A β1‐6/β1‐3 galactosidase from Bifidobacterium animalis subsp. lactis Bl‐04 gives insight into sub‐specificities of β‐galactoside catabolism within Bifidobacterium

Abstract

SummaryThe Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host-derived β-galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) β-galactosidase from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 (BlGal42A). BlGal42A displays a preference for undecorated β1-6 and β1-3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N-acetyl substituted β1-3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite + 1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite − 1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting β1-6 and β1-3 galactosides likely reflects structural differences between these substrates and β1-4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub-specificities that support metabolic specialization in the gut niche.

Structure–function relationships in Gan42B, an intracellular GH42 β-galactosidase fromGeobacillus stearothermophilus

“…Theoretical calculations conducted with PISA (Krissinel & Henrick, 2007) predict a free energy of dissociation (ÁG diss ) value of 72.3 kcal mol À1 upon dissociation of the trimeric assembly, implying considerably strong assembly interactions. In addition, a similar trimeric assembly has also been observed in the crystal structures of the three other homologous GH42 -galactosidases reported to date (Hidaka et al, 2002;Maksimainen et al, 2012;Viborg et al, 2014). The significance of such observations is strengthened considering the different crystallographic environments of these four structures, with one trimer in the crystallographic asymmetric unit of Gan42B and BlGal42A (Viborg et al, 2014), one monomer in the crystallographic asymmetric unit of A4--Gal (Hidaka et al, 2002) and two trimers in the crystallographic asymmetric unit of Bca--Gal (Maksimainen et al, 2012).…”

“…A comparison of Gan42B-WT with the other three homologous GH42 -galactosidase structures reported to date indicates relatively high structural similarities, with r.m.s.d. values of 1.98, 1.21 and 1.87 Å for A4--gal (Hidaka et al, 2002), Bca--gal (Maksimainen et al, 2012) and BlGal42A (Viborg et al, 2014), respectively (Fig. 2c).…”

“…alkalophilus (39% identity; PDB entry 3tty; Maksimainen et al, 2012) and BlGal42A from Bifidobacterium animalis subsp. lactis Bl-04 (32% identity; PDB entry 4uni; Viborg et al, 2014). In the present report, we describe the full crystallographic structural characterization of Gan42B as the fourth structure of this group of enzymes, focusing on the detailed three-dimensional structures of Gan42B-WT and Gan42B-E323A at 2.45 and 2.50 Å resolution, respectively.…”

Diversity in β-galactosidase Specificities within <i>Bifidobacterium</i>: Towards an Understanding of β-Galactoside Metabolism in the Gut Niche

“…Several GH42 β-galactosidases from B. animalis subsp. lactis BlGal42A, however targets completely different, yet structurally similar β-galactosides of plant arabinogalactan proteins (23).…”

“…In a phylogenetic analysis of GH42 (Fig. 1), including all sequences within Bifidobacterium and those structurally characterized (20,21,23) within the CAZy database (16), the enzymes segregate according to taxonomy and enzymatic function (Table 1). From the phylogenetic analysis ( Fig.…”