@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 {5183,
	title = {Catalysis and binding in L-ribulose-5-phosphate 4-epimerase: A comparison with L-fuculose-1-phosphate aldolase},
	journal = {Biochemistry},
	volume = {40},
	number = {49},
	year = {2001},
	note = {ISI Document Delivery No.: 500CJTimes Cited: 15Cited Reference Count: 15},
	month = {Dec},
	pages = {14772-14780},
	type = {Article},
	abstract = {L-Ribulose-5-phosphate (L-Ru5P) 4-epimerase and L-fuculose-1-phosphate (L-Fuc1P) aldolase are evolutionarily related enzymes that display 26\% sequence identity and a very high degree of structural similarity. They both employ a divalent cation in the formation and stabilization of an enolate during catalysis, and both are able to deprotonate the C-4 hydroxyl group of a phosphoketose substrate. Despite these many similarities, subtle distinctions must be present which allow the enzymes to catalyze two seemingly different reactions and to accommodate substrates differing greatly in the position of the phosphate (C-5 vs C-1). Asp76 of the epimerase corresponds to the key catalytic acid/base residue Glu73 of the aldolase. The D76N mutant of the epimerase retained considerable activity, indicating it is not a key catalytic residue in this enzyme. In addition, the D76E mutant did not show enhanced levels of background aldolase activity. Mutations of residues in the putative phosphate-binding pocket of the epimerase (N28A and K42M) showed dramatically higher values of K-M for L-Ru5P. This indicates that both enzymes utilize the same phosphate recognition pocket, and since the phosphates are positioned at opposite ends of the respective substrates, the two enzymes must bind their substrates in a reversed or "flipped" orientation. The epimerase mutant D120N displays a 3000-fold decrease in the value of k(cat), suggesting that Asp 120{\textquoteright} provides a key catalytic acid/base residue in this enzyme. Analysis of the D120N mutant by X-ray crystallography shows that its structure is indistinguishable from that of the wild-type enzyme and that the decrease in activity was not simply due to a structural perturbation of the active site. Previous work [Lee, L.V., Poyner, R.R., Vu, M.V., and Cleland, W.W. (2000) Biochemistry 39, 4821-4830] has indicated that Tyr229{\textquoteright} likely provides the other catalytic acid/base residue. Both of these residues are supplied by an adjacent subunit. Modeling Of L-Ru5P into the active site of the epimerase structure suggests that Tyr229{\textquoteright} is responsible for deprotonating L-Ru5P and Asp 120{\textquoteright} is responsible for deprotonating its epimer, D-Xu5P.},
	keywords = {CLASS-II, CLEAVAGE, ESCHERICHIA-COLI, MECHANISM, MUTAGENESIS, PHOSPHATE},
	isbn = {0006-2960},
	url = {<Go to ISI>://000172608100006},
	author = {Samuel, J. and Luo, Y. and Morgan, P. M. and Strynadka, N. C. J. and Tanner, M. E.}
}
@article {4298,
	title = {Epimerization via carbon-carbon bond cleavage. L-ribulose-5-phosphate 4-epimerase as a masked class II aldolase},
	journal = {Biochemistry},
	volume = {37},
	number = {16},
	year = {1998},
	note = {ISI Document Delivery No.: ZM208Times Cited: 32Cited Reference Count: 35},
	month = {Apr},
	pages = {5746-5754},
	type = {Article},
	abstract = {Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD(+)-independent enzyme epimerizes a steseocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II L-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low k(cat) values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (K-t = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.},
	keywords = {ENZYMES, ESCHERICHIA-COLI, L-FUCULOSE-1-PHOSPHATE ALDOLASE, MECHANISM, MUTAGENESIS, ORGANIC-SYNTHESIS, PCR, PURIFICATION, SEQUENCE, SITE},
	isbn = {0006-2960},
	url = {<Go to ISI>://000073515500048},
	author = {Johnson, A. E. and Tanner, M. E.}
}
@article {3922,
	title = {Properties and kinetic analysis of UDP-glucose dehydrogenase from group a streptococci - Irreversible inhibition by UDP-chloroacetol},
	journal = {Journal of Biological Chemistry},
	volume = {272},
	number = {6},
	year = {1997},
	note = {ISI Document Delivery No.: WG192Times Cited: 39Cited Reference Count: 48},
	month = {Feb},
	pages = {3416-3422},
	type = {Article},
	abstract = {UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD(+)-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (K-i, 2.7 mu M) of the enzyme. The enzyme is irreversibly inactivated by uridine 5{\textquoteright}-diphosphate chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (k(i)/K-i) was found to be 2 x 10(3) mM(-1) min(-1). Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5{\textquoteright}-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.},
	keywords = {ALDEHYDE DEHYDROGENASE, BOVINE LIVER, CAPSULE, CONSERVED CYSTEINE RESIDUES, GROUP-A STREPTOCOCCI, HISTIDINOL DEHYDROGENASE, HYALURONIC-ACID SYNTHESIS, MOLECULAR CHARACTERIZATION, MUTAGENESIS, PNEUMONIAE, SITE-DIRECTED},
	isbn = {0021-9258},
	url = {<Go to ISI>://A1997WG19200045},
	author = {Campbell, R. E. and Sala, R. F. and vandeRijn, I. V. and Tanner, M. E.}
}
