Unraveling the Substrate Recognition Mechanism and Specificity of the Unusual Glycosyl Hydrolase Family 29 BT2192 from Bacteroides thetaiotaomicron

Abstract

Glycosyl hydrolase (GH) family 29 (CAZy database) consists of retaining α-l-fucosidases. We have identified BT2192, a protein from Bacteroides thetaiotaomicron, as the first GH29 representative exhibiting both weak α-l-fucosidase and β-d-galactosidase activities. Determination and analysis of X-ray structures of BT2192 in complex with β-d-galactoside competitive inhibitors showed a new binding mode different from that of known GH29 enzymes. Three point mutations, specific to BT2192, prevent the canonical GH29 substrate α-l-fucose from binding efficiently to the fucosidase-like active site relative to other GH29 enzymes. β-d-Galactoside analogues bind and interact in a second pocket, which is not visible in other reported GH29 structures. Molecular simulations helped in the assessment of the flexibility of both substrates in their respective pocket. Hydrolysis of the fucosyl moiety from the putative natural substrates like 3-fucosyllactose or Lewis(X) antigen would be mainly due to the efficient interactions with the galactosyl moiety, in the second binding site, located more than 6-7 Å apart.

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum

“…infantis and B. thetaiotaomicron, with C␣ r.m.s.d. values of 2.9, 2.9, and 3.5 Å of 299, 305, and 304 residues aligned, respectively (PDB codes 3MO4, 3EYP, and 3GZA) (19,61). 3 Both the overall folds of the N-terminal domains and active site residues are well conserved between FgFCO1 and bacterial homologues (Fig.…”

The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains

“…The catalytic N-terminal domain exhibits the familiar TIMbarrel fold reported for other GH29 enzymes (Guillotin et al, 2014;Lammerts van Bueren et al, 2010). However, -strands 5 and 6 are offset, with only one intra-main-chain hydrogen bond between them.…”

“…Only six X-ray structures of bacterial GH29 enzymes have been deposited in the Protein Data Bank (PDB). These include three proteins from Bacteroides thetaiotaomicron [BT2970 (PDB entry 2wvs), BT2192 (PDB entry 3eyp) and BT3798 (PDB entry 3gza); Lammerts van Bueren et al, 2010;Guillotin et al, 2014], one protein from Bifidobacterium longum subsp. infantis (Blon_2336; PDB entry 3ues; Sakurama et al, 2012), one protein from Thermotoga maritima (TM0306; PDB entry 1hl8; Sulzenbacher et al, 2004) and one protein from Bacteroides ovatus ATCC8483 (BACOVA_04357; PDB entry 4zrx; Joint Center for Structural Genomics, unpublished work).…”

“…The GH29-family enzymes BT2192 from B. thetaiotaomicron (PDB entry 3eyp) and Blon_2336 from B. longum subsp. infantis (PDB entry 3ues) have been characterized as belonging to the GH29-B subfamily (Guillotin et al, 2014;Sakurama et al, 2012). There are currently no published data characterizing the activity of either the enzyme BT3798 from B. thetaiotaomicron (PDB entry 3gza) or the enzyme BACOVA_04357 from B. ovatus (PDB entry 4zrx).…”

Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major

“…47 Enzymatic reaction was carried out at 37°C for 2 hours in a mixture containing 100 nM BfrA and 1 mM substrate, buffered with phosphate (10 mM, pH 7.5). Reaction was stopped using Na2CO3 (0.5 M), and free pNP was detected at 405 nm.…”