Enzymatic hydrolysis of glycosides can occur by one of two elementary mechanisms identified by the stereochemical outcome of the reaction, inversion or retention. The key active-site residues involved are a pair of carboxylic acids in each case, and strategies for their identification and for probing the details of their roles in catalysis have been developed through detailed kinetic analysis of mutants. Similarly the roles of other active-site residues have also been probed this way, and mutants have been developed that trap intermediates in catalysis, allowing the determination of the three-dimensional structures of several such key species. By manipulating the locations or even the presence of these carboxyl side chains in the active site, the mechanisms of several glycosidases have been completely changed, and this has allowed the development of "glycosynthases," mutant glycosidases that are capable of synthesizing oligosaccharides but unable to degrade them. Surprisingly little progress has been made on altering specificities through mutagenesis, although recent results suggest that gene shuffling coupled with effective screens will provide the most effective approach.

Many bacterial pathogens express lipooligosaccharides that mimic human cell surface glycoconjugates, enabling them to attach to host receptors and to evade the immune response. In Neisseria meningitidis, the galactosyltransferase LgtC catalyzes a key step in the biosynthesis of lipooligosaccharide structure by transferring alpha-d-galactose from UDP-galactose to a terminal lactose. The product retains the configuration of the donor sugar glycosidic bond; LgtC is thus a retaining glycosyltranferase. We report the 2 A crystal structures of the complex of LgtC with manganese and UDP 2-deoxy-2-fluoro-galactose (a donor sugar analog) in the presence and absence of the acceptor sugar analog 4'-deoxylactose. The structures, together with results from site-directed mutagenesis and kinetic analysis, give valuable insights into the unique catalytic mechanism and, as the first structure of a glycosyltransferase in complex with both the donor and acceptor sugars, provide a starting point for inhibitor design.

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Lipopolysaccharyl-␣-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 ␣-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K m for UDP-galactose is not significantly altered, the k cat was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 Å away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k cat value 3000-fold lower than that of the wild type enzyme. A 2.6-Å crystal structure of the Q189E mutant with bound uridine 5 -diphospho-2-deoxy-2-fluoro-␣-D-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.Oligosaccharides on glycoproteins and glycolipids distributed on cell surfaces and within extracellular matrices are known to play key roles in normal cell functions including cell growth and differentiation, recognition by the immune system, and cell-cell interactions (1-3). Changes in the composition of these glycoconjugates are often associated with disease states, including the metastasis of cancerous cells and autoimmune responses (4 -7). They are also known to modulate interactions with viral and bacterial pathogens leading to infection and are involved in mechanisms to evade host immune responses (8 -10). Glycosyltransferases, the anabolic enzymes responsible for the highly specific construction of these carbohydrate structures, therefore, not only represent an attractive class of therapeutic targets but also are important tools for the enzymatic synthesis of this synthetically challenging class of therapeutic agents. Of central importance to both of these applications is a detailed understanding of the mechanisms by which this c...

A highly sensitive and continuous protein tyrosine phosphatase (PTPase) assay using 3,6-fluorescein diphosphate (FDP) is described. Leukocyte phosphatase CD45 (leukocyte common antigen), protein tyrosine phosphatase-lB, and leukocyte common antigen-related protein LAR preferentially hydrolyze FDP to fluorescein monophosphate (FMP) with Vmax and Km values comparable with those of phosphotyrosine peptide substrates. Further hydrolysis of FMP to fluorescein was less efficient because of increased Km values compared with those of FDP. FMP absorbs strongly at 445 nm and fluoresces intensely near 515 nm, both of which are insensitive to pH perturbations above pH 6. Its high catalytic efficiency, coupled with the highly sensitive dual detection in the visible wavelength region and wider pH operating range, make FDP the substrate of choice for PTPase inhibitor screening in HTS format and assay miniaturization.