine oxidase enzymes that react with abstraction of the pro-S hydrogen from C-1 of substrates do not exhibit a solvent exchange pathway. In contrast, this exchange occurs in lysyl oxidase and SSAO reactions. The organic cofactor in all three enzyme types is a quinone; however, the spectral features of phenylhydrazine and p-nitrophenylhydrazine-derivatized SSAO differ from those reported for all known topaquinone-containing enzymes. Cofactor identification is further complicated by the lack of the characteristic topa motif, Asn-Tyr-Asp/Glu, in lysyl oxidase and the absence of any sequence information for SSAO.

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

Effects of amine modifiers on the separation of tetramethylrhodamine-labeled mono- and oligosaccharides by capillary zone electrophoresis.

In this work, nine tetramethylrhodamine (TMR) labeled isomeric oligosaccharide derivatives of betaGal(1 --> 4) betaGlcNAc-O-TMR were separated by capillary zone electrophoresis coupled with laser-induced fluorescence detection. Charged species were created in situ by complexation with borate and phenylborate. Micellar separation was achieved by addition of 10 mM sodium dodecylsulfate to the running buffer. We have investigated the effects of adding a homologous series of monoamine modifiers on the separation efficiency of these oligosaccharides. The separation was significantly improved in the presence of the organic modifiers methyl- and ethylamines, but worsened in the presence of propyl- and butylamines. Possible mechanisms of the amine additives are discussed.

Analysis by capillary electrophoresis-laser-induced fluorescence detection of oligosaccharides produced from enzyme reactions.

Six structurally similar, fluorescently labeled oligosaccharides were baseline resolved by capillary electrophoresis (CE); laser induced fluorescence (LIF) detection gave detection limits of 50 molecules for the oligosaccharides. A simple design of the LIF detector that incorporates the advantages of high sensitivity, stability and ease of operation is described. The system was used to monitor enzyme products formed during the incubation of yeast cells with alpha-D-Glc(1-->2)alpha-D-Glc(1-->3)alpha-D-glc-O(CH2)8CONHCH2CH2NHCO - tetramethylrhodamine. This fluorescent trisaccharide is enzymatically hydrolyzed to fluorescent disaccharide, monosaccharide and the free linker arm that is used to conjugate the saccharides with the fluorophore tetramethylrhodamine.

Study of the enzymatic transformation of fluorescently labeled oligosaccharides in human epidermoid cells using capillary electrophoresis with laser-induced fluorescence detection.

Isomeric oligosaccharides of both beta Gal(1-->3)beta GlcNAc (type I) series and beta Gal(1-->4)beta GlcNAc (type II) series were studied by using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. A mixture of phenylboronic acid and sodium tetraborate was used in the CE running buffers to improve the electrophoretic separation of the oligosaccharides. Both series of the tetramethylrhodamine (TMR)-labeled substrates [beta Gal(1-->3)beta GlcNAc-O-TMR and beta Gal(1-->4)beta GlcNAc-O-TMR) and their potential enzymatic products were baseline resolved using CE. The high resolution provided by CE and the excellent detection limit (8.10(-23) mol, or 50 molecules) by LIF allowed for the determination of minor enzyme products in the presence of excess unreacted substrate. The action of competing enzymes acting on the common type I sequence was monitored after the incubation of human epidermoid carcinoma cells (A431) with a fluorescent substrate (beta Gal(1-->3)beta GlcNAc-O-TMR). The CE-LIF analyses showed the formation of both synthetic and hydrolytic products, suggesting the actions of glycosyltransferases and glycosidases in the cells.

Transfer of D-galactosyl groups to 6-O-substituted 2-acetamido-2-deoxy-D-glucose residues by use of bovine D-galactosyltransferase.

Bovine D-galactosyltransferase was found to transfer D-galactose from UDP-galactose to 6-O-substituted 2-acetamido-2-deoxy-beta-D-glucopyranosides. The resulting 6-O-substituted N-acetyllactosamines were readily synthesized in milligram amounts and conveniently isolated on a reverse-phase support when prepared as the 8-methoxycarbonyloctyl glycosides. The 6-O-substitution tolerated by the enzyme include an alpha-L-fucopyranosyl group and the methyl ester of alpha-linked N-acetylneuraminic acid, but not the free acid itself. The product trisaccharides were characterized by 1H-n.m.r. spectroscopy and fast-atom-bombardment mass spectrometry.

Chemical synthesis and kinetic characterization of UDP-2-deoxy-D-lyxo-hexose("UDP-2-deoxy-D-galactose"), a donor-substrate for beta-(1-->4)-D-galactosyltransferase.

Bovine beta-(1-->4)-galactosyltransferase (GalT) transfers galactose from UDP-galactose to beta-D-GlcpNAc-terminating oligosaccharides to produce N-acetyllactosamine sequences. We report here the chemical synthesis, structural characterization and enzymatic evaluation of the very labile UDP-2-deoxy-D-lyxo-hexose ("UDP-2-deoxy-galactose," 2) as an alternate donor for GalT. Donor 2 had kinetic parameters, including a Km value of 51 microM, almost identical to those for the natural substrate UDP-galactose when beta-D-GlcpNAc-O(CH2)8COOMe was used as the acceptor. The product of the enzymatic transfer was isolated and confirmed to have the expected 2'-deoxy-N-acetyllactosamine sequence.

Use of the "core-2"-N-acetylglucosaminyltransferase in the chemical-enzymatic synthesis of a sialyl-LeX-containing hexasaccharide found on O-linked glycoproteins.

A simple preparation of the "core-II" N-acetylglucosaminyltransferase (UDP-D-GlcpNAc:beta-D-Galp-(1-->3)-alpha-D-GalpNAc (GlcNAc to GalNAc) beta-(1-->6)-GlcNAc-transferase, GlcNAcT, EC 2.4.1.102) from commercial mouse kidney acetone powder is reported. The enzyme obtained in a single step of affinity chromatography is suitable for use in preparative oligosaccharide synthesis. In conjunction with previously described preparations of beta-(1-->4)-galactosyltransferase (EC 2.4.1.22), alpha-(2-->3)-sialytransferase (EC 2.4.99.6) and alpha-(1-->3/4)-fucosyltransferase (EC 2.4.1.65), the GlcNAcT was used in the first step of a sequence which converted the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-OR into the sialyl-LeX-containing structure alpha-D-NeupAc-(2-->3)-beta-D-Galp- (1-->4)-[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc-(1-->6)-[beta-D-Galp - (1-->3)]-alpha-D-GalpNAc-OR (5), where R = (CH2)8CO2Me. Hexasaccharide 5, thus assembled in only one week once the enzymes were prepared, was characterized by 1H and 13C NMR spectroscopy and fast-atom bombardment mass spectrometry, as were all intermediate oligosaccharides. The core II GlcNAcT thus joins the expanding repertoire of readily available reagents for the rapid assembly of oligosaccharides.

Synthesis and enzymatic evaluation of modified acceptors of recombinant blood group A and B glycosyltransferases.

The disaccharide alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> O)-Octyl (1) is an acceptor for the human blood group A and B glycosyltransferases. Seven analogues of 1, containing deoxy, methoxy and arabino modifications of the Fuc residue, were chemically synthesized and kinetically evaluated in radioactive enzymatic assays. Both the enzymes tolerate modification of the 3'-OH on the fucose residue. The 2'-OH was found to be key to the recognition of the acceptors by these enzymes. The arabino derivative was recognized as an acceptor by the A transferase (Km of 200 microM), but not the B transferase and is the first synthetic acceptor capable of distinguishing between the two enzyme activities.

Enzymic synthesis of oligosaccharides terminating in the tumor-associated sialyl-Lewis-a determinant.

The isomeric sialyl-Lea-terminating pentasaccharide derivatives, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]-beta- D-GlcpNAc-(1----3)-beta-D-Galp-O(CH2)8COOMe and alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]- beta-D-GlcpNAc-(1----6)-beta-D-Galp-O(CH2)8COOMe, have been prepared by the action in sequence of a porcine submaxillary (2----3)-alpha-sialyltransferase and a human-milk (1----3/4)-alpha-fucosyltransferase on the chemically synthesized trisaccharides beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)- and -(1----6)-beta-D-Galp- O(CH2)8COOMe, respectively.

Synthesis of a di-O-methylated pentasaccharide for use in the assay of N-acetylglucosaminyltransferase III activity.

The biantennary oligosaccharide analogue beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->2)-alpha- D-Man p-(1-->6)]-beta-D-man p-O(CH2)8COOMe (3) is a potential substrate for N-acetylglucosaminyltransferases (GlcNAcTs) III-V which are present in mammalian cells. The di-O-methylated analogue of 3, beta-D-Glc pNAc-(1-->2)-[4-O-methyl-alpha-D-Man p]-(1-->3)-[beta-D-Glc pNAc-(1-->2)-[6-O-methyl-alpha-D-Man p]-(1-->6)]-beta-D-Man p-O-(CH2)8COOMe (5), was prepared by a block synthesis approach involving sequential addition of two O-methylated disaccharide donors to a protected central beta-D-Man residue. The OH groups acted on by GlcNAcT-IV and -V are protected from glycosylation in 5 since they are present as their methyl ethers. Pentasaccharide 5 was found to be an excellent substrate for GlcNAcT-III (EC 2.4.1.144) from rat kidney with Km = 0.15 mM. The product formed by incubation of 5 with a rat kidney extract, in the presence of UDP-GlcNAc, was isolated, structurally characterized by NMR spectroscopy and confirmed to be the expected di-O-methyl hexasaccharide where a beta-D-Glc pNAc residue had been added to OH-4 of the central beta-D-Man p unit.

Acceptor-substrate recognition by N-acetylglucosaminyltransferase-V: critical role of the 4"-hydroxyl group in beta-D-GlcpNAc-(1-->2)-alpha-D-Manp(1-->6)-beta-D-Glcp-OR.

The enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) transfers GlcNAc from UDP-GlcNAc to the OH-6' group of oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp (or Manp)-OR (5, R = (CH2)7CH3) to yield the sequence beta-D-GlcpNAc-(1-->2)-[beta-D-GlcpNAc-(1-->6)]-alpha-D-Manp-(1--> 6)- beta-D-Glcp (or Manp)-OR. Biosynthetically, if beta-(1-->4)-galactosyltransferase acts first on 5, the product beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-be ta-D-Glcp (or Manp)-OR (7) is no longer a substrate for GlcNAcT-V even though it retains the active OH-6' group. The reason for this loss in activity is examined in this paper. Six analogues of the acceptor trisaccharide 5, all with the reducing-end D-gluco configuration, were chemically synthesized. A key feature of the synthetic scheme is the use of 1,2-diaminoethane for the efficient removal of N-phthalimdo protecting groups. In these analogues OH-4 of the terminal sugar unit, the site of galactosylation by GalT in the normal GlcNAc-terminating trisaccharide 5, was systematically replaced by OMe, F, NH2, NHAc, and H, as well as inverted to the galacto configuration. The interactions of the resulting trisaccharide analogues with GlcNAcT-V from hamster kidney were then evaluated kinetically. All six compounds were found to be essentially inactive either as acceptors or as inhibitors of GlcNAcT-V. The conclusion is reached that galactosylation of natural acceptors for GlcNAcT-V destroys acceptor activity, not by introduction of the steric bulk of an added sugar residue, but by destroying an important hydrogen-bonding interaction of terminal OH-4 of the GlcNAc residues with the enzyme. This OH-4 group is therefore designated as a key polar group for GlcNAcT-V.

Enzymatic synthesis of blood group A and B trisaccharide analogues.

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.

Synthesis and evaluation of eight aminodeoxy trisaccharide inhibitors for N-acetylglucosaminyltransferase-V.

N-Acetylglucosaminyltransferase-V is an important enzyme controlling the branching pattern of N-linked oligosaccharides. This enzyme recognizes the trisaccharide octyl 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->2)-alpha-D-mannopyranosyl -(1-->6)-beta-D-glucopyranoside (5) as a substrate and adds a beta-linked GlcNAc residue to OH-6 of the central alpha-Man unit. Eight analogs of 5 were chemically synthesized where C-6 of the alpha-Man residue in 5 was deoxygenated, and structurally diverse modifications were introduced at C-4 of the same residue. The key intermediate prepared for this purpose was octyl 2-acetamido-2-deoxy-beta-D- glucopyranosyl-(1-->2)-4-amino-4,6-dideoxy-alpha-D-mannopyranosyl- (1-->6)-beta-D-glucopyranoside (7a) where the original 4'-amino group was readily derivatized on the unprotected sugar. The eight analogs 7a-7h were evaluated as inhibitors for GlcNAcT-V, both isolated (from hamster kidney) and cloned (from rat kidney). All of the compounds were found to be competitive inhibitors with Ki in the range of 3-106 microM. The conclusion of this work is that recognition of acceptor 5 does not involve contact of the C-6--C-4 end of the alpha-Man residue with the protein in the E-I (or E-S) complex.

Acceptor hydroxyl group mapping for calf thymus alpha-(1-->3)-galactosyltransferase and enzymatic synthesis of alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta D-GlcpNAc analogs.

The epitope of the acceptor substrate for alpha-(1-->3)-galactosyltransferase from calf thymus has been mapped by using a series of mono-deoxygenated and mono-O-alkylated Type II (beta-D-Ga1p-(1-->4)-beta-D-G1cpNAc) disaccharides. The 4-OH group of the beta-D-galactopyranosyl residue is a key polar group essential for glycosyl transfer, tolerating neither deoxygenation nor O-alkylation. Substitution at positions 6 and 6' by a variety of polar alkyl substituents was readily tolerated, allowing the preparative enzymatic synthesis of a series of trisaccharide derivatives carrying polar substituents on each of these hydroxyl groups. These new analogs are potential inhibitors of Clostridium difficile toxin A and of a human anti-alpha-Gal antibody.