N-glycosyltransferase is an enzyme in prokaryotes which transfers individual hexoses onto asparagine sidechains in substrate proteins, using a nucleotide-bound intermediary, within the cytoplasm. They are distinct from regular N-glycosylating enzymes, which are oligosaccharyltransferases that transfer pre-assembled oligosaccharides. Both enzyme families however target a shared amino acid sequence asparagine—-any amino acid except proline—serine or threonine (N–x–S/T), with some variations. Such enzymes have been found in the bacteria Actinobacillus pleuropneumoniae (whose N-glycosyltransferase is the best researched member of this enzyme family) and Haemophilus influenzae, and later in other bacterial species such as Escherichia coli. N-glycosyltransferases usually target adhesin proteins, which are involved in the attachment of bacterial cells to epithelia (in pathogenic bacteria); glycosylation is important for the stability and function of the adhesins.
History and definition N-glycosyltransferase activity was first discovered in 2003 by St. Geme et al. in Haemophilus influenzae and identified as a novel type of glycosyltransferase in 2010. The Actinobacillus pleuropneumoniae N-glycosyltransferase is the best researched enzyme of this family. Initially, protein glycosylation was considered to be a purely eukaryotic process before such processes were discovered in prokaryotes, including N-glycosyltransferases.
Biochemistry N-glycosyltransferases are an unusual type of glycosyltransferase which joins single hexoses to the target protein. Attachment of sugars to the nitrogen atom in an amide group — such as the amide group of an asparagine — requires an enzyme, as the electrons of the nitrogen are delocalized in a pi-electron system with the carbon of the amide. Several mechanisms have been proposed for the activation. Among these are a deprotonation of the amide, an interaction between a hydroxyl group in the substrate sequon with the amide (a theory which is supported by the fact that the glycosylation rates appear to increase with the basicity of the second amino acid in the sequon) and two interactions involving acidic amino acids in the enzyme with each hydrogen atom of the amide group. This mechanism is supported by x-ray structures and biochemical information about glycosylation processes; the interaction breaks the delocalization and allows the electrons of the nitrogen to perform a nucleophilic attack on the sugar substrate. N-glycosyltransferases from Actinobacillus pleuropneumoniae and Haemophilus influenzae use an asparagine-amino acid other than proline-serine or threonine sequences as target sequences, the same sequence used by oligosaccharyltransferases. The glutamine-469 residue in the Actinobacillus pleuropneumoniae N-glycosyltransferase and its homologues in other N-glycosyltransferases is important for the selectivity of the enzyme. The enzyme activity is further influenced by the amino acids around the sequon, with beta-loop structures especially important. At least the Actinobacillus pleuropneumoniae N-glycosyltransferase can also hydrolyze sugar-nucleotides in the absence of a substrate, a pattern frequently observed in glycosyltransferases, and some N-glycosyltransferases can attach additional hexoses on oxygen atoms of the protein-linked hexose. N-glycosylation by Actinobacillus pleuropneumoniae HMW1C does not require metals, consistent with observations made on other GT41 family glycosyltransferases and a distinction from oligosaccharyltransferases.
Classification Structurally N-glycosyltransferases belong to the GT41 family of glycosyltransferases and resemble protein O-GlcNAc transferase, a eukaryotic enzyme with various nuclear, mitochondrial and cytosolic targets. Regular N-linked oligosaccharyltransferases belong to a different protein family, STT3. The Haemophilus influenzae N-glycosyltransferase has domains with homologies to glutathione S-transferase and glycogen synthase. The N-glycosyltransferases are subdivided into two functional classes, the first (e.g several Yersinia, Escherichia coli and Burkholderia sp.) is linked to trimeric autotransporter adhesins and the second has enzymes genomically linked to ribosome and carbohydrate metabolism associated proteins (e.g Actinobacillus pleuropneumoniae, Haemophilus ducreyi and Kingella kingae).
Functions N-linked glycosylation is an important process, especially in eukaryotes where over half of all proteins have N-linked sugars attached and where it is the most common form of glycosylation. The processes are also important in prokaryotes and archaeans. In animals for example protein processing in the endoplasmic reticulum and several functions of the immune system are dependent on glycosylation. The principal substrates of N-glycosyltransferases are adhesins. Adhesins are proteins that are used to colonize a surface, often a mucosal surface in the case of pathogenic bacteria. N-glycosyltransferase homologues have been found in pathogenic gammaproteobacteria, such as Yersinia and other pasteurellaceae. These homologues are very similar to the Actinobacillus pleuropneumoniae enzyme and can glycosylate the Haemophilus influenzae HMW1A adhesin. N-glycosyltransferases may be a novel glycoengineering tool, considering that they do not require a lipid carrier to perform their function. Glycosylation is important for the function of many proteins and the production of glycosylated proteins can be a challenge. Potential uses of glycoengineering tools include the creation of vaccines against protein-bound polysaccharides.
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