Crystal structure ofActinobacillus pleuropneumoniaeHMW1C glycosyltransferase

Abstract

The Haemophilus influenzae HMW1 adhesin is an N-linked glycoprotein that mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. HMW1 is glycosylated by HMW1C, a novel glycosyltransferase in the GT41 family that creates N-glycosidic linkages with glucose and galactose at asparagine residues and di-glucose linkages at sites of glucose modification. Here we report the crystal structure of Actinobacillus pleuropneumoniae HMW1C (ApHMW1C), a functional homolog of HMW1C. The structure of ApHMW1C contains an N-terminal all αdomain (AAD) fold and a C-terminal GT-B fold with two Rossmannlike domains and lacks the tetratricopeptide repeat fold characteristic of the GT41 family. The GT-B fold harbors the binding site for UDPhexose, and the interface of the AAD fold and the GT-B fold forms a unique groove with potential to accommodate the acceptor protein. Structure-based functional analyses demonstrated that the HMW1C protein shares the same structure as ApHMW1C and provided insights into the mechanism of HMW1C glycosylation of HMW1.

Phase-Variable Glycosylation in Nontypeable Haemophilus influenzae

“…50 The Actinobacillus pleuropneumoniae HMW-C glycosyltransferase has been reported to catalyze both N-and O-linked glycosyltransferase reactions. 51 NTHi HMW-A is modified with monohexose or dihexose glycan structures, suggesting that HMW-C is also capable of forming hexose−hexose O-glycosidic bonds. 45,49,51 Some bacteria, such as A. pleuropneumoniae, contain "orphan" HMW-Cs, in which hmwC and the gene encoding the target adhesin or autotransporter protein are at unlinked locations in the genome.…”

“…51 NTHi HMW-A is modified with monohexose or dihexose glycan structures, suggesting that HMW-C is also capable of forming hexose−hexose O-glycosidic bonds. 45,49,51 Some bacteria, such as A. pleuropneumoniae, contain "orphan" HMW-Cs, in which hmwC and the gene encoding the target adhesin or autotransporter protein are at unlinked locations in the genome. 52,53 In NTHi, hmwC is adjacent to the hmwAB operon, although expression of hmwAB and hmwC are controlled by separate promoters.…”

Chemoenzymatic Methods for the Synthesis of Glycoproteins

“…396 Shortly afterward the same group solved the crystal structure of ApHMW1C and found that this HMW1Clike enzyme shared the same structure with HMW1C but harbored a unique catalytic activity different from other members in the GT41 family. 397 Aebi and co-workers reported the expression of the HMW1Clike N-glycosyltransferase (ApNGT) in E. coli and performed a detailed biochemical analysis of the enzyme. 99,101 These studies confirmed that ApNGT could transfer a glucose or galactose moiety from the corresponding UDP-sugars to a peptide containing the N-X-S/T sequence to form an N-linked Glc or Gal-peptide.…”

“…The crystal structure of the NGT from A. pleuropneumoniae (ApNGT) was recently solved, which shows an N-terminal αhelical domain fold and a C-terminal GT-B fold with two Rossmann-like domains. 397 Based on this structure, Peng George Wang and co-workers carried out site-directed mutagenesis around the domain involved in the recognition of the Nglycosylation site aiming to broaden the acceptor substrate specificity of ApNGT. 400 Screening of the mutants against a was able to transfer a glucosamine (GlcN) residue from UDP-GlcN to a peptide (Scheme 31).…”