@article {1279,
	title = {The enzymes of sialic acid biosynthesis},
	journal = {Bioorganic Chemistry},
	volume = {33},
	number = {3},
	year = {2005},
	note = {ISI Document Delivery No.: 931CJTimes Cited: 36Cited Reference Count: 59},
	month = {Jun},
	pages = {216-228},
	type = {Review},
	abstract = {The sialic acids are a family of nine carbon a-keto acids that play a wide variety of biological roles in nature. In mammals, they are found at the distal ends of cell surface glycoconjugates, and thus are major determinants of cellular recognition and adhesion events. In certain strains of pathogenic bacteria, they are found in capsular polysaccharides that mask the organism from the immune system by mimicking the exterior of a mammalian cell. This review outlines recent developments in the understanding of the two main enzymes responsible for the biosynthesis of the sialic acid, N-acetylneuraminic acid. The first, a hydrolyzing UDP-N-acetyl-glucosamine 2-epimerase, generates N-acetylmannosamine and UDP from UDP-N-acetylglucosamine. The second, sialic acid synthase, generates either N-acetylneuraminic acid (bacteria) or N-acetylneuraminic acid 9-phosphate (mammals) in a condensation reaction with phosphoenolpyruvate. An emphasis is placed on an understanding of the mechanistic and structural features of these enzymes. (c) 2005 Elsevier Inc. All rights reserved.},
	keywords = {2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE, BIFUNCTIONAL KEY ENZYME, biosynthesis, ESCHERICHIA-COLI K1, FIRST 2, KDO8P SYNTHASE, MECHANISM, N-ACETYLGLUCOSAMINE 2-EPIMERASE, N-acetylmannosamine, N-ACETYLNEURAMINIC ACID, NEISSERIA-MENINGITIDIS, PHOSPHATE, phosphoenolpyruvate, RAT-LIVER, sialic acid synthase, STEPS, SYNTHASE GENE, UDP-GlcNAc 2-epimerase},
	isbn = {0045-2068},
	url = {<Go to ISI>://000229463100007},
	author = {Tanner, M. E.}
}
@article {972,
	title = {Active site mutants of the "non-hydrolyzing" UDP-N-acetylglucosamine 2-epimerase from Escherichia coli},
	journal = {Biochimica Et Biophysica Acta-Proteins and Proteomics},
	volume = {1700},
	number = {1},
	year = {2004},
	note = {ISI Document Delivery No.: 833VCTimes Cited: 4Cited Reference Count: 26},
	month = {Jul},
	pages = {85-91},
	type = {Article},
	abstract = {The "non-hydrolyzing" bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible interconversion of UDP-N-acetylglucosarnine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This homodimeric enzyme is allosterically activated by its substrate, UDP-GlcNAc, and it is thought that one subunit plays a regulatory role, while that of the other plays a catalytic role. In this work, five active site mutants were prepared (D95N, E117Q, E131Q, K15A, and H213N) and analyzed in terms of their effects on binding, catalysis, and allosteric regulation. His213 appears to play a role in UDP binding and may also assist in catalysis and/or regulation, but is not a key catalytic residue. Lys15 appears to be quite important for binding. All three of the carboxylate mutants showed dramatic decreases in the value of k(cat) but relatively unaffected values of Km. Thus, these residues are playing key roles in catalysis and/or regulation. In the case of E117Q, the reaction intermediates are released into solution at a rate comparable to that of the overall catalysis. This may indicate that Glu117 plays the role as an acid/base catalyst in the second step of the UDP-GlcNAc epimerization reaction. All three carboxylate mutants were found to exhibit impaired allosteric control. (C) 2004 Elsevier B.V. All rights reserved.},
	keywords = {2-acetamidoglucal, 2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE, ACID BIOSYNTHESIS, allosteric regulation, enterobacterial common antigen, EPIMERIZATION, HOMOLOGY, MECHANISM, MUTAGENESIS, RAT-LIVER, SEQUENCE, sialic acid, STREPTOCOCCUS-PNEUMONIAE, TRANSFERASES, UDP-N-acetylmannosamine},
	isbn = {1570-9639},
	url = {<Go to ISI>://000222367400011},
	author = {Samuel, J. and Tanner, M. E.}
}
@article {950,
	title = {Identification and mechanism of a bacterial hydrolyzing UDP-N-acetylglucosamine 2-epimerase},
	journal = {Biochemistry},
	volume = {43},
	number = {44},
	year = {2004},
	note = {ISI Document Delivery No.: 868UVTimes Cited: 13Cited Reference Count: 38},
	month = {Nov},
	pages = {14290-14298},
	type = {Article},
	abstract = {This paper reports the first identification of a fully functional hydrolyzing UDP-N-acetylglucosamine 2-epimerase from a bacterial source. The epimerase (known as SiaA or NeuC from Neisseria meningitidis MC58 group B is shown to catalyze the conversion of UDP-GlcNAc into ManNAc and UDP in the first step of sialic acid (N-acetylneuraminic acid) biosynthesis. The mechanism is proposed to involve an anti elimination of UDP to form 2-acetamidoglucal as an intermediate, followed by the syn addition of water. The observation that the alpha-anomer of ManNAc is the true product and that solvent deuterium is incorporated at C-2 is consistent with this mechanism. The use of the O-18-labeled substrate confirms that the overall hydrolysis reaction proceeds via cleavage of the C-O bond. Furthermore, the putative intermediate 2-acetamidoglucal is shown to serve as a catalytically competent substrate and is enzymatically hydrated to give ManNAc exclusively. Isotope effect studies show that cleavage of the C-H bond is not rate limiting during catalysis. Mutagenesis studies show that three active site carboxylate residues are crucial for catalysis. In two of the mutants that were studied (E122Q and D131N), 2-acetamidoglucal was released from the active site during catalysis, providing direct evidence that the enzyme is capable of catalyzing the anti elimination of UDP from UDP-GlcNAc.},
	keywords = {2-EPIMERASE/N-ACETYLMANNOSAMINE, biosynthesis, CELL-INTERACTIONS, ENZYME, ESCHERICHIA-COLI K1, KINASE, POLYSIALIC ACID, PURIFICATION, RAT-LIVER, SIALIC-ACID, STEREOCHEMISTRY},
	isbn = {0006-2960},
	url = {<Go to ISI>://000224939400045},
	author = {Murkin, A. S. and Chou, W. K. and Wakarchuk, W. W. and Tanner, M. E.}
}
@article {1023,
	title = {The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase},
	journal = {Journal of Bacteriology},
	volume = {186},
	number = {3},
	year = {2004},
	note = {ISI Document Delivery No.: 766HUTimes Cited: 18Cited Reference Count: 43},
	month = {Feb},
	pages = {706-712},
	type = {Article},
	abstract = {The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-C-14]acetamidoglucal and [N-C-14]acetylmannosamine (ManNAc) from UDP-[C-14]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.},
	keywords = {2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE, ACETYLNEURAMINIC ACID SYNTHETASE, EXPRESSION, GENE-PRODUCT, NEISSERIA-MENINGITIDIS, POLYSACCHARIDE BIOSYNTHESIS, POLYSIALIC ACID, RAT-LIVER, SEQUENCE, SIALIC-ACID},
	isbn = {0021-9193},
	url = {<Go to ISI>://000188371600014},
	author = {Vann, W. F. and Daines, D. A. and Murkin, A. S. and Tanner, M. E. and Chaffin, D. O. and Rubens, C. E. and Vionnet, J. and Silver, R. P.}
}
@article {593,
	title = {Sialic acid biosynthesis: Stereochemistry and mechanism of the reaction catalyzed by the mammalian UDP-N-acetylglucosamine 2-epimerase},
	journal = {Journal of the American Chemical Society},
	volume = {125},
	number = {9},
	year = {2003},
	note = {ISI Document Delivery No.: 649YPTimes Cited: 19Cited Reference Count: 44},
	month = {Mar},
	pages = {2455-2461},
	type = {Article},
	abstract = {The bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/ManNAc kinase, catalyzes the first two steps in the biosynthesis of the sialic acids in mammals. The epimerase domain converts UDP-GlcNAc into ManNAc and UDP. This paper demonstrates that a-ManNAc is the first formed anomer and therefore the reaction proceeds with a net retention of configuration at C-1. Studies in deuterated buffer show that solvent-derived deuterium is quantitatively incorporated into the C-2 position of the product during catalysis, but it is not incorporated into the remaining pool of substrate. This indicates that the inversion of stereochemistry is ultimately brought about by the removal and replacement of a proton at C-2 and is consistent with a two-base mechanism. Studies with O-18-labeled UDP-GlcNAc show that the anomeric oxygen of the glycosyl phosphate bond departs with the UDP product and therefore the net hydrolysis reaction involves C-O bond cleavage. Incubation of the putative intermediate, 2-acetamidoglucal, with the enzyme resulted in a slow hydration reaction to give the product, ManNAc. Additional kinetic isotope effect and positional isotope exchange (PIX) experiments address the nature of the rate-determining step of the reaction and show that C-H bond cleavage is not rate limiting. Overall, these results support a reaction mechanism involving an anti-elimination of UDP to give 2-acetamidoglucal, followed by a syn-addition of water.},
	keywords = {2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE, BIFUNCTIONAL ENZYME CATALYZES, BINDING, CELL-SURFACE SIALYLATION, EPIMERIZATION, FIRST 2 STEPS, GLUTAMATE RACEMASE, POSITIONAL ISOTOPE EXCHANGE, RAT-LIVER},
	isbn = {0002-7863},
	url = {<Go to ISI>://000181235800030},
	author = {Chou, W. K. and Hinderlich, S. and Reutter, W. and Tanner, M. E.}
}
@article {4750,
	title = {The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases},
	journal = {Biochemistry},
	volume = {39},
	number = {49},
	year = {2000},
	note = {ISI Document Delivery No.: 383GTTimes Cited: 58Cited Reference Count: 48},
	month = {Dec},
	pages = {14993-15001},
	type = {Article},
	abstract = {Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 Angstrom) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta -glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site-located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.},
	keywords = {2-EPIMERASE/N-ACETYLMANNOSAMINE KINASE, ACETYLNEURAMINIC ACID BIOSYNTHESIS, BETA-GLUCOSYLTRANSFERASE, BIFUNCTIONAL ENZYME CATALYZES, CELL-SURFACE SIALYLATION, DNA MODIFYING, enterobacterial common antigen, ENZYME, FIRST 2 STEPS, GLYCOGEN-PHOSPHORYLASE, RAT-LIVER},
	isbn = {0006-2960},
	url = {<Go to ISI>://000165875300001},
	author = {Campbell, R. E. and Mosimann, S. C. and Tanner, M. E. and Strynadka, N. C. J.}
}
@article {4074,
	title = {Eliminations in the reactions catalyzed by UDP-N-acetylglucosamine 2-epimerase},
	journal = {Journal of the American Chemical Society},
	volume = {119},
	number = {43},
	year = {1997},
	note = {ISI Document Delivery No.: YD815Times Cited: 33Cited Reference Count: 65},
	month = {Oct},
	pages = {10269-10277},
	type = {Article},
	abstract = {Mechanistic studies have been carried out on the bacterial enzyme UDP-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). This enzyme is interesting because it epimerizes a stereocenter that does not bear an acidic proton, and therefore it cannot utilize a simple deprotonation/reprotonation mechanism. A coupled enzyme assay employing UDP-ManNAc dehydrogenase has been developed. The epimerization in D2O is found to be accompanied by the incorporation of deuterium into the C-2 {\textquoteright}{\textquoteright} position of both epimers, supporting a mechanism that ultimately involves a proton transfer at this position. The epimerization of [2 {\textquoteright}{\textquoteright}-H-2]UDP-GlcNAc is slowed by a primary kinetic isotope effect indicating that C-H bond cleavage is occurring during a rate-determining step of the reaction. A positional isotope exchange (PM) experiment shows that an O-18 label in the sugar-UDP bridging position will scramble into nonbridging diphosphate positions during enzymatic epimerization. These observations are consistent with a mechanism that proceeds via cleavage of the anomeric C-O bond, with 2-acetamidoglucal and UDP as enzyme-bound intermediates. Additional evidence for this mechanism is found in the unusual observation that during extended incubations, the intermediates are gradually released from the enzyme and accumulate in solution. These intermediates are formed by an anti elimination of UDP from UDP-GlcNAc and a syn elimination of UDP from UDP-ManNAc. It is likely that El-like eliminations via oxocarbenium intermediates are involved in the reaction. Further experiments show that 3 {\textquoteright}{\textquoteright}-deoxy-UDP-GlcNAc is not a substrate for the enzyme and that the enzyme does not contain a tightly bound NAD(+) cofactor.},
	keywords = {BACILLUS-SUBTILIS, D-GLUCOSAMINE 2-EPIMERASE, enterobacterial common antigen, ESCHERICHIA-COLI, FATTY-ACID OXIDATION, GALACTOSE 4-EPIMERASE, POSITIONAL ISOTOPE EXCHANGE, RAT-LIVER, STAPHYLOCOCCUS-AUREUS-H, TEICHOIC-ACIDS, W23},
	isbn = {0002-7863},
	url = {<Go to ISI>://A1997YD81500003},
	author = {Morgan, P. M. and Sala, R. F. and Tanner, M. E.}
}
@article {4157,
	title = {Evidence for a covalently bound form of microcystin-LR in salmon liver and dungeness crab larvae},
	journal = {Chemical Research in Toxicology},
	volume = {10},
	number = {4},
	year = {1997},
	note = {ISI Document Delivery No.: WU385Times Cited: 56Cited Reference Count: 55},
	month = {Apr},
	pages = {463-469},
	type = {Article},
	abstract = {The chemically unique nature of the C-20 beta-amino acid (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoi c acid (Adda) portion of the microcystins has been exploited to develop a strategy to analyze for the total microcystin-1R (1; see Figure 1) burden in salmon liver and crab larvae tissues. Lemieux oxidation of microcystin-LR (1) gives 2-methyl-3-methoxy-4-phenylbutanoic acid (2), a unique marker for the presence of microcystins. The butanoic acid 2 can be isolated and detected by GC/MS from the livers of Atlantic salmon that received an ip injection of microcystin-1R (1) and from tissues of wild-caught crab larvae. The Lemieux oxidation-GC/MS results are compared with those from MeOH extraction-PPase analysis. Only similar to 24\% of the total microcystin-1R (1) burden in salmon liver tissue is found to be extractable with MeOH. Similarly, the Lemieux oxidation-GC/MS method detected 10 000-fold greater microcystin concentrations in Cypress Island Dungeness crab larvae than did the MeOH extraction-PPase method. The disparity in microcystin concentrations measured by the two methods is taken as direct evidence for the existence of covalently bound microcystins in vivo.},
	keywords = {BLUE-GREEN-ALGAE, CYCLIC, DIARRHETIC SHELLFISH TOXINS, DIHYDROMICROCYSTIN-LR, HEPATOCELLULAR, OKADAIC ACID, PEPTIDE TOXIN, PROTEIN PHOSPHATASE INHIBITORS, RAT-LIVER, TISSUE DISTRIBUTION, TUMOR PROMOTION, UPTAKE},
	isbn = {0893-228X},
	url = {<Go to ISI>://A1997WU38500017},
	author = {Williams, D. E. and Craig, M. and Dawe, S. C. and Kent, M. L. and Holmes, C. F. B. and Andersen, R. J.}
}
@article {3777,
	title = {A biomimetic study of cytochrome P450 related oxidations of toluenes using synthetic hemin},
	journal = {Bulletin of the Chemical Society of Japan},
	volume = {69},
	number = {12},
	year = {1996},
	note = {ISI Document Delivery No.: WB844Times Cited: 6Cited Reference Count: 68},
	month = {Dec},
	pages = {3513-3521},
	type = {Article},
	abstract = {A mechanistic study of the oxidation of toluene and its derivatives in a model system designed to mimic cytochrome P450 (P450) using iron tetrakis(2,6-dichlorophenyl)porphyrin chloride (FeTPPC8Cl) and pentafluoroiodosylbenzene (PFIB:C(6)F(5)lO) in dichloromethane is reported. The oxidation products were the corresponding benzyl alcohols and aldehydes; no further oxidation products were observed. The relative reaction rates were obtained in competitive reactions including the use of deuterated analogues. In the present model system, Hammett plots and isotope effects indicated conclusively that the rate-determining step was a hydrogen atom abstraction to the oxoiron intermediate which was then followed by a rebound of an OH group. The concerted mechanism and the rebound mechanism were discussed in terms of the differences in reactivities between the model system and natural P450 enzymes and similar monooxygenation enzymes.},
	keywords = {ALIPHATIC, AROMATIC-COMPOUNDS, CRYSTAL-STRUCTURE, DEPENDENT MONO-OXYGENASES, HYDROGEN-PEROXIDE, HYDROXYLATION, INDUCED, IRON(III) PORPHYRINS, LIVER MICROSOMAL CYTOCHROME-P-450, METABOLISM, MODEL SYSTEMS, RAT-LIVER},
	isbn = {0009-2673},
	url = {<Go to ISI>://A1996WB84400017},
	author = {Nakano, T. and Agatsuma, N. and Kodama, S. and Kakuda, H. and Dolphin, D.}
}
@article {3640,
	title = {Molecular mechanisms underlying the interaction of motuporin and microycystins with type-1 and type-2A protein phosphatases},
	journal = {Biochemistry and Cell Biology-Biochimie Et Biologie Cellulaire},
	volume = {74},
	number = {4},
	year = {1996},
	note = {ISI Document Delivery No.: VX367Times Cited: 61Cited Reference Count: 35},
	pages = {569-578},
	type = {Article},
	abstract = {Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase-toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and nodularin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.},
	keywords = {BLUE-GREEN-ALGAE, CATALYTIC SUBUNIT, cyanobacteria, IDENTIFICATION, MICROCYSTIN-LR, microcystins, motuporin, nodularin, OKADAIC ACID, PHOSPHORYLATION, POTENT INHIBITOR, PROTEIN, PROTEIN PHOSPHATASES, RAT-LIVER, TOXINS},
	isbn = {0829-8211},
	url = {<Go to ISI>://A1996VX36700017},
	author = {Craig, M. and Luu, H. A. and McCready, T. L. and Williams, D. and Andersen, R. J. and Holmes, C. F. B.}
}
