Roles of Individual Enzyme−Substrate Interactions by α-1,3-Galactosyltransferase in Catalysis and Specificity ,

Glycogenin is a glycosyltransferase that functions as the autocatalytic initiator for the synthesis of glycogen in eukaryotic organisms. Prior structural work identified the determinants responsible for the recognition and binding of UDP-glucose and the catalytic manganese ion and implicated two aspartic acid residues in the reaction mechanism for self-glucosylation. We examined the effects of substituting asparagine and serine for the aspartic acid residues at positions 159 and 162. We also examined whether the truncation of the protein at residue 270 (⌬270) was compatible with its structural integrity and its functional role as the initiator for glycogen synthesis. The truncated form of the enzyme was indistinguishable from the wild-type enzyme by all measures of activity and could support glycogen accumulation in a glycogenin-deficient yeast strain. Substitution of aspartate 159 by either serine or asparagine eliminated self-glucosylation and reduced trans-glucosylation activity by at least 260-fold but only reduced UDP-glucose hydrolytic activity by 4-14-fold. Substitution of aspartate 162 by either serine or asparagine eliminated self-glucosylation activity and reduced UDP-glucose hydrolytic activity by at least 190-fold. The trans-glucosylation of maltose was reduced to undetectable levels in the asparagine 162 mutant, whereas the serine 162 enzyme showed only an 18-30-fold reduction in its ability to trans-glucosylate maltose. These data support a role for aspartate 162 in the chemical step for the glucosyltransferase reaction and a role for aspartate 159 in binding and activating the acceptor molecule.

Section: Fig 2 Subunit Relationships Among Glycogenin Crystal Formssupporting

confidence: 74%

Section: Fig 2 Subunit Relationships Among Glycogenin Crystal Formsmentioning

confidence: 99%

Requirements for Catalysis in Mammalian Glycogenin

Hurley

Stout

Miner

et al. 2005

“…Some belong to other CAZy families, such as bovine ␣-galactosyltransferase (24,25), a representative of family 6. N. meningitidis LgtC is a logical comparison for E. coli WaaJ, because both are family 8 enzymes that add a single sugar onto the growing core OS chain (13).…”

Section: Discussionmentioning

confidence: 99%

The C-terminal Domain of the Escherichia coli WaaJ Glycosyltransferase Is Important for Catalytic Activity and Membrane Association

Maecker

Kaniuk²,

Whitfield

2007

The waaJ gene encodes an ␣-1,2-glucosyltransferase involved in the synthesis of the outer core region of the lipopolysaccharide of some Escherichia coli and Salmonella isolates. WaaJ belongs to glycosyltransferase CAZy family 8, characterized by the GT-A fold, a DXD motif, and by retention of configuration at the anomeric carbon of the donor sugar. Detailed kinetic and structural information for bacterial family 8 glycosyltransferases has resulted from studies of Neisseria meningitidis LgtC. As many as 28 amino acids could be deleted from the C terminus of LgtC without affecting its in vitro catalytic behavior. This C-terminal domain has a high ratio of positively charged and hydrophobic residues, a feature conserved in WaaJ and some other family 8 representatives. Unexpectedly, deletion of as few as five residues from the C terminus of WaaJ resulted in substantially reduced in vivo activity. With deletions of 15 residues or less, activity was only detected when levels of expression were elevated. No in vivo activity was detected after the removal of 20 amino acids, regardless of expression levels. Longer deletions (20 residues and greater) compromised the ability of WaaJ to associate with the membrane. However, the reduced in vivo activity in enzymes lacking 5-12 C-terminal residues also reflected a dramatic drop in catalytic activity in vitro (a 294-fold decrease in the apparent k cat /K m , LPS ). Deletions removing 20 or more residues resulted in a protein showing no detectable in vitro activity. Therefore, the C-terminal domain of WaaJ plays a critical role in enzyme function.

“…The kinetic analysis of the D190N variant may, therefore, be interpreted as consistent with the role of this residue as the catalytic nucleophile in a double-displacement mechanism. Recently, it was found that mutation of the proposed nucleophile of ␤-galactosyl-␣-1,3-galactosyltransferase (Glu-317, structurally equivalent to Gln-189 of LgtC) to glutamine reduced the observed k cat for galactosyltransferase activity 2400-fold (37). However, a detailed study of the binding affinities of wild type and E317Q enzyme for donor and acceptor substrates led the authors to propose that this residue is required for proper acceptor substrate orientation and does not play the role of a catalytic nucleophile (37).…”

Section: Discussionmentioning

confidence: 99%

Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue

Lairson

Chiu

et al. 2004

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...