Structure of UDP Complex of UDP-galactose:β-Galactoside-α-1,3-galactosyltransferase at 1.53-Å Resolution Reveals a Conformational Change in the Catalytically Important C Terminus

Kre2p/Mnt1p is a Golgi ␣1,2-mannosyltransferase involved in the biosynthesis of Saccharomyces cerevisiae cell wall glycoproteins. The protein belongs to glycosyltransferase family 15, a member of which has been implicated in virulence of Candida albicans. We present the 2.0 Å crystal structures of the catalytic domain of Kre2p/Mnt1p and its binary and ternary complexes with GDP/Mn 2؉ and GDP/Mn 2؉ /acceptor methyl-␣-mannoside. The protein has a mixed ␣/␤ fold similar to the glycosyltransferase-A (GT-A) fold. Although the GDPmannose donor was used in the crystallization experiments and the GDP moiety is bound tightly to the active site, the mannose is not visible in the electron density. The manganese is coordinated by a modified DXD motif (EPD), with only the first glutamate involved in a direct interaction. The position of the donor mannose was modeled using the binary and ternary complexes of other GT-A enzymes. The C1؆ of the modeled donor mannose is within hydrogen-bonding distance of both the hydroxyl of Tyr 220 and the O2 of the acceptor mannose. The O2 of the acceptor mannose is also within hydrogen bond distance of the hydroxyl of Tyr 220 . The structures, modeling, site-directed mutagenesis, and kinetic analysis suggest two possible catalytic mechanisms. Either a double-displacement mechanism with the hydroxyl of Tyr 220 as the potential nucleophile or alternatively, an S N i-like mechanism with Tyr 220 positioning the substrates for catalysis. The importance of Tyr 220 in both mechanisms is highlighted by a 3000-fold reduction in k cat in the Y220F mutant.

“…This is in keeping with the ordered bi-bi kinetics suggested for other retaining glycosyltransferases (15,17).…”

Section: Protein Expression Of Wild-type and Mutant Proteins-duringsupporting

confidence: 63%

Section: Protein Expression Of Wild-type and Mutant Proteins-duringmentioning

confidence: 99%

See 1 more Smart Citation

Structure of Kre2p/Mnt1p

Lobsanov

Romero

Sleno

et al. 2004

“…Recent findings have, however, brought into question the applicability of this mechanism to retaining nucleoside diphosphate-utilizing glycosyltransferases since good candidates for the catalytic nucleophile are generally lacking, and in no case has an intermediate been trapped and characterized. The claimed observation of a galactosyl-enzyme intermediate on the retaining ␣-1,3-galactosyltransferase (EC 2.4.1.151) (17), despite low resolution and poor structure factors, is undermined by the more recent redetermination of that structure at a higher resolution (18). This new structure revealed that a large portion of the active site adopts a different conformation that is inconsistent with prior proposals.…”

contrasting

confidence: 53%

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...

“…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.