Abstract
FlgJ is a glycoside hydrolase (GH) enzyme belonging to the Carbohydrate Active enZyme (CAZy) family GH73. It facilitates passage of the bacterial flagellum through the peptidoglycan (PG) layer by cleaving the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid sugars that comprise the glycan strands of PG. Here we describe the crystal structure of the GH domain of FlgJ from bacterial pathogen Salmonella typhimurium (StFlgJ). Interestingly, the active site of StFlgJ was blocked by the C-terminal α-helix of a neighbouring symmetry mate and a β-hairpin containing the putative catalytic glutamic acid residue Glu223 was poorly resolved and could not be completely modeled into the electron density, suggesting it is flexible. Previous reports have shown that the GH73 enzyme Auto from Listeria monocytogenes is inhibited by an N-terminal α-helix that may occlude the active site in similar fashion. To investigate if the C-terminus of StFlgJ inhibits GH activity, the glycolytic activity of StFlgJ was assessed with and without the C-terminal α-helix. The GH activity of StFlgJ was unaffected by the presence or absence of the α-helix, suggesting it is not involved in regulating activity. Removal of the C-terminal α-helix did, however, allow a crystal structure of the domain to be obtained where the flexible β-hairpin containing residue Glu223 was entirely resolved. The β-hairpin was positioned such that the active site groove was fully solvent-exposed, placing Glu223 nearly 21.6 Å away from the putative general acid/base residue Glu184, which is too far apart for these two residues to coordinate glycosidic bond hydrolysis. The mobile nature of the StFlgJ β-hairpin is consistent with structural studies of related GH73 enzymes, suggesting that a dynamic active site may be common to many GH73 enzymes, in which the active site opens to capture substrate and then closes to correctly orient active site residues for catalysis.
Highlights
Glycoside hydrolase (GH) enzymes have been classified into families based on primary amino acid sequence [1]
GH family 73 (GH73) enzymes are associated with bacterial pathogenicity, for example, LytB being critical for pneumococcal nasal colonization and cell division [13], Auto for pathogenic listeriosis caused by Listeria monocytogenes [10], and FlgJ for biogenesis and function of the flagellum, which is a major virulence factor of motile pathogenic bacteria [14]
The first mechanism involves two-steps where, initially, one carboxyl group acts as a general acid to protonate the glycosidic oxygen while the other acts as a nucleophile that attacks the anomeric carbon of the glycosidic bond
Summary
Glycoside hydrolase (GH) enzymes have been classified into families based on primary amino acid sequence (http://www.cazy.org/) [1]. Removing the C-terminal α-helix, allowed us to determine a 2.1 Å crystal structure of the domain in which the extended β-hairpin, known to accommodate a second putative catalytic carboxyl group, was well ordered and revealed that the active site of StFlgJ seems capable of opening widely, possibly aiding in substrate capture and turnover.
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