The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

Abstract

Background: β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8).

Results: BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan and displays the highest catalytic efficiency reported to date for a GH5 mannanase owing to a very high k cat (1828 ± 87 s(-1)) and a low K m (1.58 ± 0.23 g · L(-1)) using locust bean galactomannan as substrate. The novel CBM of BlMan5_8 mediates increased binding to soluble mannan based on affinity electrophoresis. Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs present only in mannanases. Members of this new subfamily lack an aromatic residue mediating binding to insoluble cellulose in canonical CBM10 members consistent with the observed low mannan affinity.

Conclusion: BlMan5_8 is evolved for efficient deconstruction of soluble mannans, which is reflected by an exceptionally low K m and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules. These features highlight fine tuning of catalytic and binding properties to support specialization towards a preferred substrate, which is likely to confer an advantage in the adaptation to competitive ecological niches.

Fig. 1

Initial products from INM and LBG. Manno-oligosaccharide products formed after 30 s of a INM and b LBG hydrolysis by BlMan5_8 as measured using HPAEC-PAD analysis. The chromatograms from BlMan5_8-ΔCBM10 (I), BlMan5_8 (II) and the substrate before hydrolysis (III)

Fig. 2

Product formation from M_5. Products after 20 min of reaction of 2 mM M₅ with BlMan5_8 (solid line) compared to the substrate before hydrolysis (dashed line) as analyzed by HPAEC-PAD. The solid line is offset by 10 nC on the y axis for clarity

Fig. 3

Transglycosylation product formation. Mass-spectrum (MALDI-TOF MS) of the product formation by BlMan5_8 after 2 h of incubation with 5 mM M₅. The inserted mass-spectrum is a close that shows the mass-spectrum from m/z 950 to1350

Fig. 4

Relative productive binding frequencies for M₅ to BlMan5_8. Percentages are calculated by combining HPAEC-PAD product quantification with analysis of ¹⁸O labeling using MALDI-TOF MS. Numbers from −4 to +4 designate substrate-binding subsites. The arrow indicates the scissile bond hydrolyzed by the enzyme. The grey sugar unit indicates the reducing end of M₅

Fig. 5

Modeling of putative subsite residues in BlMan5_8. Conserved aromatic residues in the active site of BlMan5_8 are visualized (green) by homology modeling using the structure from TfMan5A (PDB ID: 1BQC) as template (grey). A semi-transparent molecular surface is shown to depict the topology of the active site of TfMan5A. The conserved aromatic residues are designated with BlMan5_8 numbering and the co-crystallized M₃, accommodated at subsites –4 through –2 in TfMan5A, is shown in white

Fig. 6

Phylogenetic tree and sequence alignment of CBM10 sequences. Phylogenetic tree constructed based on sequence alignment of available CBM10 sequences (a) and alignment of the CBM10 of BlMan5_8 and putative CBM10 sequences of characterized enzymes as predicted by dbCAN (b). The sub-tree containing BlMan5_8 is highlighted in black. The characterized enzymes BlMan5_8, SlMan5A [54], CjMan5A [22] and CjXyn10A [55] are indicated. The dbCAN tool (http://csbl.bmb.uga.edu/dbCAN/index.php) was used to obtain putative CBM10 sequences and the sequence of the SlMan5A CBM10 was added manually. Multiple sequence alignment of CBM10 from the five characterized enzymes is shown. The proposed binding residues Tyr⁸, Thr¹⁰, Trp²², Trp²⁴ and Gln³⁹ in the CjXyn10A CBM10 structure [52] are indicated by arrows. The unique insertion in the BlMan5_8 clade preceding the binding residue Trp²² is indicated with a horizontal parenthesis