Crystal structures of a glycoside hydrolase family 20 lacto-N-biosidase from Bifidobacterium bifidum

Article abstract of DOI:10.1074/jbc.M112.420109

Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-β 1, 3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 β-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-β1, 3-GlcNAc-thiazoline) at 1.8-A resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique β-trefoil-like fold. Compared with other β-N-acetyl-hexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a - 2 subsite specific for β-1, 3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near ⁴E, whereas that of LNB-thiazoline is in a ⁴C₁ conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed.

Full text of DOI:10.1074/jbc.M112.420109

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 17, pp. 11795–11806, April 26, 2013
2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
©
Crystal Structures of a Glycoside Hydrolase Family 20
□
S
*
Lacto-N-biosidase from Bifidobacterium bifidum
Received for publication, September 15, 2012, and in revised form, March 11, 2013 Published, JBC Papers in Press, March 11, 2013, DOI 10.1074/jbc.M112.420109
¶1 §2 ʈ3 ‡4
‡
§
Tasuku Ito , Takane Katayama , Mitchell Hattie , Haruko Sakurama , Jun Wada , Ryuichiro Suzuki ,
‡
§
¶5
‡6
Hisashi Ashida**, Takayoshi Wakagi , Kenji Yamamoto , Keith A. Stubbs , and Shinya Fushinobu
From the Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the Research
Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan, the School of
‡
§
¶
Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia, the
ʈ
Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan, and the **Department of
Science and Technology on Food Safety, Faculty of Biology-oriented Science and Technology, Kinki University, Kinokawa,
Wakayama 649-6493, Japan
Background: Infant gut-associated bifidobacteria possess lacto-N-biosidase, which releases lacto-N-biose I (LNB) from
human milk oligosaccharides.
Results: The crystal structures of lacto-N-biosidase complexed with LNB and LNB-thiazoline were determined.
Conclusion: The intermediate analog complex allows the proposal of a conformational reaction coordinate.
Significance: The structures of a key enzyme in the colonization of human commensal bacteria provided its structural basis and
insight into the development of inhibitors.
Human milk oligosaccharides contain a large variety of oligo- dase consists of three domains, and the C-terminal domain has a
saccharides, of which lacto-N-biose I (Gal-␤1,3-GlcNAc; LNB) unique ␤-trefoil-like fold. Compared with other ␤-N-acetyl-
predominates as a major core structure. A unique metabolic hexosaminidases, lacto-N-biosidase has a wide substrate-bind-
pathway specific for LNB has recently been identified in the ing pocket with a ؊2 subsite specific for ␤-1,3-linked Gal, and
human commensal bifidobacteria. Several strains of infant gut- the residues responsible for Gal recognition were identified.
associated bifidobacteria possess lacto-N-biosidase, a mem- The bound ligands are recognized by extensive hydrogen bonds
brane-anchored extracellular enzyme, that liberates LNB from at all of their hydroxyls consistent with the enzyme’s strict sub-
the nonreducing end of human milk oligosaccharides and plays strate specificity for the LNB moiety. The GlcNAc sugar ring of
4
a key role in the metabolic pathway of these compounds. Lacto- LNB is in a distorted conformation near E, whereas that of
4
N-biosidase belongs to the glycoside hydrolase family 20, and its LNB-thiazoline is in a C conformation. A possible conforma-
1
reaction proceeds via a substrate-assisted catalytic mechanism. tional pathway for the lacto-N-biosidase reaction is discussed.
Several crystal structures of GH20 ␤-N-acetylhexosaminidases,
which release monosaccharide GlcNAc from its substrate, have
been determined, but to date, a structure of lacto-N-biosidase is
Human milk contains various oligosaccharides collectively
7
unknown. Here, we have determined the first three-dimensional termed human milk oligosaccharides (HMOs) (1, 2). HMOs
structures of lacto-N-biosidase from Bifidobacterium bifidum function as prebiotics, promoting the growth of bifidobacteria
JCM1254 in complex with LNB and LNB-thiazoline (Gal- in the gastrointestinal tracts of breastfed infants, which in turn
␤1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosi- promotes optimal health (3, 4). Most HMOs contain a lactose
moiety (Gal-␤1,4-Glc) at their reducing end, which is elongated
by ␤1,3-linked lacto-N-biose I (Gal-␤1,3-GlcNAc; LNB, to give
*
This work was supported in part by the Program for the Promotion of Basic
Research Activities for Innovative Bioscience, Japan (to S. F., K. Y., and T. K.),
type I HMOs) and/or ␤1,3/6-linked N-acetyllactosamine
by Japan Society for the Promotion of Science KAKENHI Grant 24380053 (Gal-␤1,4-GlcNAc; LacNAc, to give type II HMOs). A unique
(to S. F. and H. A.), and by the Australian Research Council. Part of this work
feature of the composition of HMOs is the predominance of
type I over type II oligosaccharides. Indeed, such a composition
was performed under the Priority Program for Disaster-affected Quantum
Beam Facilities Proposals PF 2011G080 and SPring-8 2011A1908.
S
□
This article contains supplemental Tables S1 and S2, Figs. S1–S4, and addi- has not been observed in milk oligosaccharides from other
tional references.
mammals, including anthropoids (5). Further elongation of
The atomic coordinates and structure factors (codes 4H04 and 4JAW) have been
deposited in the Protein Data Bank (http://wwpdb.org/).
1
7
Supported by an Australian Postgraduate Award from the University of
The abbreviations used are: HMO, human milk oligosaccharide; LNB, lacto-
N-biose I, (Gal-␤1,3-GlcNAc); LacNAc, N-acetyllactosamine, (Gal-␤1,
4-GlcNAc); LNFP I, lacto-N-fucopentaose I (Fuc-␣1,2-Gal-␤1,3-GlcNAc-␤1,
3-Gal-␤1,4-Glc); LNDFH I, lacto-N-difucohexaose I (Fuc-␣1,2-Gal-␤1,3-(Fuc-
␣1,4-)GlcNAc-␤1,3-Gal-␤1,4-Glc); LNT, lacto-N-tetraose (Gal-␤1,3-GlcNAc-
␤1,3-Gal-␤1,4-Glc);GNB, galacto-N-biose(Gal-␤1,3-GalNAc);LNBase, lacto-
N-biosidase; BbLNBase, LNBase from B. bifidum JCM1254; GH, glycoside
hydrolase; CBM, carbohydrate-binding module; ␤-HexNAcase, ␤-N-acetyl-
hexosaminidase; pNP, p-nitrophenyl; PA, pyridylamino; r.m.s.d., root mean
square deviation; SpHex, ␤-HexNAcase from Streptomyces plicatus; PUGNAc,
O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate.
Western Australia and a Jean Rogerson Postgraduate Scholarship.
Present address: Graduate School of Agriculture, Kyoto University, Kitashi-
2
rakawa, Sakyo-ku, Kyoto, 606-8502, Japan.
Present address: Kyoto Municipal Institute of Industrial Technology and Cul-
3
ture, Chudoji Awatacho, Shimogyo-ku, Kyoto 600-8815, Japan.
Present address: Dept. of Biological Production, Faculty of Bioresource Sci-
4
ences, Akita Prefectural University, Akita 010-0195, Japan.
Recipient of funding from the Australian Research Council.
To whom correspondence should be addressed. Tel.: 81-3-5841-5151; Fax:
5
6
8
1-3-5841-5151; E-mail: asfushi@mail.ecc.u-tokyo.ac.jp.
APRIL 26, 2013•VOLUME 288•NUMBER 17
JOURNAL OF BIOLOGICAL CHEMISTRY 11795

Structure of Lacto-N-biosidase
these core structures is made by the addition of fucose and sialic
LNBases are members of the GH20 family of glycoside
acid residues via ␣1,2/3/4- and ␣2,3/6-linkages, respectively. hydrolases (20) along with ␤-N-acetylhexosaminidases (␤-
HMOs are composed of more than 130 different oligosaccha- HexNAcases); however, they exhibit very low sequence homol-
ride structures that account for 2% of the solid components of ogy. For example, BbLNBase exhibits less than 24% amino
dried human milk. Of note is that four oligosaccharides consti- sequence identity to all the characterized ␤-HexNAcases.
tute 25–33% of total HMOs (6) as follows: 2Ј-fucosyl-lactose GH20 enzymes cleave the glycosidic linkage at the reducing end
(
Fuc-␣1,2-Gal-␤1,4-Glc); lacto-N-fucopentaose I (Fuc-␣1, of GlcNAc via a retaining substrate-assisted catalytic mecha-
nism in which the 2-acetamido group of the substrate acts as
the catalytic nucleophile (21–23). Whereas ␤-HexNAcases
release a monosaccharide (GlcNAc), LNBase releases a disac-
charide (LNB), implying that the latter has an extended Ϫ2
subsite. The crystal structures of multiple GH20 ␤-HexNA-
cases have been reported to date (21, 24–32); however, the
three-dimensional structure of LNBase is not yet available.
Here, we report two crystal structures of BbLNBase com-
plexed with LNB and LNB-thiazoline, a potent inhibitor of
2
-Gal-␤1,3-GlcNAc-␤1,3-Gal-␤1,4-Glc; LNFP I); lacto-N-
difucohexaose I (Fuc-␣1,2-Gal-␤1,3-(Fuc-␣1,4-)GlcNAc-␤1,
3-Gal-␤1,4-Glc; LNDFH I), and lacto-N-tetraose (Gal-␤1,
-GlcNAc-␤1,3-Gal-␤1,4-Glc; LNT). Three of these oligosac-
3
charides (LNFP I, LNDFH I, and LNT) contain an LNB unit,
highlighting the importance of this component in these
systems.
In 2005, a novel metabolic pathway specific to LNB and
galacto-N-biose (Gal-␤1,3-GalNAc; GNB) was uncovered in
bifidobacteria (7) and was termed the GNB/LNB pathway (8).
Considering the living environment of bifidobacteria (the gas-
trointestinal tract of infants), LNB and GNB are hypothesized
to originate from HMOs and intestinal mucin glycoproteins,
respectively. Proteins related to this pathway have been actively
investigated, and crystallographic analyses (9) have uncovered
the structures of an extracellular endo-␣-N-acetylgalac-
tosaminidase that releases GNB from mucin-type O-glycans
BbLNBase (K ϭ 125 Ϯ 8 nM at pH 4.5) (33). Using the sugar
i
ring conformations of LNB and LNB-thiazoline molecules
found in the active site, the reaction mechanism and possible
conformational changes of the substrate are discussed.
EXPERIMENTAL PROCEDURES
Protein Production and Purification—The overexpression
vector for N-terminally His -tagged BbLNBase (residues
6
4
1–663) was constructed by inserting the PCR-amplified frag-
(
10), a solute-binding protein of an ABC transporter specific to
ment of the lnbB gene (17) into the NdeI and EcoRI sites of the
GNB and LNB (11), and an intracellular phosphorylase that
cleaves both GNB and LNB (12). In addition, it has been dem-
onstrated that LNB is the “bifidus factor” that selectively pro-
motes growth of infant gut-associated bifidobacteria (13, 14),
but interestingly, a free form of LNB has not been found in
HMOs (1).
r
pET28b plasmid (kan ; Novagen, Madison, WI). The primers
used were 5Ј-gggaattccatatggggtacagtgccacggctccc-3Ј and
5
Ј-ccggaattctcagtcgctgaccaggtcag-5Ј (restriction sites are
underlined). The plasmid was introduced into Escherichia coli
BL21 CodonPlus (DE3)-RIL (Stratagene, La Jolla, CA) for native
protein expression. For selenomethionine-labeled protein
expression, an NcoI-XhoI fragment of the pET28b-based
Lacto-N-biosidase (LNBase, EC 3.2.1.140), which liberates
LNB from the nonreducing end of oligosaccharides, was first
found in the soil actinomycete Streptomyces sp. 142 (15, 16).
Since then, LNBase activity has also been found in several
strains of Bifidobacterium bifidum and Bifidobacterium
longum (17), which are particularly predominant in the intes-
tines of infants (18). Bifidobacterial LNBase is a membrane-
anchored extracellular enzyme, which suggests that it may play
a key role in the excision of LNB from HMOs to supply LNB to
the GNB/LNB pathway. Recently, the gene for LNBase from B.
bifidum JCM1254 (BbLNBase) was cloned, and its recombinant
protein was characterized in detail. BbLNBase consists of 1,112
amino acids and contains a signal sequence, a glycoside hydro-
lase (GH) family 20 domain, a carbohydrate-binding module
expression plasmid was inserted into the pET19b plasmid
r
(amp ), which was subsequently introduced into E. coli BL21
r
CodonPlus (DE3)-RIL-X (kan ; Stratagene). The transformants
were cultured in Luria-Bertani medium (native protein) or
LeMaster medium (selenomethionine-labeled protein) con-
taining 100 mg/liter kanamycin or ampicillin and 20 mg/liter
chloramphenicol at 25 °C for 20 h. Isopropyl 1-thio-␤-D-galac-
topyranoside was added to a final concentration of 1.0 mM to
induce protein expression. Following an additional incubation
at 25 °C for 20 h, the cells were harvested by centrifugation and
suspended in 50 mM HEPES-NaOH (pH 7.5). Cell extracts were
obtained by sonication followed by centrifugation to remove
cell debris. The protein was purified to homogeneity by sequen-
tial column chromatography involving nickel-nitrilotriacetic
acid superflow (Qiagen, Hilden, Germany), Mono Q 10/100
GL, and Superdex 200 pg 16/60 column chromatography (GE
Healthcare). The protein concentration was determined using
(
CBM) family 32 domain, a bacterial Ig-like domain, and a
transmembrane region (Fig. 1A). The enzyme’s activity favored
LNT as a substrate to produce LNB and lactose, and it was
found that it could not act on oligosaccharides where the LNB
unit is modified with fucose. In addition, it was found that the BCA protein assay kit (Thermo Fisher Scientific) with
BbLNBase specifically releases LNB at the nonreducing end of bovine serum albumin as a standard.
type I oligosaccharides but does not hydrolyze type II oligosac-
Crystallography—Crystals of both native and selenomethio-
charides. Therefore, a potential use for this enzyme is in iden- nine-labeled BbLNBase complexed with LNB were obtained at
tifying type I structures in glycoconjugates. Intriguingly, most 20 °C using the sitting drop vapor diffusion method, by mixing
of the cancer-associated oligosaccharide antigens (including 0.5 ␮l of protein solution containing 7 mg/ml BbLNBase and 10
a
x
sialyl Le , sialyl Le , and their derivatives) have a core structure mM LNB with an equal volume of reservoir solution (0.2 M
containing type I or type II chains (19).
1796 JOURNAL OF BIOLOGICAL CHEMISTRY
potassium sodium tartrate tetrahydrate, 0.1 M sodium citrate
1
VOLUME 288•NUMBER 17•APRIL 26, 2013

Structure of Lacto-N-biosidase
(
pH 5.6), and 2.0 M ammonium sulfate). Crystals of BbLNBase exhibited virtually the same activity as the full-length construct
complexed with LNB-thiazoline (synthesized as described pre- (31–1064, which has only the signal and membrane anchor
viously (33)) were obtained in a similar manner, except that the regions deleted). Thus, these three constructs were selected for
concentration of LNB-thiazoline used was 0.1 mM. Diffraction crystallization screening, but only diffraction-quality crystals
data were collected at 100 K using a charge-coupled device were obtained with the 41–663 construct in the presence of
camera on beamline BL17A at the Photon Factory of the High LNB. The crystal structure of BbLNBase was determined by the
Energy Accelerator Research Organization (KEK, Tsukuba, single-wavelength anomalous dispersion method using a sel-
Japan) and processed using HKL2000 (34). The selenomethio- enomethionine derivative. Subsequently, we determined the
nine sites were determined using SnB (35), and the initial crystal structure of native (nonlabeled) protein crystals (com-
phases were calculated using Solve/Resolve (36). Automated plexed with LNB and LNB-thiazoline) both at 1.8-Å resolution
model building was performed using ARP/wARP (37). Manual (Table 1 and Fig. 1B). The crystal contains two molecules in the
model rebuilding and refinement was achieved using Coot (38) asymmetric unit, and the final model contains residues from
and REFMAC5 (39). The statistics for data collection and Ser-30 to Ser-662 of the A chain and from Ser-30 to Val-661 of
refinement are provided in Table 1. Molecular graphic images the B chain. The protein has an N-terminal His tag containing
6
were prepared using PyMOL (DeLano Scientific, Palo Alto, 21 amino acid residues derived from the pET-28b vector
CA).
(MGSSHHHHHHSSGLVPRGSHM), in which 11 amino acids
Enzymatic Characterization of Wild-type and Mutant (SSGLVPRGSHM, residues 30–40) were visible in the electron
LNBases—C-terminally His -tagged proteins (residues 35 to density map. A region of the artificial tag sequence (Ser-30 to
6
1
064) were used for kinetic analysis. Purification and expres- Arg-36) contributes to the packing of the dimer in the asym-
sion of wild-type enzyme were carried out as described previ- metric unit (supplemental Fig. S2). The molecular masses of the
ously (17). H263F, D320A, D320N, and Y419F mutants were 41–663 construct of BbLNBase as deduced from the amino
constructed by using the QuikChange site-directed mutagene- acid sequence, estimated by SDS-PAGE and calibrated gel fil-
sis method (Stratagene) with the plasmid pET23b-lnbB as the tration chromatography, were 71.8, 72.4, and 64.9 kDa, respec-
template. The following primers and their complementary tively (data not shown), suggesting that it is monomeric in solu-
strands were used: 5Ј-aactccccgggcttcatgaacgtctgg-3Ј (H263F), tion. The root mean square deviations (r.m.s.d.) for the C␣
5
Ј-cacatgggcgccgcggagtacatgatc-3Ј (D320A), 5Ј-cacatgggcgc- atoms of all pairs of the four molecules (two chains in the two
caacgagtacatgatc-3Ј (D320N), and 5Ј-acccaggccctgttctggtc- crystal structures) are less than 0.5 Å. The two ligand molecules
ccgttcg-3Ј (Y419F). The entire sequence used for later manip- bound to chains A and B are also virtually identical (r.m.s.d. ϭ
ulation was sequenced to check that no base change other than 0.047 and 0.069 Å for LNB and LNB-thiazoline, respectively).
those designed had occurred. The mutants were expressed and Hence, our subsequent descriptions will refer to chain A, unless
purified using a similar procedure as described for the wild-type otherwise noted.
enzyme.
Overall Structure—The BbLNBase monomer consists of
Activity measurements of the enzymes were conducted using three domains as follows: an N-terminal domain (N-domain,
p-nitrophenyl (pNP)-LNB (Sigma) as a substrate. The assay residues 41–178); a catalytic (␤/␣) barrel domain (barrel
8
mixture (50 ␮l) contained substrate dissolved in 50 mM McIl- domain, residues 179–496), and a C-terminal domain (C-do-
vaine buffer (pH 4.5) and enzymes (13 nM for WT, 1.3 ␮M for main, residues 497–662) (Fig. 1B). The N-domain has an ␣/␤
H263F and Y419F, and 6.3 ␮M for D320A and D320N). The topology with a seven-stranded ␤-sheet exposed to the surface,
substrate concentrations were varied from 0.3- to 2-fold of the and two ␣-helices buried in the interface with the subsequent
K values of the respective enzymes. The reaction was carried barrel domain. The N-domain and the barrel domain corre-
m
out at 30 °C and stopped by adding an equal volume of 1 M spond to the two conserved domains of typical GH20 ␤-Hex-
Na CO . The amounts of liberated 4-nitrophenolate were NAcases (21, 24, 25, 27–32). The N-domain and the barrel
2
3
determined by measuring the absorbance at 400 nm.
domain of BbLNBase are structurally similar to the domains I
The hydrolysis of LNT (Dextra Laboratory, Reading, UK) by and II, respectively, of ␤-HexNAcase from Streptomyces plica-
wild-type enzyme was monitored by high performance anion- tus (SpHex) (Fig. 1C). The N-domain is conserved in most
exchange chromatography with a CarboPac PA1 column, fol- GH20 enzymes, although its function remains unknown.
lowed by pulsed amperometric detection (Dionex ICS3000).
The C-domain, however, is not common to GH20 enzymes.
The elution was performed by a linear gradient of 0–0.5 M Two examples of GH20 ␤-HexNAcases that have a distinct
sodium acetate in 125 mM NaOH at a flow rate of 1 ml/min for domain in the C-terminal region of the catalytic barrel domain
3
0 min. The kinetic parameters were calculated by curve-fitting are the ␤-HexNAcases from Serratia marcescens (chitobiase)
the experimental data using the Michaelis-Menten equation in and Streptococcus gordonii (GcnA), which have a small (67
KaleidaGraph 4.0 (Synergy Software).
amino acids) immunoglobulin-like ␤-sandwich domain
(
(
domain IV) and a large (227 amino acids) ␣-helical domain
RESULTS AND DISCUSSION
domain III), respectively (24, 28). In the case of the C-do-
Structural Determination of BbLNBase—We first con- main of BbLNBase, there is no resemblance to these GH20
structed various deletion mutants to determine a minimal C-domain structures. The C-domain of BbLNBase is located on
region that retained activity toward the substrate pNP-LNB the side of the barrel domain that is farthest from the active site.
(
supplemental Table S1 and supplemental Fig. S1). Constructs A deletion of only five residues at the C terminus of this domain
3
7–663, 41–663, and 46–663 (numbers indicate the residues) (construct 37–658) significantly reduced the catalytic activity,
APRIL 26, 2013•VOLUME 288•NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11797

Structure of Lacto-N-biosidase
TABLE 1
Data collection and refinement statistics
Dataset
Selenomethionine-labeled peak
LNB
LNB-thiazoline
Protein Data Bank entry
4H04
4JAW
Data collection statistics
Wavelength
0.97898 Å
1.00000 Å
1.00000 Å
Space group
P2 2 2
P2 2 2
P2 2 2
1
1
1
1
1 1
Unit cell
a ϭ 117.3 Å
a ϭ 116.8 Å
b ϭ 131.0 Å
c ϭ 104.4 Å
a ϭ 116.5 Å
b ϭ 131.6 Å
b ϭ 131.6 Å
c ϭ 104.6 Å
c ϭ 104.8 Å
a
Resolution
50.0 to 2.20 Å (2.28 to 20 Å)
607,378
50.0 to 1.80 Å (1.86 to 1.80 Å)
1,107,867
50.0 to 1.80 Å (1.83 to 1.80 Å)
1,106,951
Total reflections
Unique reflections
158,438
148,717
150,089
a
Completeness
100% (99.9%)
3.8% (3.8%)
100% (100%)
100% (100%)
7.4% (7.3%)
a
a
Redundancy
7.4% (7.4%)
Mean I/␴(I)
14.22% (3.41%)
10.0% (31.2%)
27.64% (4.67%)
7.9% (28.1%)
23.97% (4.85%)
10.2% (48.5%)
a
R
merge
Refinement statistics
Resolution range
47.51–1.80 Å
141,195
43.61–1.80 Å
141,665
No. of reflections
R-factor/R
15.5/19.1%
18.2/21.3%
free
r.m.s.d. from ideal values
Bond lengths
0.028 Å
2.503°
0.027 Å
2.365°
Bond angles
Average B-factor
Protein (chain A/B)
Ligand (chain A/B)
Water
16.0/15.7 Å2
11.6/11.3 Å2
2
2
11.5/10.1 Å
11.6/9.12 Å
2
2
26.6 Å
12.3 Å
2
4
Ϫ
32.3 Å2
23.8 Å2
SO
b
Ramachandran plot
Favored (chain A/B)
Allowed (chain A/B)
Outlier (chain A/B)
97.8/97.3%
1.7/2.4%
0.5/0.3%
98.3/98.4%
1.7/1.4%
0.0/0.2%
a
b
Values in parentheses are for the highest resolution shell.
Data were calculated by RAMPAGE (71).
r.m.s.d. ϭ 2.0 Å for 102 C␣ atoms), which also has a typical
-trefoil fold (41). The ␤-trefoil fold of MOA is composed of
␤
three subdomains that are similar in structure and are assem-
bled at a 3-fold axis (supplemental Fig. S3C), and each sub-
domain contains four-stranded ␤-hairpin turns (␤1–␤4) (sup-
plemental Fig. S3E). The C-domain of BbLNBase can be divided
into an ␣-subdomain (residues 522–550), ␤-subdomain (resi-
dues 551–615), and ␥-subdomain (residues 616–662) (supple-
mental Fig. S3, A and B), with each subdomain having at least
one additional ␣-helix. The ␤-subdomain retains the typical
topology of the ␤-trefoil fold with a complete set of four
␤
-strands (supplemental Fig. S3D). However, the ␣- and ␥-sub-
domains lack one or two ␤-strand(s), and the C-domain is
largely broken at the interface between these subdomains. In
the ␤-subdomain, a disulfide bond is formed between Cys-564
(
within ␤2) and Cys-589 (loop after ␤3). Interestingly, these
structural features (a disulfide bond in the ␤-subdomain and an
additional ␣-helix after ␤3) are also present in a CBM13-related
FIGURE 1. A, domain structure of BbLNBase. B, overall structure of BbLNBase arabinose-binding domain (CBM42) of the fungal GH54 ␣-L-
(
residues 41–663) complexed with LNB. C, overall structure of the wild-type
arabinofuranosidase (42). The CBM42 domain also partially
SpHexcomplexedwithGlcNAc(ProteinDataBankcode1M01). BandC, ␣-hel-
ices and ␤-strands in the catalytic (␣/␤) barrel domain are shown in red and lacks the 3-fold symmetry, and its ␣-subdomain lacks the ara-
8
yellow, respectively, and␣-helicesand␤-strandsintheN-terminaldomainare
shown in cyan and magenta, respectively. Bound ligands are shown as blue
sticks.
binose-binding site. However, the C-domain of BbLNBase
appears to be more broken than CBM42.
Active Site of BbLNBase—We observed a clear electron den-
and further deletions caused complete inactivation (supple- sity for ligands at the center of the barrel domain (Fig. 2, A and
mental Table S1). These results indicate that the C-domain of B). The pyranose ring of the GlcNAc residue, in LNB, is in the
4
BbLNBase is essential for protein stability and catalytic activity.
E conformation and that of GlcNAc-thiazoline, in LNB-thia-
4
Of interest is that the C-domain possesses a broken ␤-trefoil zoline, is in the C conformation (Fig. 2, C and D, discussed
1
fold. According to a structural similarity search using the DALI below). For LNB, GlcNAc and Gal are bound to the Ϫ1 and Ϫ2
server (40), the C-domain is slightly similar to an R-type lectin subsites, respectively, with all the hydroxyl groups of LNB
(
MOA) from Marasmius oreades (CBM13) (Z-score ϭ 11.0, forming hydrogen bonds with the surrounding amino acids.
1
1798 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure of Lacto-N-biosidase
FIGURE 2. Stereoviews of LNB (A and C, yellow) and LNB-thiazoline (B and D, cyan) bound to the active site of BbLNBase. Catalytic residues, residues
forming hydrogen bonds, and residues forming hydrophobic interactions are shown in magenta, green, and white, respectively. Hydrogen bonds are shown as
red dashed lines. A and B, ͉F ͉ Ϫ ͉F ͉ omit electron density map (contoured at 4␴) and interactions with protein atoms. C and D, GlcNAc and GlcNAc-thiazoline
o
c
sugar ring and surrounding residues.
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Structure of Lacto-N-biosidase
FIGURE 3. Comparison of the active sites of GH20 enzymes. A and B, active sites of BbLNBase (A) and SpHex (B), respectively. Inset, loop regions following ␤1
and ␤2 that determine the presence or absence of Ϫ2 subsite. C, amino acid sequence alignment of the loop regions of GH20 enzymes (two LNBases and eight
␤-HexNAcases). Residues important for the presence or absence of Ϫ2 subsites are labeled with red letters. Six-amino acid loop insertion of ␤-HexNAcases is
indicated by a magenta bar. LNBase142, Streptomyces sp. 142 LNBase; DspB, Actinobacillus actinomycetemcomitans dispersin B; GcnA, S. gordonii GcnA; HexA,
human lysosomal ␤-HexNAcase A; HexB, human lysosomal ␤-HexNAcase B; SmChb, S. marcescens chitobiase; PsHex1, Paenibacillus sp. TS12 Hex1; OfHex1, O.
furnacalis ␤-HexNAcase.
1
Such extensive recognitions give evidence for the strict sub- nitrogen of the 2-acetamido group and the O -hydroxyl,
strate specificity observed for this enzyme (17) as demonstrated respectively. Tyr-419 is a highly conserved residue in GH20
for pNP-␤-GNB, the 4-epimer of pNP-␤-LNB, that shows enzymes, and its side-chain hydroxyl group forms a hydrogen
highly reduced activity. In the BbLNBase structure, the C4-hy- bond with the carbonyl oxygen atom of the 2-acetamido group.
4
droxyl group of GlcNAc of LNB forms a hydrogen bond with Asp-467 forms bifurcated hydrogen bonds with the O - and
6
the side chain of Asp-467 (Fig. 2), and this amino acid would O -hydroxyl groups of the GlcNAc residue. The corresponding
block GalNAc-containing substrates from binding such as residue is often substituted with Glu in ␤-HexNAcases (Glu-
4
GNB. In accordance with other biochemical studies, BbLNBase 444 in SpHex), which forms a hydrogen bond with the O -hy-
does not hydrolyze fucosylated substrates such as pyridylamino droxyl group alone. The hydrogen bond with Asp-467 fixes the
6
(
PA)-LNFP I and PA-LNFP II (17), which can be rationalized as O -hydroxyl group in a gauche-gauche orientation in BbLN-
6
the active site of BbLNBase, and it has no space for the fucosyl Base (Fig. 3A), whereas the O -hydroxyl group of GlcNAc in
group at the C2-hydroxyl of Gal (blocked by Asn-259 and SpHex is in the gauche-trans orientation due to hydrogen
Asp-320).
bonds with Asp-395 and Trp-408 (Fig. 3B). In addition, His-
The substrate-binding site of BbLNBase was compared with 263, Trp-373, Trp-394, and Trp-465 form hydrophobic inter-
that of SpHex, which is one of the most well studied GH20 actions with the substrate in the active site (Fig. 2) and are
␤
-HexNAcases (Fig. 3) (21–23). Amino acids surrounding the highly conserved in GH20 (supplemental Fig. S4).
GlcNAc at Ϫ1 subsite (Asp-320, Glu-321, Tyr-419, and Asp-
To investigate the importance of these residues in the Ϫ1
4
67 in BbLNBase) are highly conserved with SpHex and among subsite, we constructed site-directed mutants D320A, D320N,
GH20 enzymes (supplemental Fig. S4). The two catalytic resi- Y419F, and H263F (Table 2). Mutations at the catalytically
dues of GH20, Asp-320 (polarizing residue) and Glu-321 (acid/ important Asp-320 residue (D320A and D320N) did not affect
base catalytic residue), form hydrogen bonds with the amide the K values, but they exhibited significantly reduced k val-
m
cat
1
1800 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure of Lacto-N-biosidase
Ϫ1
Ϫ1
ues, respectively, which is consistent with those of the corre- mined to be 626 Ϯ 23 ␮M, 42.1 Ϯ 0.8 s , and 67.2 Ϯ 1.9 s
Ϫ1
sponding mutants of SpHex (D313A and D313N) (22), human mM , respectively. The K and k values were higher com-
m
cat
HexB (D354N) (43), and Paenibacillus sp. TS12 Hex1 (D321N) pared with those for pNP-LNB (Table 2), demonstrating that
(
29). In the case of Y419F, the K and k values were both this enzyme is an exo-acting enzyme and does not have positive
m
cat
significantly reduced compared with the wild-type enzyme. subsites specific for sugar moieties. In addition, the entrance of
The mutation at His-263 also showed a reduced k value,
cat
the substrate-binding pocket of BbLNBase appears to be wider
than that of SpHex (Fig. 4). This is in agreement with the finding
that LNBase from Streptomyces sp. 142 releases LNB from the
nonreducing end of various oligosaccharides, including a large
triantennary sugar chain (15, 16).
which is consistent with this residue being critical in the hydro-
phobic interaction with GlcNAc.
As expected from the disaccharide-releasing characteristics
of BbLNBase, a Ϫ2 subsite specific for ␤-1,3-linked Gal is
clearly defined in the crystal structure (Fig. 3A). The side chains
Reaction Mechanism and Conformational Changes of the
Substrate—In the widely accepted mechanism of GH20 (Fig. 5),
which is one of substrate-assisted catalysis, the 2-acetamido
group of a substrate is polarized and oriented by a deprotonated
Asp residue that is located at the neighboring position of the
acid/base Glu residue (44). The oxygen atom of the carbonyl
group acts as the nucleophile, which attacks the anomeric car-
bon, resulting in the formation of an oxazoline or oxazolinium
ion intermediate. The protonated acid/base Glu residue facili-
tates bond cleavage by providing a proton to the glycosidic oxy-
gen. In the second step of the reaction, the acid/base Glu acti-
vates a water molecule, thereby facilitating its nucleophilic
attack at the anomeric carbon. During the course of this reac-
tion, two oxocarbenium ion-like transition states are thought to
exist one on each side of the oxazoline or oxazolinium ion
intermediate.
When investigating the catalytic mechanism of carbohy-
drate-active enzymes, the dynamic behavior of a substrate
sugar molecule during the catalytic route in the active site is of
particular interest. In this work, we observed distorted GlcNAc
sugars of LNB in the Ϫ1 subsite of the BbLNBase structure (Fig.
C), whereas the GlcNAc-thiazoline group of the LNB-thiazo-
line complex is in a C conformation (Fig. 2D). Therefore, to
4
of Gln-190 and Glu-216 form hydrogen bonds with the O - and
3
O -hydroxyl groups of Gal, respectively. Gln-190 and Glu-216
in BbLNBase are replaced by Arg-162 and His-188, respec-
tively, in SpHex (Fig. 3C) and jointly block the space for a poten-
tial Ϫ2 subsite in this enzyme (Fig. 3B). Furthermore, in most
␤
-HexNAcases, a 6-amino acid loop insertion is present just
after the His-188 residue (Fig. 3C), and a relatively conserved
Asp residue (Asp-191 in SpHex) located in the loop occupies a
position corresponding to the Ϫ2 subsite (Gal binding site) of
BbLNBase (Fig. 3B). Comparison of the molecular surfaces of
BbLNBase and SpHex (Fig. 4) illustrates pockets that are suita-
ble in size for binding disaccharide and monosaccharide units,
respectively. However, we were unable to determine the posi-
tive subsites in the BbLNBase structure. Attempts at co-crys-
tallization and soaking experiments with lactose, Gal, or Glc
were undertaken, but no electron densities for these sugars
were found (data not shown). BbLNBase has been previously
shown to efficiently hydrolyze PA-LNT as well as pNP-␤-LNB
(
17). However, the enzymatic activity against LNT, the natural
form of a major HMO component, has not been fully studied.
Thus, we measured the kinetic parameters for hydrolysis of
LNT by BbLNBase. The K , k , and k /K values were deter-
2
m
cat
cat
m
4
1
TABLE 2
gain insight into the conformations of the sugar at binding, we
analyzed the sugar ring conformations in detail using the Cre-
mer-Pople system, which is widely used to define the puckering
conformations of a pyranose (45). The Cremer-Pople parame-
ters of GlcNAc of LNB in chains A and B were ␾ ϭ 245.9°, ␪ ϭ
Kinetic parameters of wild-type and mutants of BbLNBase
Activities of pNP-LNB hydrolysis were measured in 50 mM McIlvaine buffer (pH
4.5) at 30 °C.
Enzyme
K
k
k
ϫ10
160 Ϯ 10
1.3 Ϯ 0.1
0.052 Ϯ 0.006
0.028 Ϯ 0.002
–
/K
s
m
Ϫ6
cat
sϪ1
cat
3
m
Ϫ1/M
ϫ1
M
Wild type
H263F
99 Ϯ 11
15 Ϯ 1
6
0.1°, and Q ϭ 0.595, and ␾ ϭ 252.6°, ␪ ϭ 56.7°, and Q ϭ 0.616,
120 Ϯ 20
130 Ϯ 20
380 Ϯ 50
0.15 Ϯ 0.01
0.0068 Ϯ 0.0005
0.011 Ϯ 0.001
0.086b
respectively, indicating that they are in a conformation close to
D320A
D320N
Y419F
4
E (ideally, ␾ ϭ 240° and ␪ ϭ 54.7°). On the other hand, the
a
–
a
b
parameters of GlcNAc-thiazoline of LNB-thiazoline in chains
The K
m
value was too low to be determined.
The v/[E]
0
value at 100 ␮M substrate.
A and B were ␾ ϭ 237.6°, ␪ ϭ 13.2°, and Q ϭ 0.547, and ␾ ϭ
FIGURE 4. Molecular surfaces of BbLNBase (A) and SpHex (B) showing the substrate binding pockets. According to the electrostatic potential, the surface
is colored blue for positive and red for negative charges. LNB and GlcNAc bound to the pocket are shown as green sticks.
APRIL 26, 2013•VOLUME 288•NUMBER 17
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Structure of Lacto-N-biosidase
FIGURE 5. Proposed reaction pathway of BbLNBase and possible confor-
mations of the GlcNAc sugar ring. R ϭ a leaving group.
FIGURE 6. Distribution map of sugar ring conformations observed in the
1 subsite of GH20 enzymes as analyzed by Cremer-Pople angle param-
؊
1
,4
4
eters. Cross symbols indicate ideal positions of B (␾ ϭ 240°, ␪ ϭ 90°), E (␾ ϭ
1
2
40°, ␪ ϭ 54.7°), and S (␾ ϭ 270°, ␪ ϭ 90°) conformations. Open squares
2
65.4°, ␪ ϭ 17.9°, and Q ϭ 0.597, respectively, indicating that
5
4
(GlcNAc ϩ R) include chitobiose, GlcNAc-␤1,2-Man, GMMG, NGAB2, and TMG-
chitotriomycin. Closed downward triangles (PUGNAcs) include PUGNAc and
they are in a conformation close to C .
1
The 2-acetamido group of GlcNAc in LNB is nestled in Gal-PUGNAc. Open upward triangles (thiazolines) include GlcNAc-thiazoline
and GalNAc-thiazoline. Open downward triangles (isofagomine) indicate Gal-
NAc-isofagomine. Closed squares indicate LNB-thiazoline. Details of the sugar
compound names and their parameters are listed in supplemental Table S2.
among three aromatic residues (Trp-373, Trp-394, and Trp-
4
4
65) and fixed by two hydrogen bonds with Asp-320 and Tyr-
19 (Fig. 2C). The 2-acetamido group at its fixed position ele-
vates the C1 atom of GlcNAc, and the hydrogen bond between
Various substrates that carry sugar moieties at positive sub-
4
Asp-467 and O -hydroxyl fixes the C4 atom of GlcNAc in an sites have also been observed in crystal structures of GH20
elevated position. These interactions potentially distort the ␤-HexNAcases. Complexes of a chitobiase (␤-HexNAcase)
4
conformation of GlcNAc to E, which is not intrinsically stable from S. marcescens with chitobiose (di-N-acetyl-D-glucosa-
as evidenced by crystallographic analysis and molecular simu- mine) were trapped using the mutants D537A and E540A (cat-
lation (46, 47).
alytic residues) (48) or by a simple soaking method (24). The
Currently, various substrates (e.g. chitobiose), inhibitors (e.g. complex structures of the N- and C-terminal modules (GH20A
GlcNAc-thiazoline), and reaction products (e.g. GlcNAc) of and GH20B) of the large multimodular ␤-HexNAcase StrH
␤
-HexNAcases have been observed in the crystal structures of from Streptococcus pneumoniae with disaccharide (in GH20A),
other GH20 enzymes (supplemental Table S2) (21, 22, 24, 25, tetrasaccharide (in GH20B), and bisected glycan heptasaccha-
2
7–31, 48–52). In an effort to gain insight into the global bind- ride (in GH20B) substrates were reported using their acid/base
ing of these ligands against GH20 enzymes, their Cremer-Pople residue mutants (E361Q of GH20A and E805Q of GH20B) (31).
parameters (␾ and ␪) were evaluated and plotted (Fig. 6) The GlcNAc moieties of these substrates bound to the Ϫ1 sub-
1
,4
together with those of the GlcNAc sugars of LNB and LNB- site were all in conformations close to a B conformation
thiazoline in BbLNBase.
(229° Ͻ ␾ Ͻ 254° and ␪ Ͼ 66°; ideally ␾ ϭ 240° and ␪ ϭ 90°).
GlcNAc-thiazoline and GalNAc-thiazoline are specific Moreover, the complex structure of TMG-chitotriomycin, a
inhibitors of GHs that utilize the substrate-assisted mechanism linear tetrasaccharide inhibitor (55), bound to the ␤-HexNA-
(
53, 54) because they structurally resemble the oxazoline or case from Ostrinia furnacalis was reported (30) with the N,N,N-
oxazolinium ion intermediate. A recent study using ab initio trimethyl-D-glucosamine group at the nonreducing end of
1
,4
molecular dynamic simulations predicts that the oxazoline or TMG-chitotriomycin also adopting a B-like conformation
oxazolinium ion intermediate is distorted to a conformational (␾ ϭ 244.2° and ␪ ϭ 84.5°). Therefore, the substrate of the
4
4
4
1,4
region of H / E/ H (44). However, in GH20 ␤-HexNAcases, Michaelis complex for GH20 enzymes is thought to adopt a
B
3
5
the thiazoline inhibitors always adopt conformations close to conformation.
4
2
C (␪ Ͻ 23°) (21, 25, 27, 52). Our crystallographic result also
Transition state analogs that have an sp -hybridized planar
anomeric carbon were also used to study ␤-HexNAcases and
1
4
indicates that the LNB-thiazoline in LNBase also adopt a C
1
conformation (Fig. 6). These ample crystallographic data indi- related enzymes as these compounds are found to be potent
4
cate that the oxazolinium ion intermediate adopts the C con- inhibitors of these enzymes. In the complex structure of human
1
formation (21).
lysosomal ␤-HexNAcase B, 2-acetamido-2-deoxy-D-glucono-
1
1802 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure of Lacto-N-biosidase
4
1
,5-lactone (␦-lactone) was bound in conformations close to E lus sp. TS12, the C-terminal region of one of the two ␤-Hex-
236° Ͻ ␾ Ͻ 253° and 48° Ͻ ␪ Ͻ 64°) (50). O-(2-Acetamido- NAcases (residues 503–978 of Hex1) was suggested to help the
-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate enzyme in its interaction with glycosphingolipid substrates in
PUGNAc) is a potent inhibitor of a wide range of ␤-HexNA- the absence of detergent (29). However, the three-dimensional
cases that utilize the classical double displacement mechanism structure of this domain remains unknown because only a trun-
(
2
(
(
e.g. GH3) as well as the substrate-assisted mechanism used by cated structure of Hex1 is available (deletion mutant 1–502),
GH20 enzymes. In ␤-HexNAcases from O. furnacalis and and there is no amino acid sequence homology between the
Paenibacillus sp. TS12 (Hex1), PUGNAc adopts conformations C-terminal region of Hex1 and the C-domain of BbLNBase.
4
1,4
ranging from E to B (240° Ͻ ␾ Ͻ 245° and 60° Ͻ ␪ Ͻ 78°) (29, The full-length BbLNBase has a CBM32 domain (residues
5
1). Our results strongly support the hypothesis that the con- 784–932) in a region adjacent to the C-domain (Fig. 1A).
4
formation at the transition states of GH20 enzymes is nearly E, CBM32 domains of GHs from enteric bacteria have been shown
which is in compliance with the requirement of coplanarity at to recognize a terminal Gal or GalNAc, or disaccharide motifs
the C2, C1, O5, and C5 pyranose ring atoms to attain the oxo- such as LacNAc and GlcNAc-␣1,4-Gal (59). Therefore, the
4
carbenium ion-like transition state. E-Like transition states are CBM32 domain of BbLNBase may also function in binding
also predicted for other GH enzymes that utilize the substrate- ␤-1,3-linked Gal, LNB, or other moieties of HMOs.
assisted mechanism, namely a GH18 chitinase (by a QM/MM
Biological Implications—In the type I HMO-degradation sys-
tem of B. bifidum JCM1254, BbLNBase plays a central role in
modeling study) (56), and a GH84 O-GlcNAcase (57).
According to previous knowledge regarding GH20 ␤-Hex- releasing LNB from HMOs (9, 60). However, the assistance of
NAcases and the present crystal structures, we propose a con- other enzymes that remove modifying sugars such as fucose or
formational itinerary pathway for the BbLNBase-catalyzed sialic acid is required. Many extracellular glycosidases involved
reaction that obeys the “principle of least nuclear motion” (21, in HMO degradation have been found in B. bifidum JCM1254.
1,4
4
4
5
8): B (Michaelis complex)- E (transition state 1)- C (oxa- GH95 1,2-␣-L-fucosidase (AfcA) (61, 62), GH29 1,3–1,4-␣-L-
4 4
1
zolinium ion intermediate)- E (transition state 2)- E (product fucosidase (AfcB) (63, 64), and GH33 exo-␣-sialidases (SiaBb1
4
complex) (Fig. 5). Of interest is the E product complex and SiaBb2) (65) all share roles in the complete degradation of
observed in this study. In GH20 ␤-HexNAcases, several com- ␣1,2- and ␣1,3/4-fucosylated and sialylated HMOs. In this
plexes observed with reaction products (GlcNAc or GalNAc) study, we provide a structural basis for the substrate specificity
have been reported. However, the ring conformations and of BbLNBase. This enzyme cannot accommodate any modified
binding modes of these molecules differ depending on the con- LNB moieties because of a substrate binding pocket that is con-
ditions of complex formation. In the structures of ␤-HexNA- strained to allow only LNB binding. This feature is suitable for
4
case (Hex1) from Paenibacillus sp. TS12, GlcNAc is in a C
the specificity of the solute-binding protein of the GNB/LNB
1
1,4
conformation, whereas GalNAc is in a B conformation (29). transporter (GL-BP) in the GNB/LNB pathway, which specifi-
4
In SpHex, GlcNAc is bound in a C conformation with the cally binds unmodified LNB disaccharide (11). GL-BP binds
1
wild-type enzyme, but it adopts the alternative conformations LNB and GNB with low K values (Ͻ 0.09 ␮M), whereas LNT, a
d
1,4
4
of B and C in the D313A mutant (22). In the D313N mutant, major HMO tetrasaccharide containing LNB, exhibits a signif-
1
4
the bound GlcNAc is in a C conformation but is tilted and icantly higher K value (11 ␮M). Following uptake, LNB is sub-
1
d
dropped from its normal position in the wild-type complex. In sequently metabolized by GNB/LNB phosphorylase and other
BbLNBase, we speculate that there are two major factors that enzymes, including N-acetylhexosamine kinase, UDP-glucose
4
fix LNB with its GlcNAc moiety in a E conformation. First, the hexose-1-phosphate uridylyltransferase, and UDP-glucose
Gal moiety bound to Ϫ2 subsite acts as an anchor to lock in 4-epimerase (8). The positive subsites of BbLNBase are wider
place the LNB disaccharide in the active site. This anchor is and appear to be capable of accommodating various groups,
lacking in other GH20 ␤-HexNAcases as they only have suggesting that this enzyme acts on various type I HMOs after
confined space in the active site to accommodate a the action of fucosidases and sialidases. Moreover, B. bifidum
GlcNAc/GalNAc residue. Second, the bifurcated hydrogen JCM1254 has three extracellular enzymes, including one GH2
bond between Asp-467 and the O4/O6-hydroxyls of the ␤-galactosidase (BbgIII) and two GH20 ␤-HexNAcases (BbhI
4
GlcNAc moiety strongly fix it in the E and gauche-gauche con- and BbhII) (66). Two of these enzymes (BbgIII and BbhI) are
formations. These factors are unique to LNBases and have to suggested to play essential roles in degrading type II HMOs
date not been shown in ␤-HexNAcases.
because they catalyze the complete hydrolysis of lacto-N-neo-
C-terminal Domains—In the crystal structure of BbLNBase, tetraose (Gal-␤1,4-GlcNAc-␤1,3-Gal-␤1,4-Glc) to monosac-
we observed a novel ␤-trefoil-like domain (C-domain) that is charides. BbgIII hydrolyzes ␤-1,4- and ␤-1,6-linked Gal but not
required for protein stability. One of our most striking findings ␤-1,3-linked Gal in LNB and LNT, suggesting that it is also
was that the C-domain has several features similar to those of involved in the complete degradation of type I HMOs after the
the arabinose-binding domain CBM42. However, there is cur- action of LNBase (e.g. cleavage of lactose released from LNT).
rently no evidence indicating that the C-domain in BbLNBase
In contrast to B. bifidum JCM1254, B. longum subsp. infantis
has carbohydrate-binding ability. We attempted co-crystalliza- ATCC15697 lacks the LNBase gene. A genomic analysis of B.
tion and soaking experiments with various sugars such as lac- longum subsp. infantis identified a unique gene cluster, the
tose, Gal, or Glc at high concentrations (up to 1 M), but we could HMO cluster, containing various intracellular GHs that lack
not observe any extra electron density in this domain (data not signal sequences (67, 68). A GH29 fucosidase, a GH95 fucosi-
shown). In a study examining ␤-HexNAcases from Paenibacil- dase, a GH33 sialidase, a GH20 ␤-HexNAcase, a GH2 ␤-galac-
APRIL 26, 2013•VOLUME 288•NUMBER 17
JOURNAL OF BIOLOGICAL CHEMISTRY 11803

Structure of Lacto-N-biosidase
tosidase, and at least four putative sugar transporters were
found in the HMO cluster. The GH2 ␤-galactosidase is specific
for lactose and type II HMOs (69). Furthermore, the intracellu-
lar GH42 ␤-galactosidase Bga42A, which is distant from the
HMO cluster, is highly specific for LNT and functions as the
9. Fushinobu, S. (2010) Unique sugar metabolic pathways of bifidobacteria.
Biosci. Biotechnol. Biochem. 74, 2374–2384
1
0. Suzuki, R., Katayama, T., Kitaoka, M., Kumagai, H., Wakagi, T., Shoun, H.,
Ashida, H., Yamamoto, K., and Fushinobu, S. (2009) Crystallographic and
mutational analyses of substrate recognition of endo-␣-N-acetylgalac-
tosaminidase from Bifidobacterium longum. J. Biochem. 146, 389–398
sole ␤-galactosidase acting on type I HMOs. In the HMO deg- 11. Suzuki, R., Wada, J., Katayama, T., Fushinobu, S., Wakagi, T., Shoun, H.,
Sugimoto, H., Tanaka, A., Kumagai, H., Ashida, H., Kitaoka, M., and
radation system of this strain, it was suggested that low molec-
Yamamoto, K. (2008) Structural and thermodynamic analyses of solute-
ular weight HMOs (degree of polymerization ranging from 3 to
binding protein from Bifidobacterium longum specific for core 1 disaccha-
8
) are directly imported into the cells by transporters and sub-
ride and lacto-N-biose I. J. Biol. Chem. 283, 13165–13173
sequently cleaved by intracellular GHs (70). This analysis as
well as recent research on commensal bacteria is unveiling the
different HMO consumption modes of bifidobacterial strains.
Extracellular bifidobacterial LNBases may also aid in the HMO
consumption of other bifidobacterial strains that lack endoge-
nous LNBases. Importantly, in this study, we identified residues
that are important for ␤-1,3-linked Gal recognition at the Ϫ2
subsite (viz. Gln-190 and Glu-216). Conservation of these resi-
dues is a critical marker that helps to predict putative LNBase
genes. A BLAST search against protein databases indicates that
putative LNBase genes are present in many bacteria, mainly in
the Actinobacteridae subclass. The presence (or absence) of
LNBase in the genome of microbes in an infant’s gastrointesti-
nal tract will provide important information that will advance
our understanding of how these microbes metabolize HMOs.
1
2. Hidaka, M., Nishimoto, M., Kitaoka, M., Wakagi, T., Shoun, H., and Fushi-
nobu, S. (2009) The crystal structure of galacto-N-biose/lacto-N-biose I
phosphorylase: a large deformation of a TIM barrel scaffold. J. Biol. Chem.
284, 7273–7283
1
1
3. Kiyohara, M., Tachizawa, A., Nishimoto, M., Kitaoka, M., Ashida, H., and
Yamamoto, K. (2009) Prebiotic effect of lacto-N-biose I on bifidobacterial
growth. Biosci. Biotechnol. Biochem. 73, 1175–1179
4. Xiao, J. Z., Takahashi, S., Nishimoto, M., Odamaki, T., Yaeshima, T., Iwat-
suki, K., and Kitaoka, M. (2010) Distribution of in vitro fermentation abil-
ity of lacto-N-biose I, a major building block of human milk oligosaccha-
rides, in bifidobacterial strains. Appl. Environ. Microbiol. 76, 54–59
5. Sano, M., Hayakawa, K., and Kato, I. (1992) An enzyme releasing lacto-N-
biose from oligosaccharides. Proc. Natl. Acad. Sci. U.S.A. 89, 8512–8516
6. Sano, M., Hayakawa, K., and Kato, I. (1993) Purification and characteriza-
tion of an enzyme releasing lacto-N-biose from oligosaccharides with type
1 chain. J. Biol. Chem. 268, 18560–18566
1
1
17. Wada, J., Ando, T., Kiyohara, M., Ashida, H., Kitaoka, M., Yamaguchi, M.,
Kumagai, H., Katayama, T., and Yamamoto, K. (2008) Bifidobacterium
bifidum lacto-N-biosidase, a critical enzyme for the degradation of human
milk oligosaccharides with a type 1 structure Appl. Environ. Microbiol. 74,
Acknowledgments—We thank Drs. M. Nishimoto and M. Kitaoka
(
National Food and Research Institute, National Agriculture and
3
996–4004
Food Research Organization, Japan) for providing LNB and for help-
ful discussion. We thank the staff of the Photon Factory and SPring-8
for the x-ray data collection. M. H. and K. A. S. thank the Centre for
Microscopy, Characterization, and Analysis at the University of
Western Australia, which is supported by University, State, and Fed-
eral Government funding.
1
8. Turroni, F., Peano, C., Pass, D. A., Foroni, E., Severgnini, M., Claesson,
M. J., Kerr, C., Hourihane, J., Murray, D., Fuligni, F., Gueimonde, M.,
Margolles, A., De Bellis, G., O’Toole, P. W., van Sinderen, D., Marchesi,
J. R., and Ventura, M. (2012) Diversity of bifidobacteria within the infant
gut microbiota. PLoS One 7, e36957
1
9. Magnani, J. L., Steplewski, Z., Koprowski, H., and Ginsburg, V. (1983)
Identification of the gastrointestinal and pancreatic cancer-associated an-
tigen detected by monoclonal antibody 19–9 in the sera of patients as a
mucin. Cancer Res. 43, 5489–5492
REFERENCES
2
0. Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V.,
and Henrissat, B. (2009) The Carbohydrate-Active EnZymes database
(CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 37,
D233–D238
1
. Urashima, T., Fukuda, K., Kitaoka, M., Terabayashi, T., and Kobata, A.
(
eds) (2011) Milk Oligosaccharides, Nova Science Publishers, New York
2
. Urashima, T., Asakuma, S., Leo, F., Fukuda, K., Messer, M., and Oftedal,
O. T. (2012) The predominance of type I oligosaccharides is a feature 21. Mark, B. L., Vocadlo, D. J., Knapp, S., Triggs-Raine, B. L., Withers, S. G.,
specific to human breast milk. Adv. Nutr. 3, 473S-482S
and James, M. N. (2001) Crystallographic evidence for substrate-assisted
3
4
5
. Zivkovic, A. M., German, J. B., Lebrilla, C. B., and Mills, D. A. (2011)
Human milk glycobiome and its impact on the infant gastrointestinal
microbiota. Proc. Natl. Acad. Sci. U.S.A. 108, 4653–4658
catalysis in a bacterial ␤-hexosaminidase. J. Biol. Chem. 276,
10330–10337
22. Williams, S. J., Mark, B. L., Vocadlo, D. J., James, M. N., and Withers, S. G.
(2002) Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a
critical role in substrate-assisted catalysis by orienting the 2-acetamido
group and stabilizing the transition state. J. Biol. Chem. 277, 40055–40065
. Venema, K. (2012) Intestinal fermentation of lactose and prebiotic lactose
derivatives, including human milk oligosaccharides. Int. J. Dairy 22,
1
23–140
. Urashima, T., Odaka, G., Asakuma, S., Uemura, Y., Goto, K., Senda, A., 23. Vocadlo, D. J., and Withers, S. G. (2005) Detailed comparative analysis of
Saito, T., Fukuda, K., Messer, M., and Oftedal, O. T. (2009) Chemical
characterization of oligosaccharides in chimpanzee, bonobo, gorilla,
orangutan, and siamang milk or colostrum. Glycobiology 19, 499–508
. Asakuma, S., Urashima, T., Akahori, M., Obayashi, H., Nakamura, T.,
Kimura, K., Watanabe, Y., Arai, I., and Sanai, Y. (2008) Variation of major
neutral oligosaccharides levels in human colostrum. Eur. J. Clin. Nutr. 62,
the catalytic mechanisms of ␤-N-acetylglucosaminidases from families 3
and 20 of glycoside hydrolases. Biochemistry 44, 12809–12818
24. Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., and Vor-
gias, C. E. (1996) Bacterial chitobiase structure provides insight into cata-
lytic mechanism and the basis of Tay-Sachs disease. Nat. Struct. Biol. 3,
638–648
6
4
88–494
25. Mark, B. L., Mahuran, D. J., Cherney, M. M., Zhao, D., Knapp, S., and
James, M. N. (2003) Crystal structure of human ␤-hexosaminidase B: un-
derstanding the molecular basis of Sandhoff and Tay-Sachs disease. J. Mol.
Biol. 327, 1093–1109
7
. Kitaoka, M., Tian, J., and Nishimoto, M. (2005) Novel putative galactose
operon involving lacto-N-biose phosphorylase in Bifidobacterium
longum. Appl. Environ. Microbiol. 71, 3158–3162
8
. Nishimoto, M., and Kitaoka, M. (2007) Identification of N-acetylhexo- 26. Ramasubbu, N., Thomas, L. M., Ragunath, C., and Kaplan, J. B. (2005)
samine 1-kinase in the complete lacto-N-biose I/galacto-N-biose meta-
bolic pathway in Bifidobacterium longum. Appl. Environ. Microbiol. 73,
Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase
from the periodontopathogen Actinobacillus actinomycetemcomitans. J.
Mol. Biol. 349, 475–486
6
444–6449
1
1804 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 17•APRIL 26, 2013

Structure of Lacto-N-biosidase
2
7. Lemieux, M. J., Mark, B. L., Cherney, M. M., Withers, S. G., Mahuran, D. J.,
and James, M. N. (2006) Crystallographic structure of human ␤-hexo-
saminidase A: interpretation of Tay-Sachs mutations and loss of GM2
ganglioside hydrolysis. J. Mol. Biol. 359, 913–929
48. Prag, G., Papanikolau, Y., Tavlas, G., Vorgias, C. E., Petratos, K., and Op-
penheim, A. B. (2000) Structures of chitobiase mutants complexed with
the substrate Di-N-acetyl-D-glucosamine: the catalytic role of the con-
served acidic pair, aspartate 539 and glutamate 540. J. Mol. Biol. 300,
611–617
2
8. Langley, D. B., Harty, D. W., Jacques, N. A., Hunter, N., Guss, J. M., and
Collyer, C. A. (2008) Structure of N-acetyl-␤-D-glucosaminidase (GcnA) 49. Mark, B. L., Vocadlo, D. J., Zhao, D., Knapp, S., Withers, S. G., and James,
from the endocarditis pathogen Streptococcus gordonii and its complex
with the mechanism-based inhibitor NAG-thiazoline. J. Mol. Biol. 377,
M. N. (2001) Biochemical and structural assessment of the 1-N-azasugar
GalNAc-isofagomine as a potent family 20 ␤-N-acetylhexosaminidase in-
hibitor. J. Biol. Chem. 276, 42131–42137
1
04–116
2
9. Sumida, T., Ishii, R., Yanagisawa, T., Yokoyama, S., and Ito, M. (2009) 50. Maier, T., Strater, N., Schuette, C. G., Klingenstein, R., Sandhoff, K., and
Molecular cloning and crystal structural analysis of a novel ␤-N-acetyl-
hexosaminidase from Paenibacillus sp. TS12 capable of degrading glyco-
sphingolipids. J. Mol. Biol. 392, 87–99
Saenger, W. (2003) The x-ray crystal structure of human ␤-hexosamini-
dase B provides new insights into Sandhoff disease. J. Mol. Biol. 328,
669–681
3
0. Liu, T., Zhang, H., Liu, F., Wu, Q., Shen, X., and Yang, Q. (2011) Structural 51. Liu, T., Zhang, H., Liu, F., Chen, L., Shen, X., and Yang, Q. (2011) Active-
determinants of an insect ␤-N-acetyl-D-hexosaminidase specialized as a
chitinolytic enzyme. J. Biol. Chem. 286, 4049–4058
pocket size differentiating insectile from bacterial chitinolytic ␤-N-acetyl-
D-hexosaminidases. Biochem. J. 438, 467–474
3
1. Pluvinage, B., Higgins, M. A., Abbott, D. W., Robb, C., Dalia, A. B., Deng, 52. Sumida, T., Stubbs, K. A., Ito, M., and Yokoyama, S. (2012) Gaining insight
L., Weiser, J. N., Parsons, T. B., Fairbanks, A. J., Vocadlo, D. J., and Boras-
ton, A. B. (2011) Inhibition of the pneumococcal virulence factor StrH and
molecular insights into N-glycan recognition and hydrolysis. Structure 19,
into the inhibition of glycoside hydrolase family 20 exo-␤-N-acetylhexo-
saminidases using a structural approach. Org. Biomol. Chem. 10,
2607–2612
1
603–1614
53. Knapp, S., Vocadlo, D. J., Gao, Z., Kirk, B., Lou, J., and Withers, S. G. (1996)
NAG-thiazoline, an N-acetyl-␤-hexosaminidase inhibitor that implicates
acetamido participation. J. Am. Chem. Soc. 118, 6804–6805
3
3
2. Jiang, Y. L., Yu, W. L., Zhang, J. W., Frolet, C., Di Guilmi, A. M., Zhou,
C. Z., Vernet, T., and Chen, Y. (2011) Structural basis for the substrate
specificity of a novel ␤-N-acetylhexosaminidase StrH protein from Strep- 54. Amorelli, B., Yang, C., Rempel, B., Withers, S. G., and Knapp, S. (2008)
tococcus pneumoniae R6. J. Biol. Chem. 286, 43004–43012
N-Acetylhexosaminidase inhibitory properties of C-1 homologated
GlcNAc- and GalNAc-thiazolines. Bioorg. Med. Chem. Lett. 18,
2944–2947
3. Hattie, M., Debowski, A. W., and Stubbs, K. A. (2012) Development of
tools to study lacto-N-biosidase: an important enzyme involved in the
breakdown of human milk oligosaccharides. ChemBioChem 13, 55. Usuki, H., Nitoda, T., Ichikawa, M., Yamaji, N., Iwashita, T., Komura, H.,
1
128–1131
and Kanzaki, H. (2008) TMG-chitotriomycin, an enzyme inhibitor spe-
cific for insect and fungal ␤-N-acetylglucosaminidases, produced by acti-
nomycete Streptomyces anulatus NBRC 13369. J. Am. Chem. Soc. 130,
4146–4152
3
3
4. Otwinowski, Z., and Minor, W. (1997) Processing of x-ray diffraction data
collected in oscillation mode. Methods Enzymol. 276, 307–326
5. Rappleye, J., Innus, M., Weeks, C. M., and Miller, R. (2002) SnB version 2.2:
an example of crystallographic multiprocessing. J. Appl. Crystallogr. 35, 56. Jitonnom, J., Lee, V. S., Nimmanpipug, P., Rowlands, H. A., and Mulhol-
3
74–376
land, A. J. (2011) Quantum mechanics/molecular mechanics modeling of
substrate-assisted catalysis in family 18 chitinases: conformational
changes and the role of Asp-142 in catalysis in ChiB. Biochemistry 50,
4697–4711
3
3
6. Terwilliger, T. C., and Berendzen, J. (1999) Automated MAD and MIR
structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861
7. Perrakis, A., Morris, R., and Lamzin, V. S. (1999) Automated protein
model building combined with iterative structure refinement. Nat. Struct. 57. Rao, F. V., Dorfmueller, H. C., Villa, F., Allwood, M., Eggleston, I. M., and
Biol. 6, 458–463
van Aalten, D. M. (2006) Structural insights into the mechanism and in-
hibition of eukaryotic O-GlcNAc hydrolysis. EMBO J. 25, 1569–1578
58. Sinnott, M. L. (1984) On the antiperiplanar lone pair hypothesis and its
application to catalysis by glycosidases. Biochem. J. 224, 817–821
3
8. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and
development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501
9. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of
3
macromolecular structures by the maximum-likelihood method. Acta 59. Ficko-Blean, E., and Boraston, A. B. (2012) Insights into the recognition of
Crystallogr. D Biol. Crystallogr. 53, 240–255
the human glycome by microbial carbohydrate-binding modules. Curr.
Opin. Struct. Biol. 22, 570–577
4
4
0. Holm, L., and Sander, C. (1993) Protein structure comparison by align-
ment of distance matrices. J. Mol. Biol. 233, 123–138
60. Asakuma, S., Hatakeyama, E., Urashima, T., Yoshida, E., Katayama, T.,
Yamamoto, K., Kumagai, H., Ashida, H., Hirose, J., and Kitaoka, M. (2011)
Physiology of consumption of human milk oligosaccharides by infant gut-
associated bifidobacteria. J. Biol. Chem. 286, 34583–34592
1. Grahn, E., Askarieh, G., Holmner, A., Tateno, H., Winter, H. C., Goldstein,
I. J., and Krengel, U. (2007) Crystal structure of the Marasmius oreades
mushroom lectin in complex with a xenotransplantation epitope. J. Mol.
Biol. 369, 710–721
61. Katayama, T., Sakuma, A., Kimura, T., Makimura, Y., Hiratake, J., Sakata,
K., Yamanoi, T., Kumagai, H., and Yamamoto, K. (2004) Molecular clon-
ing and characterization of Bifidobacterium bifidum 1,2-␣-L-fucosidase
(AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J.
Bacteriol. 186, 4885–4893
4
2. Miyanaga, A., Koseki, T., Matsuzawa, H., Wakagi, T., Shoun, H., and
Fushinobu, S. (2004) Crystal structure of a family 54 ␣-L-arabinofurano-
sidase reveals a novel carbohydrate-binding module that can bind arabi-
nose. J. Biol. Chem. 279, 44907–44914
4
3. Hou, Y., Vocadlo, D. J., Leung, A., Withers, S. G., and Mahuran, D. (2001) 62. Nagae, M., Tsuchiya, A., Katayama, T., Yamamoto, K., Wakatsuki, S., and
Characterization of the Glu and Asp residues in the active site of human
-hexosaminidase B. Biochemistry 40, 2201–2209
4. Greig, I. R., Zahariev, F., and Withers, S. G. (2008) Elucidating the nature
Kato, R. (2007) Structural basis of the catalytic reaction mechanism of
novel 1,2-␣-L-fucosidase from Bifidobacterium bifidum. J. Biol. Chem.
282, 18497–18509
␤
4
of the Streptomyces plicatus ␤-hexosaminidase-bound intermediate using 63. Ashida, H., Miyake, A., Kiyohara, M., Wada, J., Yoshida, E., Kumagai, H.,
ab initio molecular dynamics simulations. J. Am. Chem. Soc. 130,
Katayama, T., and Yamamoto, K. (2009) Two distinct ␣-L-fucosidases
from Bifidobacterium bifidum are essential for the utilization of fucosy-
lated milk oligosaccharides and glycoconjugates. Glycobiology 19,
1010–1017
1
7620–17628
4
4
5. Cremer, D., and Pople, J. A. (1975) A general definition of ring puckering
coordinates. J. Am. Chem. Soc. 97, 1354–1358
6. Wada, M., Kobayashi, K., Nishimoto, M., Kitaoka, M., and Noguchi, K.
64. Sakurama, H., Fushinobu, S., Hidaka, M., Yoshida, E., Honda, Y., Ashida,
H., Kitaoka, M., Kumagai, H., Yamamoto, K., and Katayama, T. (2012)
1,3–1,4-␣-L-Fucosynthase that specifically introduces Lewis a/x antigens
into type-1/2 chains. J. Biol. Chem. 287, 16709–16719
(
2009) 2-Acetamido-2-deoxy-3-O-␤-D-galactopyranosyl-D-glucose dihy-
drate. Acta Crystallogr. E 65, 1781–1782
4
7. Sattelle, B. M., and Almond, A. (2011) Is N-acetyl-D-glucosamine a rigid
4
C chair? Glycobiology 21, 1651–1662
1
65. Kiyohara, M., Tanigawa, K., Chaiwangsri, T., Katayama, T., Ashida, H.,
APRIL 26, 2013•VOLUME 288•NUMBER 17
JOURNAL OF BIOLOGICAL CHEMISTRY 11805

Structure of Lacto-N-biosidase
and Yamamoto, K. (2011) An exo-␣-sialidase from bifidobacteria involved
biol. 18, 298–307
in the degradation of sialyloligosaccharides in human milk and intestinal 69. Yoshida, E., Sakurama, H., Kiyohara, M., Nakajima, M., Kitaoka, M.,
glycoconjugates. Glycobiology 21, 437–447
Ashida, H., Hirose, J., Katayama, T., Yamamoto, K., and Kumagai, H.
(2012) Bifidobacterium longum subsp. infantis uses two different ␤-galac-
tosidases for selectively degrading type-1 and type-2 human milk oligo-
saccharides. Glycobiology 22, 361–368
6
6. Miwa, M., Horimoto, T., Kiyohara, M., Katayama, T., Kitaoka, M., Ashida,
H., and Yamamoto, K. (2010) Cooperation of ␤-galactosidase and ␤-N-
acetylhexosaminidase from bifidobacteria in assimilation of human milk
oligosaccharides with type 2 structure. Glycobiology 20, 1402–1409
7. Sela, D. A., Chapman, J., Adeuya, A., Kim, J. H., Chen, F., Whitehead, T. R.,
Lapidus, A., Rokhsar, D. S., Lebrilla, C. B., German, J. B., Price, N. P., Richard-
son, P. M., and Mills, D. A. (2008) The genome sequence of Bifidobacterium
longum subsp. infantis reveals adaptations for milk utilization within the in-
fant microbiome. Proc. Natl. Acad. Sci. U.S.A. 105, 18964–18969
70. Barboza, M., Sela, D. A., Pirim, C., Locascio, R. G., Freeman, S. L., German,
J. B., Mills, D. A., and Lebrilla, C. B. (2009) Glycoprofiling bifidobacterial
consumption of galacto-oligosaccharides by mass spectrometry reveals
strain-specific, preferential consumption of glycans. Appl. Environ. Micro-
biol. 75, 7319–7325
6
71. Lovell, S. C., Davis, I. W., Arendall, W. B., 3rd, de Bakker, P. I., Word, J. M.,
Prisant, M. G., Richardson, J. S., and Richardson, D. C. (2003) Structure
validation by C␣ geometry: ␾, ␺, and C␤ deviation. Proteins 50, 437–450
6
8. Sela, D. A., and Mills, D. A. (2010) Nursing our microbiota: molecular
linkages between bifidobacteria and milk oligosaccharides. Trends Micro-
1
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