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

A β1-6/β1-3 galactosidase from Bifidobacterium animalissubsp. lactis Bl-04 gives insight into sub-specificities ofβ-galactoside catabolism within Bifidobacterium

Alexander Holm Viborg,1 Folmer Fredslund,1

Takane Katayama,2 Stinne Kirketerp Nielsen,3

Birte Svensson,1 Motomitsu Kitaoka,4

Leila Lo Leggio3* and Maher Abou Hachem1**1Enzyme and Protein Chemistry, Department ofSystems Biology, Technical University of Denmark,DK-2800 Kgs. Lyngby, Denmark.2Ishikawa Prefectural University, Nonoichi, Ishikawa921-8836, Japan.3Department of Chemistry, University of Copenhagen,DK-2100 Copenhagen Ø, Denmark.4National Food Research Institute, National Agricultureand Food Research Organization, 2-1-12 Kannondai,Tsukuba, Ibaraki 305-8642, Japan.

Summary

The Bifidobacterium genus harbours several healthpromoting members of the gut microbiota. Bifidobac-teria display metabolic specialization by preferentiallyutilizing dietary or host-derived β-galactosides. Thisstudy investigates the biochemistry and structure of aglycoside hydrolase family 42 (GH42) β-galactosidasefrom the probiotic Bifidobacterium animalis subsp.lactis Bl-04 (BlGal42A). BlGal42A displays a prefer-ence for undecorated β1-6 and β1-3 linked galacto-sides and populates a phylogenetic cluster with closebifidobacterial homologues implicated in the utiliza-tion of N-acetyl substituted β1-3 galactosides fromhuman milk and mucin. A long loop containing aninvariant tryptophan in GH42, proposed to bind sub-strate at subsite + 1, is identified here as specificitysignature within this clade of bifidobacterial enzymes.Galactose binding at the subsite − 1 of the active siteinduced conformational changes resulting in an extrapolar interaction and the ordering of a flexible loopthat narrows the active site. The amino acid sequenceof this loop provides an additional specificity signa-ture within this GH42 clade. The phylogenetic related-

ness of enzymes targeting β1-6 and β1-3 galactosideslikely reflects structural differences between thesesubstrates and β1-4 galactosides, containing an axialgalactosidic bond. These data advance our molecularunderstanding of the evolution of sub-specificitiesthat support metabolic specialization in the gut niche.

Introduction

The human gastrointestinal tract is colonized by a complexand densely populated microbial community that is estab-lished shortly after birth and develops to an adult-likecomposition over 2–3 years (Morgan et al., 2013). Theinterplay between the human host, diet and gut microbiotahas a profound impact on human health and development,and imbalance in this community (dysbiosis) is associatedwith serious metabolic and immune-disorders, e.g. inflam-matory bowel disease, obesity and metabolic syndromesas well as colon cancer (Nicholson et al., 2012).

The saccharolytic capabilities of humans are limited tosucrose, lactose, and to a certain extent starch (Cantarelet al., 2012). Human diet, however, is rich in a wide varietyof saccharides derived from plants and animals and com-petition for these non-digestible glycans is increasinglyrecognized as an important factor in shaping the gutmicrobiota (Koropatkin et al., 2012). Metagenomic andmetaproteomic (Verberkmoes et al., 2009) studies haveunderscored the key role of glycan metabolism in micro-bial adaptation to the gut niche. Glycans or glycoconju-gates containing β-galactosidic linkages are particularlyinteresting as they are abundant in nutrition of infants, e.g.milk from humans (Han et al., 2012) and cows (Urashimaet al., 2001), as well as adults, e.g. in edible plants (veg-etables, fruits, cereals) where β-galactosides are majorcomponents in primary cell wall pectic polysaccharides(Ridley et al., 2001; Vincken et al., 2003) and arabinoga-lactan proteins (McNeil and Darvill, 1984). In addition,β-galactosides are ubiquitous in the mucin glycoproteinlayer that coats the epithelial colonocytes (Jensen et al.,2010). The abundance and diversity of β-galactosides ismatched by different microbial utilization strategies inthe gut. Dominant gut commensals from the Bacteroidesgenus target both host (mucin) and dietary polymeric

Accepted 28 September, 2014. For correspondence. *E-mail [email protected]; Tel. (+45) 35320295; Fax (+45) 35320322; **[email protected]; Tel. (+45) 45252732; Fax (+45) 45886307.

Molecular Microbiology (2014) ■ doi:10.1111/mmi.12815

© 2014 John Wiley & Sons Ltd

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β-galactosides (pectic galactan and arabinogalactan) viapolysaccharide utilization loci encoding sensory anduptake proteins as well as a variety of cell-attached andperiplasmic hydrolases (Martens et al., 2009). By con-trast, other microbiota members are considered assecondary degraders that preferentially act on smallerβ-galactoside oligomers, which either occur naturally as inhuman milk oligosaccharides (HMOs), or are acquired bycross-feeding from other microorganisms of the micro-biota. β-Galactoside oligomers of milk and plant originhave been shown to be preferentially fermented by healthstimulating probiotic bacteria from the Lactobacillusand Bifidobacterium genera (Gibson et al., 2004). β-Galactosidases assigned into glycoside hydrolase family42 (GH42) in the sequence-based classification system inthe CAZy database (Lombard et al., 2014) are instrumen-tal in the metabolism of a variety of β-galactosidesin the gut niche (Hinz et al., 2004; Goulas et al., 2009;Yoshida et al., 2012). Recently, the specificities of GH42β-galactosidases from Bifidobacterium longum subsp.infantis (Yoshida et al., 2012; Garrido et al., 2013; Viborget al., 2014), Bifidobacterium bifidum (Goulas et al.,2009), and Bifidobacterium adolescentis (Van Laereet al., 2000; Hinz et al., 2004) were explored. However,insight into the specificity of GH42 β-galactosidases fromthe pseudolongum group of bifidobacteria, that harboursimportant probiotic taxa (Ventura et al., 2007) is lacking.Bifidobacterium animalis subsp. lactis, which is the mostimportant representative of the pseudolongum group, wasoriginally isolated from fermented milk (Meile et al., 1997)and has been widely used as a probiotic due to its tech-nological robustness compared to other bifidobacteria(e.g. tolerance to oxygen and bile acids) and clinicallydocumented probiotic properties (Loquasto et al., 2013),e.g. modulation of the immune system (Rizzardini et al.,2012), reduction of colonic transit time (Marteau et al.,2002), and improved digestive comfort (Guyonnet et al.,2009).

The genome of B. animalis subsp. lactis Bl-04(Barrangou et al., 2009) encodes two GH42 putativeβ-galactosidases, but only one of which was transcription-ally upregulated upon growth of the strain on a commercialβ-galacto-oligosaccharide (GOS) mixture (Andersen et al.,2013). The responsive GH42 gene resides in an operonencoding a LacI-type transcriptional regulator and an ATP-binding cassette (ABC) transporter, which outlines a routefor uptake and catabolism of GOS in this bacterium(Andersen et al., 2013). Biochemical characterization ofthe GH42 β-galactosidase (BlGal42A) encoded by thislocus reveals a clear albeit modest preference for undeco-rated β1-6 galactosides closely followed by β1-3 counter-parts. Comparison of this first three-dimensional structureof a GH42 from Bifidobacterium with characterized ortho-logues enabled the elucidation of possible specificity deter-

minants within this β-galactosidase family associated withphysiologically important members of the gut microbiota.Altogether, these data promote our understanding of theevolution of β-galactoside sub-specificities that supportmetabolic specialization and contribute to fitness in the gutniche.

Results

Biochemical characterization

Recombinant BlGal42A was produced and purified to elec-trophoretic homogeneity in a yield of 1.4 mg g−1 cell wetweight. BlGal42A migrated as a single band in SDS-PAGEanalysis with the expected theoretically calculated molecu-lar mass of 78.241 kDa. Analytical gel filtration datashowed that BlGal42A eluted as a single peak correspond-ing to 148 kDa. This is supportive of an oligomeric organi-zation as confirmed by the crystal structure (described indetail below), albeit with a smaller size than the theoreti-cally calculated 235 kDa for the trimer. Discrepancybetween size determined by gel filtration and by crystallog-raphy for other carbohydrate active enzymes hasbeen attributed to non-specific interactions with thepolysaccharide-based chromatography resin (e.g. Ernstet al., 2006). BlGal42A displayed significant activitytowards o-nitrophenyl-β-D-galactopyranoside (ONP-Galp)and p-nitrophenyl-β-D-galactopyranoside (PNP-Galp), butno activity was detected for other PNP-glycosides. Themaximal activity towards ONP-Galp was at pH 5.5and 45°C, with an Arrhenius activation energy ofEa = 13.8 kJ mol−1 (Fig. S1). The kinetics of hydrolysis ofONP-Galp by BlGal42A were best described using anun-competitive substrate inhibition model with kcat, Km

and Kis values of 1018 ± 49 s−1, 6.5 ± 0.5 and 27 ± 3 mMrespectively. Thus, substrate inhibition was most pro-nounced at high substrate concentrations. The catalyticnucleophile (inferred from multiple sequence alignment)mutant BlGal42A-Glu324Ala showed 750 000-fold lowerspecific activity for ONP-Galp than wild-type BlGal42A,confirming the status of the glutamic acid side-chain.

Hydrolysis kinetics data for a series of β-galactosidesubstrates (Table 1) showed that BlGal42A has prefer-ence for 6-β-galactobiose (Galβ1-6Gal; kcat/Km = 74 ±5 s−1 mM−1), closely followed by 3-β-galactosylglucose(Galβ1-3Glc; kcat/Km = 56 ± 1 s−1 mM−1) and 3-β-galactobiose (Galβ1-3Gal; kcat/Km = 52 ± 1 s−1 mM−1). Allthese substrates have an equatorial galactosidic linkage(Fig. 1). By contrast, the catalytic efficiency was fivefoldlower on 4-β-galactobiose (Galβ1-4Gal; kcat/Km = 15 ±1 s−1 mM−1), which has an axial glycosidic linkage, butonly twofold lower for Galβ1-4-L-Rha (kcat/Km = 36 ±1 s−1 mM−1), owing to the high kcat value towards this sub-strate with an equatorial galactosidic linkage. BlGal42A

2 A. H. Viborg et al. ■

© 2014 John Wiley & Sons Ltd, Molecular Microbiology

hydrolysed the longer oligosaccharides assayed in thepresent study with 6- to 16-fold reduced catalytic efficiencyas compared to the corresponding disaccharides,e.g. 6-β-galactotetraose (Galβ1-6Galβ1-6Galβ1-6Gal;kcat/Km = 12 ± 1 s−1 mM−1) and β1-4 linked galactotetraose.BlGal42A showed either low (< 4% of 6-β-galactobiose)or essentially no activity on various HMOs, i.e. lactose(Galβ1-4Glc), lacto-N-tetraose (LNT; Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-neotetraose (LNnT; Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and selected fucosylatedderivatives thereof. All oligosaccharides with an N-acetylhexosamine at the penultimate position from thenon-reducing end (which would occupy subsite + 1 ifbound) were either not hydrolysed or hydrolysed with verylow efficiencies (kcat/Km < 1.5 s−1 mM−1). The same wasvalid for oligosaccharides containing fructose or mannoseat the same position (Table 1).

Determination of the BlGal42A structure in free andgalactose bound forms

Three-dimensional structures of BlGal42A in complex withgalactose (BlGal42A-Gal), the Glu324Ala catalytic nucleo-phile mutant (BlGal42A-Glu324Ala) and this mutant incomplex with galactose (BlGal42A-Glu324Ala-Gal) weredetermined by X-ray crystallography. Molecular replace-ment phases were initially obtained for a crystal ofBlGal42A with no galactose bound, but the structure couldnot be fully refined due to twinning. Refinement and Ram-achandran statistics for the three other structures aresummarized in Table 2. BlGal42A is a homo-trimer of 695residues per subunit. The first six residues (Met1–Gln6)and a part of the loop Val220–Asn225 in all chains of thegalactose-free structure BlGal42A-Glu324Ala were disor-dered and not included in the final models. The three

Table 1. Hydrolysis kinetic parameters of BlGal42A from B. animalis subsp. lactis Bl-04.

Oligosaccharide Km (mM) kcat (s−1) kcat/Km (mM−1 s−1)

6-β-galactobioseGalβ1-6Gal

5.4 ± 0.5 398 ± 12 74 ± 5

6-β-galactosyllactosea

Galβ1-6Galβ1-4Glc<5

6-β-galactotetraoseGalβ1-6Galβ1-6Galβ1-6Gal

15 ± 2 179 ± 13 12 ± 1

3-β-galactosylglucoseGalβ1-3Glc

6.3 ± 0.3 357 ± 9 56 ± 1


9.4 ± 0.3 492 ± 10 52 ± 1


Galβ1-3Galβ1-4Glc< 5

3-β-galactobiosyllactoseGalβ1-3Galβ1-3Galβ1-4Glc

n.d.

3-β-galactotriosyllactoseGalβ1-3Galβ1-3Galβ1-3Galβ1-4Glc

n.d.

LactoseGalβ1-4Glc

25 ± 6 70 ± 10 2.8 ± 0.3


19 ± 1 281 ± 11 15 ± 0.5


Galβ1-4Galβ1-4Glc< 10

4-β-galactotrioseGalβ1-4Galβ1-4Gal

12 ± 2 123 ± 12 10 ± 1

4-β-galactotetraoseGalβ1-4Galβ1-4Galβ1-4Gal

0.9 ± 0.1

3-fucosyllactoseGalβ1-4[Fucα1–3]Glc

n.d.

LactuloseGalβ1-4Fru

2.8 ± 0.1

EpilactoseGalβ1-4Man

0.1 ± 0.0

4-galactosyl-L-rhamnoseGalβ1-4-L-Rha

21 ± 6 740 ± 41 36 ± 1

a. As judged by thin-layer chromatography.There was no activity towards substrates with N-acetylhexosamine at the penultimate position: lacto-N-biose I (LNB; Galβ1-3GlcNAc), galacto-N-biose (GNB; Galβ1-3GalNAc), lacto-N-tetraose (LNT; Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose II (Galβ1-3[Fucα1–4]GlcNAcβ1-3Galβ1-4Glc), Lewis A (Galβ1-3[Fucα1–4]GlcNAc), N-Acetyl-D-lactosamine (LacNAc; Galβ1-4GlcNAc), lacto-N-neotetraose (LNnT; Galβ1-4GlcNAcβ1-3Galβ1-4Glc), Lewis X (Galβ1-4[Fucα1–3]GlcNAc), and lacto-N-fucopentaose III (Galβ1-4[Fucα1–3]GlcNAcβ1-3Galβ1-4Glc).n.d., not detected.

β-Galactoside catabolism within Bifidobacterium 3


molecules in each asymmetric unit are similar, with aroot mean square deviation (RMSD) of 0.18 Å, 0.19 Åand 0.22 Å, calculated for all aligned atoms betweenmonomers, in BlGal42A-Glu324Ala, BlGal42A-Gal, andBlGal42A-Glu324Ala-Gal respectively.

Overall structure

BlGal42A is a trimer shaped as a bowl with the widest(inner) diameter of 30 Å at the rim and a bottom closed bya zinc ion (Fig. 2A). The BlGal42A monomer has threedomains (Fig. 2A and Fig. S2); Domain A (Met1–Ala405) isa (β/α)8 barrel with eight loops (1: Met1–Ile23, 2: Ala57–Phe73, 3: His107–Ser132, 4: Gly162–Ser174, 5: Gly200–Asn228, 6: Val269–Asp277, 7: Gln325–Gly342, and 8:Gln361–S386) containing the metal binding-site and cata-lytic residues (Glu164 as the acid/base and Glu324 as thenucleophile); Domain B (Gly406–Glu627) folds into acentral seven-stranded β-sheet and five α-helices andpacks onto Domain A of the adjacent monomer (Fig. S2).The Domain B interface area of 2650 Å2 (interface freeenergy ΔiG = − 97.1 kJ mol−1) highlights the structural roleof this domain in the stability of the quaternary structure.These interface contacts are effectuated by packing ofMet545–Ser552 against the loop Phe294–Ile301 connect-

ing Aβ6 and Aα6 of Domain A in the neighbouringmonomer. Domain C (Leu628–Arg695) adopts an anti-parallel β-sandwich fold and the role of this domain iscurrently unknown.

Metal binding site

A strong peak of difference electron density was observedat the threefold non-crystallographic symmetry axis for theBlGal42A structures. Significant anomalous differencedensity was also observed at this site. Three histidines(His118), one from each monomer, and a water moleculeform the tetrahedral metal binding-site that closes thebottom part of the bowl formed by the trimer (Fig. 2B).Based on the coordination distances (Harding, 2006) andX-ray fluorescence spectra of BlGal42A-Glu324Ala crys-tals (not shown), zinc was assigned as the metal ion.According to conservation of the histidine metal ionligands, BlGal42A shares this metal binding-site with thebiochemically characterized Bga42B from B. longumsubsp. infantis ATCC 15697 (62% sequence identity)(Viborg et al., 2014) and close homologues thereof. Thismetal binding-site is only found in seven other GH42sequences from Actinobacteria, Dictyoglomi and Firmi-cutes as judged by the conservation of the histidine metalion ligands.

Active-site architecture and ligand binding

The trimer contains three active sites, each formed at theinterface of two adjacent monomers. The omit electrondensity maps clearly showed density for D-galactose in theβ- and α-anomeric forms in BlGal42A-Gal (Fig. 2C) andBlGal42A-Glu324Ala-Gal respectively. Galactose is recog-nized by hydrogen bonds mediated by six residues in eachmonomer:Arg125,Asn163, Glu324 (catalytic nucleophile),Trp332, Glu372, and His375. Additionally, Glu164 (cata-lytic acid/base) interacts with the C1-OH in BlGal42A-Galand Tyr293 interacts with the C1-OH in BlGal42A-Glu324Ala-Gal (Table S1). The conserved Phe362 estab-lishes specificity by packing against C4 of the D-galactose,which precludes binding of a pyranose ring with an equa-torial hydroxyl group (e.g. glucose or mannose) at thisposition. Although residues in direct contact with galactoseare from a single monomer, an invariant tryptophan inGH42 (Trp204) in the loop connecting Aβ4 and Aα4 fromthe neighbouring monomer forms a water mediated hydro-gen bond to the C3-OH of β-D-galactose and asserts thefunctional importance of the trimeric organization. TheDomain A loop bearing Trp204 also governs the exo-actingmode of BlGal42A (and other GH42 enzymes) on oligo-meric substrates by blocking the space at the subsite − 2that is accessible for substrate binding in monomeric endo-acting GH53 β-galactanases (Le Nours et al., 2009), which

Fig. 1. Chemical structures of the main β-galactoside substratesused and discussed in this study.



hydrolyse internal bonds in polymeric β1-4 linked plantgalactosides. Tyr293, which is invariant in GH42, forms ahydrogen bond with the catalytic nucleophile Glu324 con-sistent with its proposed role as pKa modulator (Hidakaet al., 2002) (Fig. 2C).

Comparison of the ligand free (BlGal42A-Glu324Ala)with the galactose bound structures (BlGal42A-Glu324Ala-Gal and BlGal42A-Gal) reveals a significantlydifferent conformation of the Domain A loop spanningAsn202–Gln206 (RMSD of 5.0 Å calculated for all alignedatoms). The movement of this loop, and particularly theside-chain of Phe203, eliminates steric hindrance ofGlu372 from Domain A of the adjacent monomer, bringingthis side-chain within hydrogen bonding distance of theC4-OH of the β-galactosyl ring bound at subsite − 1(Fig. 3A). Notably, electron density was missing for theDomainA loop formed by Val220–Asn225 in the galactose-free structure (BlGal42A-Glu324Ala), suggesting confor-

mational flexibility. This loop, however, was ordered upongalactose binding and was positioned close to the sub-strate binding subsite + 1 of the neighbouring monomer,which likely affects the spaciousness in the proximity ofsubstrate binding subsite + 1 (Fig. 3B).

Comparison with other GH42 structures

The structure of BlGal42A was compared to the two avail-able GH42 structures from the Gram-negative thermo-philic bacterium Thermus thermophilus A4 in complex withα-D-galactose (A4-β-gal, PDB: 1KWK) (Hidaka et al.,2002) and the Gram-positive Bacillus circulans sp. alka-lophilus in complex with α-D-galactose (Bcα-β-Gal, PDB:3TTY) (Maksimainen et al., 2012), which allowed inferringGlu164 and Glu324 as the acid/base catalyst and thenucleophile respectively. BlGal42A shares 29% and 33%sequence identity to A4-β-Gal and Bcα-β-Gal, respec-

Table 2. Data collection and structure refinement statistics.

Item BlGal42A-Gal BlGal42A-Glu324Ala BlGal42A-Glu324Ala-Gal

Resolution (Å) 2.60 2.70 2.30Outer shell (Å) 2.60–2.67 2.70–2.77 2.30–2.36Wavelength (Å) 1.04 1.00 1.00Beamline, MAX-lab 911-2 911-3 911-3Temperature (K) 100 100 100Space group C2221 P321 C2Unit cell parameters (Å) a = 139.51

b = 199.40c = 217.71

a = b = 150.26c = 237.25

a = 134.20b = 167.97c = 108.58β = 115.64o

No. of reflections 764 427 (46 623) 1 002 869 (64 458) 392 837 (29 000)No. of unique reflections 92 722 (6 470) 85 508 (6 247) 95 832 (7 068)I/σ 19.07 (2.60) 24.65 (4.40) 9.64 (2.18)R-meas (%) 10.6 (90.6) 9.3 (59.5) 16.5 (78.2)Completeness (%) 99.5 (94.8) 99.5 (100) 99.7 (99.8)R-factor (%) 17.53 18.09 18.92R-free (%) 23.41 22.67 24.81No. of atoms:

Protein atoms 16 408 16 328 16 441Water molecules 373 300 565Galactose atoms 36 0 36Zn atoms 1 1 1

RMSD bond lengths (Å) 0.015 0.009 0.016RMSD bond angles (°) 1.612 1.189 1.811Average B-factors (Å2):

Protein atoms 46.9 38.5 23.2Water 39.1 32.4 19.2Zinc 54.0 77.7 43.2Galactose 49.5 – 35.2

Ramachandran statistics (%):Favoured regions 95.1 97.4 95.6Allowed regions 4.1 2.4 3.9Disallowed regions 0.8 0.2 0.5Non-Gly and non-Pro outliers Tyr504 (chain A)

Asp510 (chains A and C)His7 (chain B)Tyr504 (chain A)

Thr201 (chain A)Ala9 (chain B)Ser328 (chain B)Tyr504 (chains B and C)

PDB code 4uni 4uoq 4uoz

Numbers in parentheses are for the outer shell.



tively, but the specificity of these two enzymes is notknown. The trimeric organization is very similar in thesethree enzymes, but the metal binding site connecting themonomers in BlGal42A is absent in the two previouslydetermined structures. Instead, a different tetrahedral zincbinding site effectuated by four cysteine ligands is presentat the bottom of the catalytic domain in each monomer.The distance of both types of zinc binding sites from theactive site precludes direct participation in catalysis, butthe role of the Zn ligand has not been experimentallytested. Incubation of BlGal42A with 100 mM EDTA for30 min at 37°C resulted in 50% reduction of activity buthad no measurable effect on the oligomeric state asjudged by analytical gel filtration, suggesting that the zincion in BlGal42A is not crucial for the gross stability of thequaternary structure. The modest 50% reduction of activ-ity upon EDTA treatment suggests, however, a local dis-ruption of the active conformation.

The domain organization of the monomer of BlGal42Ais also similar to previously characterized structures, asreflected by the RMSD for Cα atoms of the catalyticdomain of 1.6 Å (2.1 Å for entire monomer) and 1.7 Å(2.4 Å for entire monomer) for A4-β-gal and Bcα-β-Galrespectively. The recognition of galactose at subsite − 1 isessentially invariant between the three enzymes with theexception that in the Bacillus enzyme (Bcα-β-Gal), thepolar contact to C6-OH of the α-D-galactose is made byGln313 Oε1 as compared to Trp332 Nε1 in BlGal42A-Gal.

The Domain A loop spanning Gln325–Gly342 betweenAβ7 and Aα7 carries the invariant residue within GH42,

Fig. 2. Overall structure, metal and galactose binding toBlGal42A-Gal.A. A top view along the axis of the BlGal42A-Gal trimer, whichresembles a bowl with the widest diameter at the rim (outer: 105 Å,inner: 30 Å) and with the bottom closed by a zinc ion (yellowsphere). Domain A (Met1–Ala405, green) adopts a (β/α)8 TIMbarrel fold and is responsible for catalysis. Domain B(Gly406–Glu627, magenta) is a mixed β-sheet and α-helicecontaining domain, which contributes to trimerization, with a majorinterface (2650 Å2) to Domain A from the adjacent monomer.Domain C (Leu628–Arg695, purple) is a small anti-parallelβ-sandwich structure. β-D-galactose molecules bound in the activesite are shown as spheres, with the carbon atoms in orange andoxygen in red.B. Three histidines (His118) in BlGal42A, one from each monomer,and a water molecule form a tetrahedral metal binding-site at thecentre of the bottom part of the bowl. Based on anomalous signal,coordination distances and X-ray fluorescence, zinc was assignedas the metal ion.C. The omit electron density map (2F(o) − F(c) contoured at 1.0 σ)of BlGal42A-Gal showed a clear density for β-D-galactose in eachmonomer. The galactose ring stacks onto Phe362 and isrecognized by hydrogen bonding (distances in Å are shown)mediated by Arg125, Asn163, Glu164 (catalytic acid/base), Glu324(catalytic nucleophile), Trp332, Glu372 and His375 with all hydroxylgroups except C3-OH. The α-D-galactose in theBlGal42A-Glu324Ala-Gal structure has similar interactions, with theexception of a hydrogen bond between the C1 OH and Tyr293.



Fig. 3. Conformational changes induced by galactose binding in BlGal42A and comparison between BlGal42A-Gal and the two GH42structures from Bacillus circulans sp. alkalophilus and Thermus thermophilus sp. A4.A. The loop region Asn202–Gln206 from the neighbouring monomer (grey) in the galactose-free BlGal42A-Glu324Ala (dark green) assumes adifferent conformation in BlGal42A-Gal (green and white; β-D-galactose: orange) and BlGal42A-Glu324Ala-Gal (light green and dark grey;α-D-galactose: yellow). This conformational change in the galactose bound structures eliminates steric hindrance of Glu372 by the side-chainof Phe203, which allows Glu372 to stretch out and form a hydrogen bond to C4-OH of galactose.B. Surface representation highlighting the loop Val220–Asn225 from the neighbouring monomer in BlGal42A-Glu324Ala-Gal (transparentsurface), which is positioned approximately 10 Å from the C1 of the galactose. This loop is disordered in the galactose free structure(BlGal42A-Glu324Ala), but galactose binding induces ordering of the loop in a conformation that narrows the space above the active site. Thecolouring is the same as in Fig. 1 and the Trp332 residue that mediates aromatic stacking at position + 1 is coloured in cyan to illustrate theexpected location of the substrate binding subsite + 1.C. The loop Gln325–Gly342 adopts very different conformations in BlGal42A (green) as compared to Bcα-β-Gal in complex with α-D-galactose(cyan, PDB: 3TTY). The putative substrate binding residue Trp332 is also flipped indicative of a different mode of substrate binding betweenthese two enzymes. Two additional residues are present in Bcα-β-Gal (light cyan) in the loop corresponding to the Val220–Asn225 (white).The ordering transition of Val220–Asn225 is not observed in the longer loop of Bcα-β-Gal (PDB: 3TTS).D. The positioning of the Gln325–Gly342 loop in A4-β-gal (magenta) is identical to that observed in BlGal42A (green), while the loop regioncorresponding to Val220–Asn225 in BlGal42A (white) is shorter in A4-β-gal (light magenta) and adopts a different conformation. Otherconserved residues in the active site are shown to depict the proximity of this loop to the active site.



Trp332, proposed to mediate aromatic stacking onto thesubstrate at subsite + 1. The loop assumes a similar posi-tion in BlGal42A-Gal, Bcα-β-Gal, and A4-β-gal, but theTrp332 side-chain is flipped in Bcα-β-Gal as compared toboth BlGal42A-Gal (Fig. 3C) and A4-β-gal (Fig. 3D). Addi-tionally, the side-chain of this tryptophan in Bcα-β-Galmoves towards the galactose (2.5 Å) as observed whencomparing the structure of the galactose complex (PDB:3TTY) with the ligand free structure (PDB: 3TTS). Further-more, the Val220–Asn225 loop of Domain A, whichbecomes ordered upon binding of galactose in BlGal42A,adopts different conformations in both A4-β-gal andBcα-β-Gal (Fig. 3C and D). However, no differences areobserved in this loop between the free and complex struc-tures in these two enzymes.

Discussion

Bifidobacteria are among the first taxa to colonize thehuman gut (Turroni et al., 2012) and several strains ofthis genus are implicated in different physiologicallyimportant roles, e.g. maturation of the immune system,pathogen exclusion, and regulation of mood and behav-iour (Nicholson et al., 2012). The preferential metabolismof short β-galactosides of different origin by bifidobacteriais well established (Davis et al., 2011; Peacock et al.,2013). By contrast, the molecular recognition and speci-ficity determinants of β-galactosidases in Bifidobacteriumand other gut microbiota members, remain elusive. Thisstudy provides the first structural insight into the catabo-lism of short β-galactosides by GH42 β-galactosidasesabundant in bifidobacteria and other taxa from thegut microbiota, e.g. lactobacilli (Andersen et al., 2011).Comprehensive enzymatic analysis using a library ofβ-galactosides revealed that BlGal42A has an unprec-edented activity profile among hitherto characterizedGH42 enzymes, with highest activity towards undeco-rated β1-6/β1-3 galactosides.

Ecology and genetics of human gutassociated bifidobacteria

Access to metabolic fuel and competition are importantfactors in niche adaptation, especially in densely popu-lated habitats such as the human distal gut. Bifidobacteriadisplay ecological and genetic diversity, populating sixdistinct phylogenetic groups (Ventura et al., 2007). Thetwo best-studied groups, i.e. the B. adolescentis and theB. longum, along with B. bifidum are able to adhere to themucus layer coating the epithelium, thereby persisting inthe human gut (Ventura et al., 2007). By comparison, theB. pseudolongum group, members of which are isolatedfrom faeces of different animals (including humans) andfermented milk (Lee and O’Sullivan, 2010), is in general

less well studied in particular with respect to glycanmetabolism. B. animalis subsp. lactis is the most impor-tant member of the pseudolongum owing to its large-scaleuse as a probiotic (Masco et al., 2005). B. animalis subsp.lactis was recently isolated from neonatal ileum (Wallet al., 2008), although this taxon is generally consideredtransient in the gut (Turroni et al., 2009).

These ecological differences manifest in differentβ-galactosides (and other glycans) utilization profiles. Theexclusive preference of B. bifidum to host glycans from theepithelial mucin layer as compared to other bifidobacteriaexemplifies its metabolic specialization (Turroni et al.,2011). B. bifidum genomes possess loci encoding ATP-binding cassette transporters and α-L-fucosidases, whichare necessary for uptake and degradation of fucosecapped core 1 mucin glycans (Galβ1-3GalNAc), respec-tively, in addition to at least one GH42 β-galactosidase(Jensen et al., 2010). A similar specialization is observedin B. longum subsp. infantis ATCC 15697, which pos-sesses the uptake and catalytic machinery to metabolizetype 1 human milk oligosaccharides, including aGH42 β-galactosidase active on Galβ1-3GlcNAc bonds(Pokusaeva et al., 2011). B. animalis subsp. lactis shows adifferent isomer-specific utilization pattern of commercialβ-galacto-oligosaccharides as compared to B. longumsubsp. infantis and B. adolescentis (Peacock et al., 2013),which are persistent members of human infant and adultgut microbiota respectively. Thus, B. animalis subsp. lactisBB-12 is able to grow on the β1-6 linked galactoside6-β-galactosyllactose (Urashima et al., 2013) but noton the β1-4 linked counterpart 4-β-galactosyllactose(Cardelle-Cobas et al., 2011), both from bovine milk. β1-6galactosidic linkages occur abundantly in type II arabi-nogalactan side-chains of arabinogalactan proteins andrhamnogalacturonan type I pectic glycans (Caffall andMohnen, 2009). The preference for β1-6 galactosides in B.animalis subsp. lactis BB-12 is consistent with the sub-strate specificity measured for BlGal42A (Table 1). Theoperon containing BlGal42A, which has been shown tobe significantly upregulated in B. animalis subsp. lactisBl-04 cultures grown on a commercial β-galacto-oligosaccharide mixture (Andersen et al., 2013), is con-served only within the B. animalis lactis subspecies, whichoffers an opportunity to selectively promote the prolifera-tion of this probiotic taxon by supplementing it with β1-6galactosides in synbiotic preparations.

Specificities and phylogenetics of human gut associatedbifidobacterial GH42 β-galactosidases

BlGal42A displays highest activity towards β1-6 linkedgalactosides followed closely by undecorated β1-3 galac-tosides. Characterized GH42 enzymes from B. longumsubsp. infantis and B. adolescentis are specific either for



β1-3/β1-6 or for β1-4 linked β-galactosides (Hinz et al.,2004; Yoshida et al., 2012; Viborg et al., 2014) occurringin host glycans as well as in dietary milk or plant-derivedpectic galactosides. The Km values towards the preferrednatural substrates for these characterized GH42 enzymesare in the range 0.26–6.4 mM (Van Laere et al., 2000;Yoshida et al., 2012; Viborg et al., 2014), which is alsovalid for the measured value for BlGal42A towards thepreferred substrate used in this study (6-β-galactobiose,Km = 5.4 mM).

We recently demonstrated that bifidobacterial GH42enzymes cluster in four main phylogenetic groups (Viborget al., 2014) (Fig. 4). Notably, BlGal42A shares about 62%amino acid sequence identity with both Bga42A from

B. longum subsp. infantis ATCC 15697 (Yoshida et al.,2012) and BbgII from B. bifidum NCIMB41171 (Goulaset al., 2009), with all three enzymes assigned into group 3in the phylogenetic tree (Fig. 4). Bga42A is highly active onthe most common HMO galactoside lacto-N-tetraose (LNT;Galβ1-3GlcNAcβ1-3Galβ1-4Glc) (Yoshida et al., 2012),which is decorated with an N-acetyl group at the subsite + 1bound glucosyl moiety (penultimate position from the non-reducing end), while BlGal42A is essentially inactive onLNT and other oligosaccharides with an N-acetyl group atthe corresponding position (Table 1). Interestingly, BbgIIfrom B. bifidum NCIMB41171 preferred β1-6 over β1-4linked β-galactosides and lactose (Goulas et al., 2009).However, this enzyme has been suggested to be specificfor the host mucin core I structure (Galβ1-3GalNAc)(Jensen et al., 2010), based on genetic context analysis(Turroni et al., 2010). It is worth special note thatmucin core I and HMO galactosides share a structuralmotif featuring a Galβ1-3 glycosidic linkage to an N-acetylhexosamine (GalNAc in mucin core I or GlcNAc inthe type I HMO galactoside lacto-N-tetraose and its build-ing block lacto-N-biose). This shared substrate motif is alsoreflected by the close clustering of BbgII from B. bifidumadjacent to the enzyme active on HMO type I in thephylogenetic tree (Fig. 4). BlGal42A is inactive towards aGalβ1-3 galactosidic linkage to an N-acetylhexosamine,which is a unique signature distinguishing it from the othercharacterized enzymes in group 3 (Table 1). A commonfeature shared by all group 3 enzymes, however, is theirhigh activity on both β1-3 and β1-6 galactosidic linkages ascompared to β1-4 linked galactosides.

Trp332 provides aromatic stacking in GH42 and maymodulate specificity by adopting different conformations

Trp332 and Tyr293 are invariant in GH42 and it has beensuggested that Tyr293 is the pKa modulator of the nucleo-phile catalyst and Trp332 mediates aromatic stacking at aputative subsite + 1 based on the comparison of the GH42A4-β-gal from T. thermophilus A4 and the Escherichia coliGH2 (Hidaka et al., 2002). Comparison of BlGal42A withthe two reported GH42 structures reveals that the spatialpositioning of Tyr293 is identical in all three structures,while that of Trp332 is different in Bcα-β-Gal (Fig. 3C), butshared between BlGal42A and A4-β-gal (Fig. 3D).

To bring more insight into the role of Trp332, we com-pared BlGal42A and related β-galactoside active enzymesfrom clan GH-A enzymes (all sharing a common (β/α)8 foldand retaining mechanism), i.e. a GH2 β-galactosidase fromE. coli (Juers et al., 2001) and a GH53 β-galactanasefrom Bacillus licheniformis (Le Nours et al., 2009) bothin complex with β-galactoside ligands. These enzymescontain a tryptophan that occupies a similar position inspace as Trp332 in BlGal42A. The conservation of Trp332

Fig. 4. Phylogenetic analysis of bifidobacterial GH42 enzymesreveals four distinct groups. Experimental evidence has recentlyprovided specificities within groups G2 and G3, whereas thesubstrate preferences of groups G1 and G4 remain unknown.Substrates or substrate motifs that are recognized from thenon-reducing end galactosyl moiety, the galactosidic linkage andglycosyl moiety at the penultimate position, are denoted inbrackets. Group 3 (G3) contains BlGal42A and closely relatedhomologues. The preferred substrates for BlGal42A areundecorated β1-6/β1-3-linked β-galactosides while Bga42A from B.longum subsp. infantis ATCC 15697 is specific for the human milkoligosaccharide lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc).BbgII from B. bifidum NCIMB41171 shows preference forβ1-6-linked over β1-4-linked β-galactosides and lactose (Goulaset al., 2009), but the proposed substrate for this enzyme is mucincore I structures (Galβ1-3GalNAc), based on genetic colocalizationof α-L-fucosidases that catalyse the removal of the fucose cap fromcore I mucin glycans (Turroni et al., 2010).



in characterized β-galactosidase and β-galactanase ofGH-A clan, together with mutational and crystallographicdata (Huber et al., 2003) support the notion that thisresidue constitutes an aromatic platform for binding ofglycosyl moieties at subsite + 1 throughout the mentionedGH families.

Docking the energetically preferred conformations of theβ1-6, β1-3 and β1-4 galactobiose isomers in the active siteof BlGal42A was performed to assess their accom-modation. The planes of the galactosyl moiety of β1-6-galactobiose, bound at subsite + 1, and the aromatic ring ofTrp332 are almost parallel supporting aromatic stackingat this position (Fig. 5A). This is also valid for β1-3-galactobiose, which possesses an equatorial galactosidiclinkage, albeit with a shorter and less flexible bond ascompared to β1-6-galactobiose, due to an extra dihedralangle in the latter ligand (Peric-Hassler and Hansen, 2010)(Fig. 5B). In comparison, the β1-4 isomer has an axialgalactosidic bond and the ring at subsite + 1 is tilted awayfrom the plane of Trp332 (Fig. 5C), suggesting adjust-ments of the enzyme active site or substrate conformationmay be necessary for an optimal fit, consistent with the3.5-fold higher Km for this substrate. Interestingly, thisdifference in geometry between β1-3 and β1-6 linkages onthe one side and the β1-4-linkage on the other seems to bewell reflected by the clustering in the phylogenetic tree ofGH42 bifidobacterial enzymes (Fig. 4). Thus all character-

Fig. 5. Docking of β1-6 (blue), β1-3 (yellow) and β1-4 (green)galactobiose isomers into the active site of BlGal42A-Gal illustratesthe accommodation these substrates as judged by overlay of thenon-reducing galactosyl of the disaccharides with theexperimentally determined galactose bound at subsite − 1 (orange).A. The β-6-galactobiose planes of the galactosyl moiety at subsite+ 1 and Trp332 are almost parallel to allow efficient aromaticstacking. Cys167, which is strictly conserved in group 3 of GH42enzymes, is within hydrogen bonding distance of C3-OH of thegalactosyl moiety at subsite + 1.B. The β-3-galactobiose assumes a similar conformation asβ-6-galactobiose, but the distance between the two galactose ringsis shorter as compared to 6-β-galactobiose.C. The galactosyl at subsite − 1 in β-4-galactobiose (green) is notperfectly aligned to the Gal in BlGal42A as observed for β-6 andβ-3-galactobiose after docking. The galactosyl moiety at subsite + 1is tilted away from the plane of Trp332, which suggests stackingonto Trp332 requires minor conformational rearrangements of theenzyme and/or ligand. This difference is accommodation of theβ-4-galactobiose is attributed to the axial galactosidic bond ascompared to the equatorial bond in the β1-6 and β1-3 isomers,which is consistent with the fivefold lower catalytic efficiency on thissubstrate as compared to the 6-β-galactobiose.D. The superimposition of the GH2 β1-6-galactosidase (pink) fromEscherichia coli in complex with allolactose (Galβ1-6Glc; grey)demonstrates the aromatic stacking of Trp332 onto the glucosylmoiety bound at subsite + 1. This stacking interaction resemblesthat observed with the galactosyl moiety of 6-galactobiose in linewith the observed preference of BlGal42A towards β1-6-linkages.The Cys167, which is conserved in group 3 GH42 enzymes, iswithin hydrogen bonding distance to recognize the C3-OH of theglucosyl (or galactosyl) moiety.



ized members with a β1-3 or β1-6 preference populate aspecific sub-tree (group 3), whereas enzymes with prefer-ence for β1-4 linkages displaying little or no activity towardsβ1-6-galactosides (Hinz et al., 2004; Viborg et al., 2014)group separately in group 2 (Fig. 4). Functional clusteringof enzymes based on structural motifs present in preferredsubstrates is also observed within phylo-group 3, as theenzymes from B. longum subsp. infantis and B. bifidumactive on N-acetylated β1-3 linked galactosides clusterclosely together, whereas the B. animalis subsp. lactissequences with preference for undecorated β1-6 linkagessegregate in a relatively distant branch of group 3 (Fig. 4).

Additionally, the superimposition of BlGal42A-Gal andthe E. coli GH2 β-galactosidase in complex with allolac-tose (Galβ1-6Glc; PDB: 4DUW) (Wheatley et al., 2013),which is similar to the substrate preferred by BlGal42A(Fig. 1) highlighted the stacking of the glucosyl moiety inallolactose against Trp332 at a distance of 3.0 Å (Fig. 5D).The sulphur atom of Cys167 is at 3.5 Å distance from theglucosyl C3-OH of allolactose, which was not tested as asubstrate in this study. This cysteine is invariant in group3, but not conserved in the three other groups (G1: tyros-ine; G2: tryptophan; G4: glycine) and the cysteine side-chain may play a role in recognition of carbohydrate ringsbound at the + 1 subsite.

Mapping possible specificity signatures on the structureof BlGal42A

Mapping of conserved residues from Bga42A and BbgIIonto the BlGal42A structure showed that all residues atsubsite − 1 are conserved between the three enzymes(Fig. 6B), but that the loop Gln325–Gly342 (Fig. 6A;circles in Fig. S3), with Trp332 (blue circle in Fig. S3)contained large changes within the group 3 branch of thephylogenetic tree. Thus five residues within this loop weredivergent between BlGal42A and Bga42A, e.g. Glu334and Pro337 that correspond to Pro and Tyr in Bga42Arespectively. The variation in the Gln325–Gly342 loopwithin this group of GH42 enzymes suggests that the loopsequence and conformation correlate to specificity fordifferent β1-3 and β1-6 galactosides. The proximity of thisloop to the putative subsite + 1 is compatible with thisrole. Interestingly, the Gln325–Gly342 loop is intertwinedwith the neighbouring monomer where it forms hydrogenbonds and stacking interactions to several residues intwo Domain B loops (Glu430–Glu441 and Glu534–Leu549). These two loops of the adjacent monomer,however, are less conserved and do not correlate withsubstrate specificity.

Another interesting structural element in BlGal42A isthe Domain A disordered loop Val220–Asn225, whichbecomes ordered upon galactose binding and occupies aposition close to the substrate binding subsite + 1

(Fig. 3B). This loop assumes different conformations inBlGal42A and the two other GH42 structures (Fig. 3C andD). Furthermore, the loop is also variable within group 3(Fig. 6C) and contains an additional glycine in sequencesrepresented by BlGal42A as opposed to counterparts inthe two other branches within group 3 (Figs 4 and 6C).The proximity of this loop to the active site and the corre-lation of its amino acid sequence with the different spe-cificities within group 3 enzymes render it into a secondspecificity signature within group 3 bifidobacterial GH42enzymes. The usefulness, however, of this sequence sig-nature globally within GH42 requires additional biochemi-cal support.

Structural plasticity of BlGal42A

The binding of galactose induced two main changes inBlGal42A: (i) conformational difference allowing the for-mation of an extra hydrogen bond between Glu372 andthe C4-OH galactose ring at subsite − 1 (BlGal42A-Galand BlGal42A-Glu324Ala-Gal, Fig. 2C), and (ii) the order-ing of the Domain A loop Val220–Asn225, which positionsthe side-chain of Glu223 in the galactose bound struc-tures at a distance of 10 Å from C1-OH of galactose atsubsite − 1 of the neighbouring monomer (Figs 3B and6B). The extra hydrogen bond mediated by Glu372 islikely to affect the substrate affinity, whereas the orderingof the loop Val220–Asn225 narrows the active site(Fig. 3B) and may contribute to substrate recognition orsize selection. These conformational changes and thegalactose induced disorder-order transition are unique toBlGal42A and are not observed in the two other structur-ally characterized GH42 enzymes. Further experimentalwork is required to unravel the significance of thesechanges in catalysis and their possible role in regulationof enzyme activity.

Conclusions

This study presents the structure and enzymology of abifidobacterial β1-6/β1-3 galactosidase with a novel speci-ficity profile as compared to characterized counterpartsfrom GH42 and identifies structural elements andsequence motifs that are associated with GH42 enzymesthat confer utilization of different β-galactosides in bifido-bacteria. This molecular insight together with the phyloge-netic analysis and sequence mapping possess predictivepower and offer a robust framework for assigning sub-specificities of GH42 enzymes from the gut niche. Theligand induced conformational changes in two loop regionsaround the active site are unprecedented within thisenzyme family, but the significance of these observationsin catalysis merits additional experimental work. Alto-gether, this study promotes our understanding of differen-



tial β-galactoside metabolism within the physiologicallyimportant Bifidobacterium genus.

Experimental procedures

Substrates

p-nitrophenyl (PNP)-β-D-galactopyranoside (PNP-Galp),PNP-β-D-glucopyranoside (PNP-Glcp), PNP-α-L-arabinofuranoside (PNP-α-L-Araf), PNP-α-L-arabinopyranoside (PNP-α-L-Arap), PNP-β-D-fucopyranoside(PNP-Fucp), PNP-β-D-xylopyranoside (PNP-Xylp), ando-nitrophenyl (ONP)-β- D-galactopyranoside (ONP-Galp),lactose, epilactose and lactulose were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-β-galactosylglucose, 3-β-galactobiose, and 4-galactosyl-L-rhamnose, were prepared

as previously described (Nakajima et al., 2009). 4-β-galactobiose, 6-β-galactobiose, LacNAc, 3-fucosyllactose,Lewis X (Galβ1-4[Fucα1–3]GlcNAc), lacto-N-fucopentoseIII, lacto-N-fucopentose II, and Lewis A (Galβ1-3[Fucα1–4]GlcNAc) were purchased from Dextra Laboratories (Read-ings, UK). Lacto-N-biose I (LNB) and galacto-N-biose (GNB)were synthesized as described previously (Nishimoto andKitaoka, 2007; 2009). Lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) were purchased from Elicityl (Crolles,France). 4-β-galactotriose, 4-β-galactotetraose, and 6-β-galactotetraose were generous gifts from Professor MadsHartvig Clausen (Center for Nanomedicine and Theranostics,Department of Chemistry, Technical University of Denmark).3-β-galactosyllactose, 3-β-galactobiosyllactose, and 3-β-galactotriosyllactose were purified essentially as described(Messer et al., 1980; Collins and Bradbury, 1981) fromTammar Wallaby and Wombat milk (generous gifts of Profes-

Fig. 6. Conservation of the loop Gln325–Gly342, which bears the proposed Trp332 subsite + 1 aromatic platform, in related GH42β-galactosides as defined by group 3 in the phylogenetic analysis in the present study, and the loop Val220–Asn225 that is ordered upongalactose binding.A. Sequence alignment of the Gln325–Gly342 loop in the group 3 GH42 enzymes representing distinct β-galactoside specificities: Bga42Afrom in B. longum subsp. infantis ATCC 15697 hydrolyses the non-reducing β-D-galactose in LNT (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) (Yoshidaet al., 2012), BbgII is proposed to target mucin core I (Galβ1-3GalNAc) in B. bifidum NCIMB41171 and BlGal42A preferring undecoratedβ1-6/β1-3 galactosides.B. BlGal42A monomer A and Domain A loop Val220–Asn225 from the adjacent monomer, both coloured according to conservation withinphylogenetic group 3 (blue 0% conservation, red 100% conservation). Glu164 (acid/base), Glu324 (nucleophile), and the Trp332 putativesubsite + 1 aromatic platform are shown in sticks. In addition, Gln325, Pro337, Lys339, Arg333 and Asn336 residues in loop Gln325–Gly342identified as divergent within GH42 enzymes from phylogenetic group 3 are shown to highlight their proximity to the active site. The aminoacid sequence of this loop correlates with the specificities mentioned above, highlighting the loop sequence as a specificity signature withingroup 3 of GH42 enzymes. The disordered loop Val220–Asn225 in BlGal42-Gal becomes ordered upon galactose binding and occupies aposition close to the substrate binding subsite + 1 in the adjacent monomer, which may contribute to substrate recognition or size selection.C. Sequence alignment of the loop region Pro217–Asn228 flanking the loop Val220–Asn225 that is variable in bifidobacterial GH42 enzymes.The proximity of this loop to the active site and the correlation of its amino acid sequence with phylogenetic groups and specificity render itinto a suitable specificity signature of bifidobacterial GH42 enzymes.



sor Tadasu Urashima, Graduate School of Food Hygiene,Obihiro University of Agriculture & Veterinary Medicine, Japanand Professor Michael Messer, School of Molecular andMicrobial Biosciences, University of Sydney, Australia).

Expression and purification of BlGal42A

The gene encoding the putative β-galactosidase (locus tag:balac_0484, GenBank Accession No. ACS45863.1) wasamplified from B. animalis subsp. lactis Bl-04 genomic DNA byPCR using High-fidelity DNA polymerase (Fermentas, St.Leon-Rot, Germany) with the following primers: 5′-CAT GCCATG GGC TCAGCATCCACACAACAT C-3′; reverse: 5′-CTAGCT AGC TCA GCG CCT GAA CGC-3′. Amplified DNA wascloned into pET28(a) + (Novagen, Darmstadt, Germany)using the NcoI and NheI restriction sites (underlined) and theresulting plasmid pET28balac_0484 was transformed into E.coli DH5α and verified by full sequencing. The plasmid wastransformed into E. coli Rosetta for recombinant proteinexpression. Cells were grown in 6 l Luria–Bertani medium with50 μg ml−1 kanamycin and 34 μg ml−1 chloramphenicol at 37°Cto an OD600 of 0.5, and expression was induced by 100 μMisopropyl-β-thiogalactopyranoside for 24 h at 16°C. Recombi-nant BlGal42A was purified using a 6 ml ResourceQ (GEHealthcare, Uppsala, Sweden) anion exchange column with alinear gradient 15–100% 0.5 M NaCl (10 mM MES, pH 6.5).Fractions containing β-galactosidase activity were pooled andpurified by gel filtration (HiLoad 26/60 Superdex G200; GEHealthcare) in 10 mM MES, 150 mM NaCl (pH 6.5) eluted by1.2 column volume of this buffer at a flow rate of 1 ml min−1.The purity of the enzyme was analysed by SDS-PAGE. Theenzyme concentration was determined spectrophotometri-cally (ε280 = 151 425 M−1 cm−1). BlGal42A was incubated with100 mM EDTA in 50 mM HEPES, pH 7.0 for 30 min at 37°C toassess the effect of the zinc metal ligand on activity and trimerstability. The EDTA treated and untreated BlGal42A wereapplied onto a gel filtration column (HiLoad 16/60 SuperdexG200; GE Healthcare) and eluted in 10 mM MES, 150 mMNaCl, pH 6.5 against a molecular size standard proteinmixture (Gel Filtration Standard; Bio-Rad, California, USA) inthe same buffer at a flow rate of 0.75 ml min−1. The nucleophilemutant Glu324Ala was prepared using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, California, USA), usingthe following primers: 5′-CC TGG TTC CTC ATG GCG CAATCC ACC TCC-3′; reverse: 5′-GGA GGT GGATTG CGC CATGAG GAA CCA GG-3′ and prepared as described forwild-type.

Crystallization, data collection, andstructure determination

BlGal42A was concentrated to 13.3 mg ml−1 in 10 mM MESpH 6.5, 150 mM NaCl and screened for initial crystallizationconditions using an Oryx 8 protein crystallization robot with96-well trays (Douglas Instruments, UK) at room temperaturewith the JCSG + and PACT screens (Qiagen, Hilden,Germany) and the Index screen (Hampton Research, Cali-fornia, USA). Crystals were observed in the JCSG + screen[0.2 M MgCl2, 30% PEG 400 (v/v), 0.1 M HEPES pH 7.5] andPACT screen [25% PEG 1500 (v/v) 0.1 M MMT (DL-malicacid, MES and Tris) buffer pH 7, 8 or 9]. The final crystalliza-

tion condition was 27% PEG1500, 0.01 M MgCl2, 0.1 M MMTbuffer pH 8 obtained from optimization in 24-well VDX trays(Hampton Research) in hanging drops containing 3 μl proteinstock mixed with 1–10 μl water and then 1 μl reservoir atroom temperature. Glycerol was added as cryoprotectantbefore harvesting. For the galactose-soaked crystal, galac-tose was added directly to the drop and left overnight beforeharvesting. Crystals of BlGal42A-Glu324Ala were obtainedunder slightly different conditions (29% PEG1500, 0.01 mMMgCl2, 0.1 M MMT buffer pH 8).

Crystals for BlGal42A-Glu324Ala-Gal were obtained byco-crystallization in MRC 2-drop screening trays. Suitablecrystals were obtained with 0.2 M MgCl2, 0.1 M HEPESpH 7.5, 30% PEG 400 reservoir, in a 0.1 μl drop set up with a1:1 ratio of reservoir to stock solution containing 12 mg ml−1

protein and 150 mM β1,6-galactobiose.Data sets were obtained at either the I911-2 or 911-3 side

station of the Cassiopeia beamline MAX-lab, Lund, Sweden,at 100K. Processing and scaling of the data was processedwith XDS and XSCALE (Kabsch, 2010). A data set wascollected to 2.2 Å resolution of a crystal taken directly from thecrystallization drop (BlGal42A-Native) and a 2.6 Å data setwas obtained from a galactose soaked crystal (BlGal42A-Gal).The data set of the catalytically impaired mutant (BlGal42A-Glu324Ala) was collected to 2.7 Å from a drop supplementedwith 3 μl 0.3 M β1,6-galactobiose in 50% PEG400 + 50% res-ervoir just prior to harvesting. The last data set of catalyticallyimpaired mutant in complex with galactose (BlGal42A-Glu324Ala-Gal) with no addition of cryoprotectant was col-lected to a maximum resolution of 2.3 Å.

Data collection and processing statistics for BlGal42A-Native are shown in Table S2, and for data sets of BlGal42A-Gal, BlGal42A-Glu324Ala, and BlGal42A-Glu324Ala-Gal inTable 2.

The space group for BlGal42A-Native was determined to beC2221 with three molecules in the asymmetric unit accordingto the calculated Matthew coefficient. Despite poor twinningstatistics, molecular replacement (MR) was initially attemptedusing phenix.automr with this data set. The structure of theA4-β-Gal trimer from T. thermophilus (PDB 1KWG) was usedas search model after modification with phenix.sculptor(Bunkóczi and Read, 2011). Following automatic model build-ing with phenix.autobuild (Terwilliger et al., 2008) and manualrebuilding in Coot (Emsley et al., 2010) combined with refine-ment with phenix.refine, a partly built model with R-free 39.2%was obtained, which was not finally refined due to poor twin-ning statistics.Adata set was acquired of BlGal42A-Gal, whichhad similar cell parameters and space group, but better twin-ning statistics. Final refinement statistics are shown in Table 2.

Several crystals of BlGal42A-Glu324Ala were tested, manyessentially isomorphous to wild-type crystals previouslyobtained. However, crystals belonging to a trigonal spacegroup and showing no sign of twinning were chosenfor refinement. The BlGal42A-Glu324Ala and BlGal42A-Glu324Ala-Gal data sets were built using the BlGal42A-Gal astemplate by MR and the structures were thereafter refined.No evidence was seen for binding of 6-β-galactobiose;however, α-galactose was bound at the − 1 subsite (BlGal42A-Glu324Ala-Gal).

The quality of the structures was verified by MolProbity,and PyMOL v1.7 (Schrödinger, LLC, New York) was used for



structural analysis and rendering of molecular graphics. Thesystematic comparisons between structural elements weredone using the structural analysis package ProSMART andcalculations of free energies and surface areas was performedwith PISA. Carbohydrate ligands for docking were preparedwith the online linear carbohydrate builder GLYCAM06(Kirschner et al., 2008). AutoDockTools v1.5.6 (Morris et al.,2009) was used to prepare BlGal42A-Gal as receptor mol-ecules (the Gal molecules at subsite − 1 were removed) and todefine the search space for docking. The carbohydrate ligandswere docked into the receptor molecule using AutoDock Vina(Trott and Olson, 2010) and the best nine energetically favour-able docking solutions were visualized in PyMOL v1.7(Schrödinger, LLC, New York). The optimal ligand conforma-tion was chosen based on the best visual alignment of thenon-reducing end galactose of the docked ligand to the experi-mentally determined subsite − 1 Gal.

Bioinformatic analysis

The amino acid sequences of GH42 members were extractedfrom the CAZy database (Lombard et al., 2014) and alignedusing MUSCLE v3.8 with default settings. The alignment wasused to render a phylogenetic tree by ClustalW2 with defaultsettings, which was visualized in Dendroscope 3.0.

Enzyme assays

β-Galactosidase activity was assayed in a mixture (50 μl)of 12.5 mM ONP-Galp, 100 mM sodium citrate, 0.0125%Triton X-100, pH 6.5 and added enzyme at 37°C for 10 minand the reaction was stopped by 1 M Na2CO3 (200 μl). Theamount of released ONP was measured spectrophotometri-cally at A410 using ONP (0–2 mM) as standard. One unitof activity was defined as the amount of enzyme thatreleased 1 μmol of ONP min−1. The pH dependence of β-galactosidase activity was determined in the range pH 3–12in 40 mM Britton-Robinson’s universal pH buffer at theabove standard assay conditions except for the buffer com-ponent. The effect of temperature on enzyme activity wasdetermined by performing the above assay at in the tem-perature range 30–70°C.

The kinetic parameters were determined from initial rates ofONP-Galp (0.5–20.0 mM) hydrolysis in 40 mM sodium citratepH 6.5, 0.005% Triton X-100, with 3 nM enzyme at 37°C for16 min. kcat and Km were obtained by non-linear regression fitof the Michaelis-Menten model with substrate inhibition,v = kcat × [E] × S/[Km + S × (1 + S/Kis)], to the data from tripli-cate experiments (GraphPad Prism 5, La Jolla, USA), whereKis is the inhibition equilibrium constant. Activity of BlGal42Awas screened towards PNP-glycosides (see above) usingthe same procedure as for ONP-Galp and towards2 mM 3-β-galactosyllactose, 4-β-galactosyllactose, and 6-β-galactosyllactose in 50 mM sodium phosphate pH 6.5 at 30°Cfor 20 and 60 min. The catalytic efficiency towards substrateswith at least two adjacent galactose residues was analysedusing a continuous coupled assay using galactose mutar-otase, galactokinase, UDP-glucose hexose-1-phosphateuridylyltransferase, phosphoglucomutase, and glucose 6-phosphate dehydrogenase (Nihira et al., 2007; Viborg et al.,

2014) performed in 40 mM sodium citrate pH 6.5, 0.005%Triton X-100 (50 μl) at 37°C with 6-β-galactobiose, 6-β-galactotetraose, 3-β-galactobiose, 3-β-galactobiosyllactose,3-β-galactotriosyllactose, 4-β-galactobiose, 4-β-galactotriose,or 4-β-galactotetraose (2–20 mM). Reactions were initiated byaddition of pre-incubated enzyme to final concentrations in therange 0.008–18 nM at 37°C in 384-well plates and releasedgalactose was monitored every 30 s for up to 90 min bycolorimetric quantification of α-D-galactose 1-phosphate asdescribed (Nihira et al., 2007). All other substrates wereassayed under the same condition but using galactose dehy-drogenase as the only intermediate enzyme.

Accession numbers

Coordinates and structure factors have been deposited in theProtein Data Bank with accession numbers 4uni, 4uoq, and4uoz.

Conflict of interest

None declared.

Funding

This work is supported by the FøSu grant from the DanishStrategic Research Council to the project ‘Gene discoveryand molecular interactions in prebiotics/probiotics systems.Focus on carbohydrate prebiotics’, and a PhD stipend(AHV) from The Technical University of Denmark. The IdellaFoundation and the Scandinavia-Japan Sasakawa Founda-tion funded travel support to Japan (AHV). The DanishMinistry of Higher Education and Science through theInstrument Center DANSCATT and the European Commu-nity’s Seventh Framework Programme (FP7/2007-2013)under BioStruct-X (grant agreement N°283570) fundedtravel to synchrotrons.

Acknowledgements

Dorthe Boelskifte and Jens-Christian Navarro Poulsen (Uni-versity of Copenhagen) are thanked for technical assistance.Mamoru Nishimoto (National Food Research Institute) isthanked for help with production and purification of 3-β-galactosylglucose and 3-β-galactobiose. MAX-lab and theassociated staff are acknowledged for provision of synchro-tron beam time and assistance. We are thankful to Dr MichaelMesser (School of Molecular and Microbial Biosciences, Uni-versity of Sydney, Australia) and Professor Tadasu Urashima(Graduate School of Food Hygiene, Obihiro University ofAgriculture & Veterinary Medicine, Japan) for the generous giftof mammalian milk samples containing 3-β-galactosyllactose,3-β-galactobiosyllactose, and 3-β-galactotriosyllactose. Thegroup of Professor Mads Hartvig Clausen (Center for Nano-medicine and Theranostics, Department of Chemistry, Tech-nical University of Denmark) is gratefully acknowledged for theproduction of 6-β-galactobiose, 6-β-galactotetraose, 4-β-galactotriose, and 4-β-galactotetraose.



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