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Configured for the Human Gut Microbiota: Molecular Mechanisms

of Dietary β‑Glucan Utilization
Benedikt Golisch,⊥ Zhenhuan Lei,⊥ Kazune Tamura, and Harry Brumer*

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ABSTRACT: The β-glucans are a disparate group of structurally diverse polysaccharides, whose members are widespread in human
diets as components of the cell walls of plants, algae, and fungi (including yeasts), and as bacterial exopolysaccharides. Individual β-
glucans from these sources have long been associated with positive effects on human health through metabolic and immunological
effects. Remarkably, the β-configured glucosidic linkages that define these polysaccharides render them inaccessible to the limited
repertoire of digestive enzymes encoded by the human genome. As a result, the various β-glucans become fodder for the human gut
microbiota (HGM) in the lower gastrointestinal tract, where they influence community composition and metabolic output, including
fermentation to short chain fatty acids (SCFAs). Only recently, however, have the specific molecular systems that enable the
utilization of β-glucans by select members of the HGM been fully elucidated by combined genetic, biochemical, and structural
biological approaches. In the context of β-glucan structures and their effects on human nutrition and health, we summarize here the
functional characterization of individual polysaccharide utilization loci (PULs) responsible for the saccharification of mixed-linkage
β(1→3)/β(1→4)-glucans, β(1→6)-glucans, β(1→3)-glucans, β(1→2)-glucans, and xyloglucans in symbiotic human gut bacteria.
These exemplar PULs serve as well-defined biomarkers for the prediction of β-glucan metabolic capability in individual bacterial taxa
and across the global human population.

■ INTRODUCTION
Glucose is the fundamental monosaccharide building block from
humans (and other mammals), because our genomes do not
encode the required β-glucanases. What a difference a bond
makes!
photosynthesis that is polymerized into plant energy storage and
In fact, the β-glucans comprise a disparate group of
cell wall polysaccharides via two possible anomeric config-
polysaccharides with varied backbone linkages and side chain
urations, alpha and beta.1,2 Of these, α-glucans (i.e., starch,
composition. The inherent recalcitrance of β-glucans to human
comprising amylose and amylopectin) in common fruits and
digestive enzymes makes them fodder for the human gut
vegetables, especially tubers and cereal grains, are readily
microbiota (HGM), particularly in the lower gastrointestinal
depolymerized by human digestive enzymes after cooking and
tract where they are deconstructed and fermented along with
thus constitute a major, rapidly accessible energy source in diets
other components of “dietary fiber”.7−9 We are thus holobionts,10
worldwide.3 On the other hand, our genomes do not equip us
with the enzymes necessary to digest the diverse β-glucans,
which are likewise common in our diets as structural Special Issue: Chemical Glycobiology
components of plant and fungal (including yeast) cell walls.4,5 Received: July 20, 2021
This dichotomy is best exemplified by the linear homopolymers Accepted: October 5, 2021
amylose (α(1→4)-glucan) and cellulose (β(1→4)-glucan, Published: October 28, 2021
Figure 1): Amylose is digestible by human amylases and α-
glucosidases,6 whereas cellulose is inherently undigestible by

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Figure 1. Representative structures of common dietary α- and β-glucans. Monosaccharide residues are represented using the Consortium for
Functional Glycomics symbol nomenclature,34 as summarized in the key.

who are critically dependent on our resident microbes for the
metabolism of β-glucans and other nonstarch polysaccharides
(NSPs). One on hand, this relationship has long been
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appreciated by microbiologists and nutritionists.8,11−20 On the
other, recent years have witnessed the elucidation of highly
specialized molecular systems dedicated to the recognition,
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hydrolysis, and uptake of β-glucans by individual members of the
HGM, which underpins the role of these polysaccharides in
nutrition and health.21
■ A PRIMER ON DIETARY β-GLUCAN STRUCTURES
The β-glucans are, by definition, polysaccharides comprising a
backbone of D-glucopyranosyl residues linked via β-glycosidic
bonds.22,23 β-glucans are widespread in nature, from the
extracellular polysaccharides of bacteria24 to the cell walls of
fungi,25,26 algae and seaweed,27 and terrestrial plants,2 all of
which may constitute dietary sources for humans. The inherent
diversity of potential glycosidic linkages, branching patterns, and
degrees of polymerization28 enables a wide variation in β-glucan
structures among sources. In addition, environmental factors
during biosynthesis,25 as well as extraction and purification
methods, can influence β-glucan molar mass and substitution
patterns.29 In turn, these molecular properties, as well as the
organization of β-glucans into higher-order composite struc-
tures in cell walls, will influence their accessibility to the HGM.30
The structural intricacies and diverse sources of β-glucans
have been revealed through a rich history of study.29,31−33
Despite this, the exact nature of these polysaccharides in specific
contexts is often obfuscated by the use of the generic term “β-
glucan” in scientific articles, popular media, and product labels.
In the following sections, we provide a primer on representative
β-glucan structures of particular relevance to the HGM.
β(1→4)-Glucans. Cellulose (β(1→4)-glucan, Figure 1) is
the primary load-bearing component of all terrestrial plant cell
walls, as well as many algae, and is therefore abundant in a well-
balanced diet. Additionally, some bacteria, especially Komaga-
taeibacter xylinus (previously Acetobacter xylinum, Gluconaceto-
bacter xylinus), produce cellulose as an extracellular poly-
saccharide,35 which forms the basis of the gelatinous dessert nata
de coco. The linear, unbranched structure of cellulose enables
ordered packing of cellulose chains into semicrystalline
structures,2 which are essentially undigestible by the HGM.
On the other hand, the incorporation of branching along the
β(1→4)-glucan chain by some organisms disfavors ordered
packing, yielding soluble polysaccharides. For example, xanthan
(Figure 1), a complex extracellular polysaccharide produced by
the bacterium Xanthomonas campestris, is a widely used food
rheology modifier (E415).36 Likewise, the xyloglucans (XyGs)
comprise a family of ubiquitous fruit, vegetable, and grain cell
wall matrix polysaccharides, the amorphous structures of which
result from frequent and extensive branching of the β(1→4)-
glucan backbone via α(1→6)-xylosyl and other residues (Figure
1).2,8,37
β(1→3)/β(1→4)-Glucans (Mixed-Linkage β-Glucans). A
further way in which nature avoids the self-assembly of β(1→4)-
glucans into insoluble, self-assembled structures is through the
introduction of alternate linkages in the backbone. Thus,
“mixed-linkage β-glucans (MLGs)”, viz. β(1→3)/β(1→4)-
glucans, have a kinked structure composed of randomly
distributed β(1→4)-linked cellotriosyl (Glc3) and cellotetraosyl
(Glc4) units that are linked via β(1→3)-glycosidic bonds. MLGs
are found particularly in the starchy endosperm and aleurone
layer of common dietary cereals, in which the composition of
β(1→3)- and β(1→4)-glycosidic bonds and random block
structure differs between species. The following Glc3/Glc4 ratios
are observed: wheat, 3.0−4.5; barley, 1.8−3.5; rye, 1.9−3.0; and
oat, 1.5−2.3.38,39 Lichens produce the eponymous MLG
lichenan, which is predominantly composed of Glc3 units.40
Although this MLG is more relevant to the diet of reindeer than
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humans,41 lichenan finds widespread use as a commercially
available substrate for glycosidase enzymology.
β(1→3)-Glucans. Polysaccharides comprising exclusively
β(1→3)-linked poly(glucosyl) backbones are widely distributed
in nature in both linear and branched forms. Bacteria, yeast,
microalgae, and terrestrial plants are known to produce linear
β(1→3)-glucans (Figure 1), which fulfill different biological
roles and are historically referred to by source-specific names, for
example, curdlan (Agrobacterium sp.), zymosan (Saccharomyces
cerevisiae), paramylon (Euglena gracilis), and callose (Viridi-
plantae).42−45 Of these, curdlan is an approved food gelling
agent (E424),46 while callose is produced in limited amounts by
plants, including food crops, during particular developmental
stages and in response to wounding or pathogen attack.2,45
Branched β(1→3)-glucans are widespread in fungi and algae,
in which they are key components of the cell wall. Both between
and within these taxa, β(1→3)-glucans are distinguished by the
frequency and structures of the pendant side chains. In general,
β-glucans from fungi have comb-like structures comprising a
β(1→3)-linked backbone that is branched by β-(1→6)-linked
glucosyl side chains.47,48
Pachyman, from the fungus Poria cocos, represents perhaps the
most minimalist example, with a branching ratio of single β(1→
6)-glucosyl residues of ca. 1:60.49,50 Other well-known examples
of branched β(1→3)-glucans include lentinan from Lentinula
edodes (shiitake mushroom), grifolan from Grifola f rondosa
(maitake), pleuran from Pleurotus ostreatus (oyster mushroom),
schizophyllan from Schizophyllum commune (split gill), and
scleroglucan from Sclerotium species.25,42,51,52 The first four of
these originate in fungi that are consumed by humans in
different regions of the world and are likely to be generally
representative of β-glucans in other edible fungi.53 All five
contain single β(1→6)-Glc branches and are distinguished by
small differences in the degree of branching,51 typically one
branch for every three to four backbone residues.54−58 Yeasts
contain more elaborate branched β(1→3)-glucans in their cell
walls. In Saccharomyces cerevisiae, the most widely recognized
β(1→3)-glucan has long β(1→3)-Glc side chains extended
from β(1→6) branch points59 (Figure 1). An additional β(1→
3)-glucan component has side chains of at least four β(1→6)-
Glc residues.60,61
β(1→3)-glucans from brown algae are known as laminarins,
the β(1→6)-branched structures of which are species dependent
(Figure 1). Laminarins from the genus Laminaria invariably
have short branches,33 as exemplified by L. digitata laminarin,
which contains single β(1→6)-glucosyl branches in a ratio of 1:7
versus backbone residues.33,62 In contrast, the laminarin from
Eisenia bicyclis exhibits a higher molecular weight and a 2:3 ratio
of β(1→6) to β(1→3) linkages.62 Notably, the side chains of
Eisenia bicyclis laminarin comprise up to three β(1→6)-glucosyl
residues per branch.42 Other unique β(1→3)-glucans include
the capsular polysaccharide of the bacterium Streptococcus
pneumoniae Type 37, which contains Glcβ(1→2)−Glcβ(1→6)
side chains,63 and the cyclic, branched mixed-linkage β(1→3)/
β(1→6)-glucan from Bradyrhizobium japonicum.64
β(1→6)-Glucans. Linear β(1→6)-glucans are exemplified
by pustulan from the lichen Lasallia pustulata, which serves as a
commercial source of this polysaccharide (Figure 1). Linear
β(1→6)-glucan has also been isolated from the lichen
Umbilicaria caroliniana40 and the fungus Guignardia citricarpa.65
Dietary yeasts such as Saccharomyces cerevisiae also produce a
linear β(1→6)-glucan, which is cross-linked with other cell wall
components.59,66 Other fungi also contain branched β(1→6)-
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glucans. For example, Agaricus blazei (almond mushroom) and
an edible hybrid of Pleurotus f lorida and Lentinula edodes contain
β(1→6)-glucans with single β(1→3)-linked glucosyl branches
every three to four backbone residues.67,68
β(1→2)-Glucans. Although β(1→4)-, β(1→3)-, and β(1→
6)-glucans are widely distributed in nature and found in the
human diet, β(1→2)-glucans are rare. Thus far, only cyclic
β(1→2)-glucans comprising ca. 20 residues are known from
bacteria, where they function as osmolytes and virulence
factors69−71 (Figure 1).
■ THE β-GLUCAN/HGM NEXUS
The United Nations Food and Agriculture Organization (FAO)
estimates that over 871 million tonnes of fruits, 1.1 billion
tonnes of vegetables, and 2.9 billion tonnes of cereals were
produced worldwide in 2018.72 These agricultural products are
indispensable parts of a well-balanced diet and are thus regular
sources of β-glucans. Dicotyledonous plants, which include most
fruits and vegetables, are abundant in cellulose and xyloglucans,
the latter of which can comprise up to one-fourth of the dry
weight of the cell wall.2,8 Monocotyledonous plants of the
taxonomic Family Poaceae, which includes cereal crops, are
excellent sources of MLGs,2 which comprise nearly three-
quarters of the soluble, nonstarch glycan content of the
endosperm.8,73 Although they are not a staple per se, the fungi
(e.g., edible mushrooms and baker’s yeast) are regular dietary
sources of β(1→3)- and β(1→6)-glucans, which constitute up
to 85% of cell walls.33 Not least, bacterial β-glucans, such as
curdlan (E424) and xanthan (E415), are widely used in food
applications due to their physical−chemical effects on
thickening, whippability, emulsification, and gelation. 29
Although it is difficult to estimate the total mass of β-glucans
consumed worldwide due to widely varying diets across cultures
and regions, national health agencies generally recommend a
daily intake by adults of 20−40 g of total dietary fiber. Moreover,
daily consumption of 1−4 g of cereal (e.g., oat or barley) MLGs
is associated with reduced circulating cholesterol levels and
mitigation of rapid increases in postprandial blood glucose
levels.74,75
The diverse β-glucans in our diet resist digestion in the upper
gastrointestinal tract and hence transit to the large intestine,
where they can be partially or completely metabolized by the
HGM.76 The fermentation of complex carbohydrates such as β-
glucans by the HGM directly affects our nutrition and health by
generating short-chain fatty acids (SCFAs) that enter the
circulatory system and contribute up to 10% of our daily caloric
intake.18−20 Butyrate, in particular, is the primary energy source
of the colonic endothelium and is critical to maintaining a
healthy gut barrier, including the prevention of cancer.12,17 β-
glucans and their partial hydrolysis products may themselves
mediate physiological effects,77 including immune system
stimulation, through direct interaction with cell-surface
receptors.78−81 Furthermore, dietary β-glucans can modulate
the composition and metabolism of the HGM to quench
reactive oxygen species (ROS) and promote carcinogen
egestion.47
The HGM is a complex and dynamic ecosystem comprising
trillions of cells and diverse taxa, the composition of which
fluctuates depending upon daily dietary composition and other
factors.82−87 The HGM is predominantly comprised of bacteria
from the phyla Bacteroidetes (e.g., Bacteroides, Prevotella) and
Firmicutes (e.g., Clostridium, Roseburia, Lactobacillus), which
constitute ca. 90% of taxa, together with lesser amounts of
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Actinobacteria (e.g. Bif idobacterium), Proteobacteria (e.g.,
Escherichia), Fusobacteria (Fusobacterium), and Verrucomicro-
bia (Akkermansia).88 Due to their indigestibility by human
digestive enzymes, the β-glucans comprise “prebiotics” that can
modulate the growth and activity of beneficial gut bacteria.11
Thus, there is a long history of fundamental microbiological
studies to identify key β-glucan-utilizing taxa, which predates
more recent innovations in human gut (meta)genomics.89−93
β(1→4)-Glucans. Cellulose breakdown in the human gut
has been studied since the 19th century.13 In the early 20th
century, the disappearance of over 50% of cellulose extracted
from carrots and cabbage in the stool of subjects implicated
colonic microorganisms in cellulose degradation, although
notably, purified cellulose remained indigestible.94,95 Subse-
quently in the late 20th century, individual cellulolytic bacterial
strains were successfully isolated from human feces, including
Ruminococcus, Clostridium, Eubacterium, and Enterococcus
species.14,96−99 More recently, cellulolytic Bacteroides species
have been identified from the human gut.100 The effects of
amorphous plant cell wall matrix glycans, for example,
xyloglucan, in facilitating cellulose degradation by human fecal
samples has also been recently demonstrated.101
Considering the branched dietary β(1→4)-glucans, the ability
of Bacteroides ovatus, Bif idobacterium infantis, and several
Clostridium species, as well as crude human fecal slurries, to
degrade xyloglucan polysaccharide has been long known.102
More recently, the ability of several strains of Prevotella copri
(phylum Bacteroidetes) and Ruminococcus bicirculans (phylum
Firmicutes) to grow on xyloglucan has been demonstra-
ted.103,104 A wider range of Bif idobacterium species are able to
ferment xyloglucan oligosaccharides,102,105 which might be
liberated by primary degraders, such as the Bacteroidetes. It has
been recently shown that xyloglucans exert a significant impact
on human fecal community structures in vitro, favoring the
expansion of Bacteroides,106,107 which are known to possess
specific xyloglucan utilization loci (vide infra).108 Apart from
their use as a food gelling agent, xyloglucans have been explored
to resolve intestinal maladies.109,110 Although little is presently
known about the interaction of xanthan with the HGM, exciting
new work has revealed a keystone Ruminococcaceae and a cross-
feeding Bacteroides that can utilize xanthan and its hydrolysis
products, respectively.111
β(1→3)/β(1→4)-Glucans. β-glucans from cereals, that is,
MLGs, have been widely advertised as health-promoting by the
food industry, due to cholesterol lowering properties112,113 and
beneficial effects on postprandial glucose levels and insulin
response.114 The cholesterol-lowering effect is caused primarily
by the viscosity of β-glucan and the entrapment of bile acids in
the upper gastrointestinal tract. The resulting reduction triggers
an increase in bile acid biosynthesis, which consumes
cholesterol.115,116 The HGM also contributes to the cholester-
ol-lowering effect of MLGs through the production of
propionate; an increased propionate-to-acetate ratio in the
peripheral serum reduces cholesterol biosynthesis.117
The viscosity of MLGs also modulates postprandial glucose
levels by enclosing the food bolus in a thick layer, with
corresponding implications for hyperglycemia and type 2
diabetes.15,118,119 On one hand, this bulk physical phenomenon
results in less mixing between food and enzymes in the
stomach.120 On the other, it slows down diffusion and
adsorption of glucose in the intestine, leading to a more
constant uptake. These phenomena extend the feeling of
satiety.121 In addition, SCFA production by the HGM
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Figure 2. Glucan-specific polysaccharide utilization loci. (a) Genetic arrangement of exemplar Bacteroides β-glucan utilization loci (β-GULs) with
reference to the archetypal starch (α-glucan) utilization system (Sus). Boundary gene locus tags are indicated under each PUL. Proteins with
homologous functions are colored identically: blue, glycoside hydrolase (GH); green, cell-surface glycan-binding protein (SGBP); orange, TonB-
dependent transporter (TBDT); purple, sensor-regulator (SusR-like or hybrid-two component system (HTCS)). (b) Cellular organization of β-GUL-
encoded proteins that hydrolyze β-glucan homopolysaccharides, colored as in panel a.

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Figure 3. Cellular organization and structural biology of the XyGUL-encoded proteins that saccharify the heteropolysaccharide xyloglucan. The
XyGUL is shown in Figure 2a. GH and SGBP structures are represented by the following PDB IDs: BoGH5 (PDB ID 3ZMR108), BoGH9 (PDB ID
6DHT171), BoGH31 (PDB IDs 5JOU and 5JOV172), BoGH3B (PDB ID 5JP0172), BoGH43A (PDB IDs 5JOW, 5JOX, and 5JOY172), and BoGH43B
(PDB ID 5JOZ172).

modulates postprandial glucose levels by increasing the
production of peptide YY in intestinal cells, which is involved
in the regulation of appetite and insulin mediated glucose uptake
in muscle and adipose tissue.122 However, dietary MLG only
shows a small impact on long-term effects such as body
weight.123
Motivated by the health-promoting effects of β(1→3)/β(1→
4)-glucans, the fermentation of cereal MLGs by endogenous
members of the HGM has received significant attention since
the late 1900s. For example, in vitro analyses revealed that
selected Bacteroidetes (Bacteroides and Prevotella) and Firmi-
cutes (Ruminococcus and Coprococcus) isolates grew on barley
MLG (polysaccharide), whereas Lactobacillus, Bif idobacterium,
or Enterococcus species or E. coli strains did not.103,104,124,125 On
the other hand, there is evidence to suggest that Lactobacillus
and Bif idobacterium species can utilize MLG hydrolysis
products,126 which likely accounts for growth observed in
mixed cultures127 and changes in the ratio of Bacteroidetes to
Firmicutes.128,129 Such effects may arise from “sharing” of
“public goods” in the complex ecosystem of the HGM.130
Surveys of individual Bacteroides species have indicated that
growth on MLG is not ubiquitous across the genus but is
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determined by the presence of specific polysaccharide utilization
loci in genomes (vide infra).131−133
β(1→3)-Glucans. Whereas cereal MLGs are generally
associated with metabolic health,119,134 fungal β(1→3)-glucans
are more commonly implicated in effects on the immune
system.51,135 Research in this area can be traced back to the
beginning of the 20th century, when the immunomodulating
function of yeast cells was discovered and later linked to the
crude cell wall preparation zymosan, which contains β(1→3)-
glucans.26,136 Over time, it became apparent that branched
β(1→3)[β(1→6)]-glucans also play important roles in immune
responses directly. In particular, M cells of Peyer’s patches in the
small intestine can mediate the transport of fungal β-glucans
through the epithelium,47 followed by binding to different
receptors on phagocytic and cytotoxic innate immune cells,
which in turn triggers signaling pathways leading to immune
responses.135 Prominent receptors such as dectin-1 and
complement receptor 3 (CR3) are able to bind β(1→
3)[β(1→6)]-glucans and thereby enhance the release of
proinflammatory cytokines.79,137 Furthermore, β(1→3)[β(1→
6)]-glucans are able to induce the expression of receptors such as
Toll-like receptor 2 and can also shift the balance between T
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helper 1 (Th1) cells and T helper 2 (Th2) cells toward Th1,
which is related to allergic responses.47,138
Fungal β(1→3)[β(1→6)]-glucans, including lentinan, grifo-
lan, scleroglucan, and schizophyllan, have also received attention
as potential antitumor compounds.17 Here, fungal β-glucans are
first internalized by macrophages and transported to the bone
marrow where they are degraded into oligosaccharides, which
bind to the CR3 receptor of neutrophils. The resulting activated
granulocytes are able to kill iC3b-coated tumor cells.135,139,140
Thus, fungal β(1→3)[β(1→6)]-glucans play an important role
in the regulation of lymphocyte, neutrophil, and natural killer
cells in the innate immune system.139
The ability of bacterial species in the HGM to utilize β(1→3)-
glucans was defined through pioneering work by Abigail Salyers
and co-workers in the late 1970s.141 Nine of 11 Bacteroides
species, represented by ca. 200 strains in total, fermented
laminarin (and many terrestrial polysaccharides).142 In contrast,
a survey of 154 strains from 22 species of human colonic
bacteria, including Bif idobacterium, Peptostreptococcus, Lactoba-
cillus, Ruminococcus, Coprococcus, Eubacterium, and Fusobacte-
rium species, revealed no laminarin fermenters, except for a
single Peptostreptococcus productus strain.142 More recently, the
ability of Lactobacillus and Bif idobacterium species to utilize
laminarin for growth has been demonstrated.143 Notably,
growth of Bacteroides species on laminarin was found to induce
specifically β(1→3)-glucanase activity,144 portending the
discovery of β(1→3)-glucan-specific polysaccharide utilization
loci in select Bacteroides and other Bacteroidetes (vide infra).133
These results collectively underpin the interest in using
laminarin as a prebiotic.145
■
MOLECULAR SYSTEMS FOR β-GLUCAN
UTILIZATION
Despite the long-standing appreciation that the HGM is able to
saccharify β-glucans to glucose (and in the case of xyloglucan,
other monosaccharides; Figure 1) to feed primary metabolism,
detailed molecular insight into this process has only recently
been achieved through combined microbiological, biochemical,
and structural biological studies on β-glucan-specific poly-
saccharide utilization loci (PULs)146 in exemplar Bacteroides
species. In this context, it is worth noting that Bacteroidetes are
particularly prodigious utilizers of complex glycans, with
numbers of glycoside hydrolases (GHs) and polysaccharide
lyases (PLs) collectively numbering in the hundreds in some
species, which far exceeds other bacteria in the HGM.4 Despite
the organization of many of these enzymes into families in the
carbohydrate-active enzymes (CAZy) classification, sequence
analysis alone is often insufficient to confidently assign
molecular function at the level of substrate and product
specificity.147,148 Hence, detailed structure−function character-
ization is necessitated.
Canonical PULs are multigene clusters found in Bacteroidetes
that encode molecular systems of carbohydrate-binding,
-cleaving, and -transporting proteins, the expression of which
is under the control of a glycan-specific sensor or regulator that
recognizes intermediate breakdown products.131,146,149−151 The
archetypal PUL encodes the starch (α-glucan) utilization system
elucidated by Abigail Salyers and colleagues in the late 1980s and
early 1990s152 (comprising susABCDEFG/susR, locus tags
BT3698−3705, Figure 2a). Hallmarks of complete PULs are a
core pair of SusC/SusD homologues constituting a TonB-
dependent transporter (TBDT) and cell-surface glycan-binding
protein (SGBP) complex153 and a cohort of GHs and PLs, the
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number of which scales with the linkage complexity of the
substrate154,155 (Figure 2, Figure 3). The general steps of PUL-
mediated glycan saccharification are as follows:
(1) substrate binding by one or more SGBPs at the bacterial
cell surface,
(2) polysaccharide backbone cleavage by cell-surface anch-
ored endo-acting enzymes,
(3) oligosaccharide import into the periplasm through the
TBDT complex,
(4) ultimate conversion to monosaccharides (sometimes
disaccharides) for transport into the cytosol, through
the concerted action of one or more exo-acting enzymes.
The following sections will describe notable molecular
features of PULs responsible for the utilization of β(1→3)/
β(1→4)-glucans, β(1→6)-glucans, β(1→3)-glucans, β(1→2)-
glucans, and xyloglucan in the context of this paradigm. We will
specifically highlight the crucial role that the combined use of
specific substrates and inhibitors, analytical biochemistry, and
structural biology has played in detailed SGBP and GH
structure−function studies.
Mixed-Linkage Glucan Utilization Loci. The mixed-
linkage β-glucan utilization locus (MLGUL) of B. ovatus (locus
tags BACOVA_02740−02745) encodes, in order, a hybrid two-
component sensor (HTCS), a glycoside hydrolase family 16
(GH16) mixed-linkage endo-β-glucanase (MLGase), a TBDT/
SGBP (SusC/SusD-homologue) pair, an additional sequence-
divergent SGBP, and a glycoside hydrolase family 3 (GH3) β-
glucosidase (Figure 2a).132,156 Initial reverse genetics (gene
deletion) experiments established that this locus was exclusively
responsible for the growth of B. ovatus on MLGs. Subsequent
biochemical analysis revealed that the vanguard BoGH16MLG
(PDB ID 5NBO) hydrolyzes MLGs with much higher specificity
than the β(1→3)-glucans curdlan, laminarin, or yeast β-glucan.
Product analysis demonstrated that this endo-acting enzyme
hydrolyzes MLGs into only two distinct oligosaccharides,
G4G3G and G4G4G3G (where “G” represents a β-glucosyl
residue and the number indicates the linkage regiochemistry),
which reflects the specific hydrolysis of β(1→4) linkages
immediately preceding β(1→3)-linked glucosyl residues.
Further substrate analysis with chromogenic oligosaccharides
showed that a β(1→3) linkage spanning enzyme subsites −2
and −1 is necessary for catalysis, whereas β(1→4)-Glc binding
in the −3 and −4 subsites contributes to substrate recognition
(see ref 157 for an explanation of sugar-binding subsites). These
kinetic data were rationalized by the tertiary structures of
BoGH16MLG in complex with the product G4G4G3G and
structurally related covalent inhibitors (PDB IDs 5NBP, 6VHO,
7KR6), which illustrated the existence of three well-defined
negative subsites and a fourth weakly interacting negative subsite
in the active-site cleft.132,158 BoGH16MLG possessed a β-jellyroll
fold characteristic of the family147 and was outer-membrane-
anchored by N-terminal lipidation.
Biochemical analysis showed that the exo-β-glucosidase
GH3MLG can completely degrade the BoGH16MLG-derived
oligosaccharides, G4G4G3G and G4G3G, into glucose by
sequential hydrolysis from the nonreducing end, after import
into the periplasm (Figure 2b). Initial-rate kinetics using series
of gluco-oligosaccharides with different linkages and degrees of
polymerization demonstrated the preference of BoGH3MLG for
β(1→3)- and β(1→4)-linkages over β(1→6). Furthermore,
BoGH3MLG hydrolyzes oligosaccharides longer than trisacchar-
ides with similar catalytic efficiencies due to the presence of one
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negative subsite and two positive subsites that contribute to
catalysis. In the absence of an experimental tertiary structure,
homology modeling indicated that the (β/α)8-barrel (triose
phosphate isomerase (TIM)-barrel) and the α/β sandwich
domain define a narrow active-site cleft typical of GH3
members.159
Detailed analysis of substrate recognition by the SGBP-A
(SusD homologue) and SGBP-B, using pull-down assays,
affinity gel electrophoresis, and isothermal titration calorimetry,
revealed particular affinities of both proteins for mixed-linkage
glucans over all β(1→4)-glucans (e.g., cellulose, xyloglucan).
Commensurate with roles in recognition of MLG polysacchar-
ide chains at the cell surface, both SGBPs bound longer
oligosaccharides, such as the hexasaccharide G4G3G4G4G3G
and the heptasaccharide G4G4G3G4G4G3G but not the limit-
digest products of BoGH16MLG, G4G3G and G4G4G3G.156
The SGBP-A (PDB IDs 6E60, 6DMF, 6E61) has a tertiary
structure based upon four tetratricopeptide repeat (TPR) that is
typical of SusD homologues, whereas the SGBP-B (PDB IDs
6E57, 6E9B) has an extended, multimodular domain structure
that presents the binding site distal to the membrane-anchored
N-terminus. Reverse genetics demonstrated that SGBP-A has an
irreplaceable function for the growth of B. ovatus on MLGs and
therefore plays a key role in carbohydrate uptake by the TBDT.
In contrast, the SGBP-B was dispensable for growth but
increased the lag phase, suggesting an important complementary
role in MLG recognition.156
Systems-based approaches such as those described above,
which incorporate microbiological, biochemical, and structural
data, underpin definition of the MLGUL as a reference to
identify homologous PULs in other Bacteroidetes, for example,
Bacteroides, Prevotella, Dysgonomonas, and Xylanibacter132
Furthermore, these PULs serve as valuable molecular markers
for the predictive analysis of specific polysaccharide utilization in
bacterial genomes and metagenomes132 (see ref 103 for a recent
survey of a large number of Prevotella copri isolates). For
example, screening of 121 Bacteroides strains revealed that only 7
were able to utilize MLGs, and all possessed a syntenic MLGUL.
Subsequent analysis of publicly available human gut meta-
genome data revealed, in turn, that MLG utilization is
ubiquitous in human populations, commensurate with diverse
cereals forming a stable part of the diet.132 Although Firmicutes
systems are much less studied, individual CAZyme families (e.g.,
GH16, GH9, and GH5) have been implicated in MLG
utilization by ‘omics and biochemical approaches,104,125 which
may have similar predictive value.
β(1→6)-Glucan Utilization Loci. Like the MLGUL, the
β(1→6)-glucan utilization locus of B. thetaiotaomicron (locus
tags BT3309−3314 (“PUL1,6‑β‑glucan” in ref 66, here “β1,6-GUL”
for consistency160) represents another minimalist PUL compris-
ing only two GHs, viz. a GH30 and a GH3 member (Figure 2). A
syntenic PUL from B. ovatus was originally predicted to target
MLGs.131 However, exemplary work by Lowe, Gilbert, and
colleagues revealed instead that the B. thetaiotaomicron β1,6-
GUL is specifically upregulated during growth on pustulan and
yeast cell wall extract, which contains β(1→6)-glucans.66
Moreover, these authors demonstrated that the encoded
SGBPs and GHs were highly specific for β(1→6)-glucans and
the corresponding oligosaccharides. In total, the β1,6-GUL
encodes a SusR homologue, a SusC/SusD-like TBDT/SGBP
pair, a GH30 subfamily 3 (GH30_3) member, an additional
SGBP, and a GH3 member. The overall organization of this PUL
is notable in two ways. First, the auxiliary SGBP is not
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immediately downstream of the SusD-like SGBP, as is common
among most PULs. Second, the SusR-like regulator, which is
strictly conserved in syntenic β1,6-GULs,66 is comparatively rare
versus HTCS and ECF regulators in Bacteroides PULs; less than
10% of Bacteroidetes regulators are SusR-like.151,161
Commensurate with a role in initiating substrate capture, both
the SusD-like SGBP-A and the additional SGBP-B selectively
bind to β(1→6)-glucan and show no affinity toward β(1→3)-
nor β(1→4)-glucans by affinity gel electrophoresis analysis.
Isothermal titration calorimetry further showed that the SGBP-B
targets the polysaccharide with higher affinity than the SusD-like
SGBP-A and that both proteins are likely to present at least six
glycosyl-binding subsites.66 Thus, in the canonical model of
PUL-encoded systems (Figure 2b), SGBP-B likely provides
general affinity of the bacterium to the substrate, whereas SGBP-
A assists in feeding β(1→6)-glucan fragments generated by the
GH30_3 endo-glucanase into the TBDT through a “pedal-bin”
action.153
Indeed, BtGH30_3 (PDB IDs 5NGK, 5NGL66) was revealed
by kinetic analyses to be a highly specific endo-β(1→6)-
glucanase with activity on pustulan, but not on β(1→3)-glucan,
β(1→4)-glucan, nor β(1→6)-galactan. The BtGH30_3-cata-
lyzed hydrolysis of pustulan generated a distribution of
oligosaccharides of different chain lengths, typical of endo-acting
GHs and consistent with the observed release of these
oligosaccharides from the cell surface under nonlimiting growth
conditions. Further kinetic and product analyses using a range of
β(1→6)-gluco-oligosaccharides (Glc2 to Glc8) suggested that
BtGH30_3 requires three subsites, from −2 to +1, to be
occupied for catalytic activity; gentibiose (Glcβ(1→6)-Glc) was
not hydrolyzed by the enzyme and thus constituted the limit-
digest product in vitro. Solution of the tertiary structure of
BtGH30_3 (PDB ID 5NGK) revealed a classic (α/β)8 TIM
barrel fold typical of the family.162 A complex with the bespoke
inhibitor β-glucosyl-1,6-deoxynojirimycin (GlcDNJ; PDB ID
5NGL), suggested that the active-site cleft presented four
subsites, −2 to +2, versus the three subsites indicated by the
biochemical data. The authors explain this distinction by
suggesting that the oligosaccharides used for kinetic analysis
do not fully recapitulate the bent conformation of the native
polysaccharide, resulting in suboptimal occupation of the four
subsites in the deep U-shaped cleft of the enzyme.66
Final saccharification of β(1→6)-gluco-oligosaccharides in
the periplasm appears to be mediated by the exo-β-glucosidase
BtGH3, which was 30-fold more specific for β(1→6)-glycosidic
linkages than the corresponding β(1→3) and β(1→4) linkages.
The authors note that the gene encoding this β-glucosidase,
BT3314, was transcribed at a 10-fold lower level than the other
genes of the β1,6-GUL and further speculate that this may allow
the cognate oligosaccharide ligand of the SusR-like regulator to
maintain a significant steady-state level in the periplasm, thereby
sustaining upregulation.66 Because the commercially available
pustulan was used for all biochemical work, it is presently
unknown how BtGH30_3 and BtGH3 (and the other β1,6-GUL
components) accommodate the β(1→3) branches that would
be typically encountered on fungal cell wall β(1→6)-glucans. At
the same time, the non-negligible activity of BtGH3 toward the
β(1→6) linkage suggests that this enzyme may be sufficient to
fully degrade all incoming oligosaccharides from more complex
substrates.
In this context, it is interesting to note that yeast β(1→6)-
glucan is believed to be cross-linked to α-mannans and
mannoproteins in the cell wall, suggesting that the β1,6-GUL
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may synergize with α-mannan-specific PULs. Yet, a broad survey
of Bacteroidetes indicated that syntenic β1,6-GULs are more
widely distributed in a strain-specific manner than the α-mannan
PULs. Like the MLGUL, β1,6-GULs are found in human gut
resident Bacteroides, Prevotella, and Dysgonomonas strains, thus
indicating a widespread importance of dietary β(1→6)-glucan
metabolism.66
β(1→3)-Glucan Utilization Loci. Déjean et al. recently
presented the functional characterization of three homologous
β(1→3)-glucan utilization loci (β1,3-GULs) from B. uniformis
ATCC 8492 (locus tags BACUNI_01484−01490), B. f luxus
YIT 12057 (locus tags HMPREF9446_00611−00616), and B.
thetaiotaomicron NLAE-zl-H207 (locus tags
H207DRAFT_02223−02229), which revealed that the ability
of individual taxa to utilize different β(1→3)- and β(1→3)/
β(1→4)-glucans arises through a combination of the individual
specificities of the cognate SGBPs and cell-surface GHs.133 Each
of these β1,3-GULs contain a syntenic TBDT, SGBP-A (SusD
homologue), SGBP-B, and GH3 member as a core set of
conserved genes yet notably differ in the nature of the outer-
membrane endo-glucanase. Specifically, B. thetaiotaomicron
possesses a GH16 member, B. f luxus possesses a GH158
member, and B. uniformis possesses one of each (Figure 2a).
Detailed enzyme kinetic studies on BuGH16 and BtGH16
demonstrated that both possessed broad substrate specificity,
that is, the ability to hydrolyze L. digitata laminarin (LdLam,
which contains single β(1→6)-linked glucosyl branches on the
β(1→3)-glucan backbone, Figure 1), Saccharomyces cerevisiae
(yeast) β-glucan (yBG, which contains longer β(1→3)-linked
side chains, Figure 1), and barley MLG (bMLG, with a β(1→
3)/β(1→4)-linked backbone, Figure 1). In contrast, BuGH158
and Bf GH158 were highly specific for LdLam. Thus, despite
possessing an SGBP-A capable of binding both LdLam and yBG,
and an SGBP-B capable of binding LdLam, yBG, and bMLG, the
limited substrate range of Bf GH158 only permitted B. f luxus to
grow on the laminarin.133
In comparison, B. thetaiotaomicron could utilize both LdLam
and the more extensively branched yBG for growth, because
BtGH16 could hydrolyze both of these β(1→3)-glucans, which
were also bound by the cognate BtSGBP-A and BtSGBP-B. On
the other hand, B. thetaiotaomicron was incapable of growth on
bMLG, despite this being a substrate for BtGH16, because
neither BtSGBP-A nor BtSGBP-B recognized the β(1→3)/
β(1→4)-linked backbone. Finally, B. uniformis grew on all three
polysaccharides, viz. LdLam, yBG, and bMLG, due to the
combined specificities of BuGH16 (hydrolyzing LdLam, yBG,
and bMLG), BuGH158 (hydrolyzing LdLam), and BuSGBP-B
(binding LdLam, yBG, and bMLG). Notably, no binding affinity
for these polysaccharides was observed for BuSGBP-A, although
site-directed mutagenesis studies on other PUL systems have
demonstrated that a lack of substrate binding by SusD
homologues can be compensated by a functional SGBP-B.156,163
While the reader is referred to the original publication for a
detailed description of the biochemical analyses of all of the B.
uniformis, B. thetaiotaomicron, and B. fluxus GHs and SGBPs,
some points regarding the structure and function of BuGH16
and BuGH158 deserve special mention. Michaelis−Menten
kinetics analysis revealed that BuGH16 was most active toward
yBG and exhibited lower, similar activity against laminarins with
different branching frequency, as well as MLG. BuGH16
hydrolyzes the β(1→3)-glucans LdLam and yBG to glucose,
laminaribiose, and a branched trisaccharide, while bMLG is
completely converted into the oligosaccharides G4G3G and
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G4G4G3G. Concordant with this specificity, phylogenetic
analysis places BuGH16 in the “β-bulge laminarinase/MLGase”
clade, corresponding to GH16 subfamily 3.147 This subfamily
contains both laminarinases with secondary MLGase activity
and specific MLGases,132 which require a β(1→3)-linkage
between subsites −1 and −2 for substrate binding and catalysis,
yet are tolerant of diverse linkages and branching in other
subsites.133
In contrast to the broader substrate repertoire of BuGH16,
BuGH158 is a highly specific endo-β(1→3)-glucanase, with a
strong preference for limited backbone branching. Family
GH158 was recently discovered in 2019, and BuGH158 (PDB
ID 6PAL) serves as the first structurally and mechanistically
characterized example. The tertiary structure of BuGH158
revealed a (β/α)8 TIM barrel domain that places GH158 in the
large Clan GH-A and makes this the fourth family in GH-A
known to contain endo-β(1→3)-glucanases (along with GH17,
GH128, and GH148). Superposition and analysis of the active-
site cleft vis-à-vis other endo-β(1→3)-glucanases revealed a
pocket formed by aromatic residues adjacent to the position of
6-OH of the glucose unit in the −1 subsite, suggesting a binding
site for a single β(1→6)-linked glucosyl branching residue
typical of LdLam.133
Finally, Michaelis−Menten kinetics for a range of gluco-
oligosaccharides indicated that BuGH3 is a specific exo-β(1→
3)-glucosidase with a two-orders-of-magnitude higher kcat/Km
value for G3G disaccharide over G4G and G6G disaccharides.
Despite this specificity, BuGH3 has the ability to completely
hydrolyze into glucose all oligosaccharides produced by
BuGH16 and BuGH158 from MLG and laminarin. It is also
possible that other periplasmic glucosidases encoded outside of
the β1,3GUL assist with alternate linkage degradation.133
Using the β1,3GULs as markers of HGM metabolic potential,
Déjean et al. also surveyed publicly available gut metagenomes
from nearly 2500 adults on five different continents, viz. North
America, South America, Africa, Europe, and Asia. The
distribution of β1,3GULs was generally not restricted to any
particular geographic region or population, which likely reflects
the ubiquity of edible fungi and yeast fermentation products in
diets worldwide. Strikingly, however, β1,3GULs were not
detected in gut metagenomes of the indigenous Hadza and
Yanomami tribes, perhaps due to the prevalence of Prevotella
over Bacteroides in these populations.133
β(1→2)-Glucan Utilization Loci. The extent to which
members of the HGM may utilize β(1→2)-glucans has not been
explicitly studied. Intriguingly, “PUL61” from B. thetaiotaomi-
cron VPI-5482 (locus tags BT3566−3569) was first identified by
Martens et al. in 2008 as part of a genomic census, but at that
time, its substrate was unknown and difficult to predict based on
a limited knowledge of the GH complement (see Document S2,
Figure S5 in ref 164). PUL61 is now known to encode both a
GH3 member that is specific for β(1→2)-gluco-oligosacchar-
ides (PDB IDs 5XXL, 5XXM, 5XXN, 5XXO),165 and a GH144
member (Figure 2a).150 GH144 was recently established as a
family of endo-β(1→2)-glucanases following seminal biochem-
ical and structural work in 2017 on a Chitinophaga pinensis
homologue.166 Thus far, no Bacteroides homologues have been
biochemically characterized, although unliganded tertiary
structures of B. f ragilis (PDB ID 3EU8), B. uniformis (PDB ID
4GL3), and B. caccae (PDB ID 4QT9) have been deposited as
part of a structural genomics initiative.167 Biochemical and
structural characterization of a GH144 member from Para-
bacteroides distasonis revealed this enzyme to be an exo-sophoro-
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biohydrolase (i.e., releasing Glcβ(1→2)-Glc), the specificity of
which is dictated by the presence of a loop blocking one end of
the active site (PDB ID 5Z06).168 With the benefit of hindsight
and experimental data on orthologous GH144 members, it is
highly probable that PUL61 and related PULs in other taxa are
responsible for β(1→2)-glucan utilization in the HGM.
Xyloglucan Utilization Loci. The xyloglucans, represented
by (fucogalacto)xyloglucan, (arabinogalacto)xyloglucan, and
other variants, are demonstrably the structurally most complex
of the β-glucans (Figure 1). Correspondingly, xyloglucan
utilization loci (XyGULs) comprise a greater number of GHs
than other β-GUL to address the monosaccharide diversity in
these plant cell wall polysaccharides169 (Figure 2a). The
characterization of an exemplar XyGUL from B. ovatus (locus
tags Bacova_02644−02659) was reported in 2014.108 This
study utilized a combination of bacterial genetic, biochemical,
and structural biological approaches, which would be recapitu-
lated in subsequent PUL functional studies,170 including those
described above.
Foremost, genetic deletion of the XyGUL established its
unique requirement for growth on xyloglucan.108 Individual
deletion of both SGBPs the GH5 endo-xyloglucanase defined the
essential roles of these XyGUL gene products in capturing the
polysaccharide and initiating chain cleavage at the cell surface,
respectively.108,163 Curiously, an additional cell-surface endo-
glucanase, BoGH9, which is unique to B. ovatus among
homologous XyGULs, exhibited feeble endo-xyloglucanase
activity and was not required for growth on xyloglucan. A
subsequent study revealed that this enzyme is in fact more
specific for MLGs (including a requirement for calcium for
optimal activity) and may coordinate with the SGBPs for
substrate capture.108,171
Tauzin et al. specifically revealed the structural basis of the
SusD homologue (SGBP-A; PDB IDs 5E75, 5E76) and the
accessory SGBP (SGBP-B; PDB IDs 5E7G, 5E7H) in the
noncatalytic recognition of xyloglucan by B. ovatus (Figure
3).163 Both SGBPs exhibit specificity for XyG and XyG
oligosaccharides, while SGBP-A also exhibits limited affinity to
cellohexaose, glucomannan, and MLGs. Thus, both SGBPs bind
the β-glucan backbone residues, with increased affinity resulting
from XyG side-chain recognition. Oligosaccharide-complexed
structures of SGBP-A and SGBP-B rationalized these bio-
chemical observations. The SGBP-A possessed the canonical
SusD-fold, yet displayed an extended linear binding platform of
aromatic residues that accommodated seven Glc residues. In
contrast, SGBP-B had a distinct four-domain extended
architecture, which presented a single binding platform that
specifically accommodated five Glc resides on the C-terminal
domain. These features distinguish the SGBP-B from the
noncatalytic, multimodular amylose-binding proteins SusE and
SusF, which are highly sequence-divergent despite their similar
genomic organization within the Sus vis-à-vis the XyGUL.
Detailed reverse genetics experiments revealed that the presence
of the SGBP-A but not its XyG-binding ability was strictly
essential to enable growth on XyG, commensurate with a tight
association to the TBDT. Whereas the SGBP-B was
independently dispensable, its presence improved foraging of
shorter oligosaccharides, which might be released by other gut
bacteria.163
Detailed enzyme kinetics and product analysis of the GHs
encoded by the XyGUL enabled a complete pathway for the
saccharification of (arabinogalacto)xyloglucan to be established
in B. ovatus. Operating at the cell surface, the vanguard endo-
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xyloglucanase BoGH5, possibly with the assistance of BoGH9,
hydrolyzes the XyG backbone specifically at unbranched Glc
units to generate shorter fragments. Following import into the
periplasm by the TBDT/SGBP-A (SusC/SusD-homologue)
complex, the six exo-acting GHs deconstruct the resulting
oligosaccharides in a concerted manner (Figure 3): Two GH43
α-L-arabinofuranosidases and one GH2 β-galactosidase remove
side chain capping Arafα(1→2) and Galpβ(1→2) residues,
respectively, to free underlying Xylpα(1→6) residues. Hydrol-
ysis of the side chain Xyl residue at the nonreducing end by the
GH31 α-xylosidase frees the corresponding backbone Glc
residue for removal by the GH3 β-glucosidases. This α-
xylosidase/β-glucosidase cycle repeats to fully hydrolyze the
xyloglucan fragment.108
In addition to the novel structures of the SGBPs described
above, structural biology of the XyGUL revealed specific insight
into substrate specificity of the GHs through the solution of free
and complexed structures of BoGH5 (PDB ID 3ZMR), BoGH9
(PDB ID 6DHT), BoGH31 (PDB IDs 5JOU, 5JOV), BoGH3B
(PDB ID 5JP0), BoGH43A (PDB IDs 5JOW, 5JOX, 5JOY), and
BoGH43B (PDB ID 5JOZ). While a full description of these
structures is beyond the scope of this review, particularly notable
features include the following:
• The endo-xyloglucanase BoGH5 possesses an N-terminal
PFAM PF13004 domain, which has previously been
referred to as a “BACON (Bacteroidetes-associated
carbohydrate-binding),” although no experimental evi-
dence for a carbohydrate-binding function has been
observed in this or other PF13004-containing pro-
teins.108,133 The BoGH5 structure represents the first
tertiary structure of a PF13004 domain, and the conserved
N-terminal positioning and linker flexibility suggest that
these domains act as spacers to distance the correspond-
ing GH module from the cell surface.108
• The endo-(xylo)glucanase BoGH9 has an N-terminal Ig-
like fold domain and an (α/α)6 catalytic domain with a
comparatively narrow active-site cleft that is typical of
GH9 endo-glucanases (cellulases). Superposition with
other GH−oligosaccharide complexes, including
BoGH5:XXXG, suggests that the binding of xyloglucan
is likely to be disfavored due to active-side clashes,
whereas unbranched MLG is better accommodated,
commensurate with kinetic analyses.171
• The tertiary structures of the α-L-arabinofuranosidases
BoGH43A and BoGH43B, which share limited sequence
identity (41%), were typical of GH43, comprising two
domains: a 5-bladed β-propeller domain embracing the
catalytic active site at the N-terminus and a β-sandwich
domain at the C-terminus. A complex of BoGH43A with
the bespoke inhibitor 1,4-dideoxy-1,4-imino-L-arabinitol
(“arabino-deoxynojirimycin”, AraDNJ) revealed key
substrate-binding interactions in the active-site pocket
and the positions of the catalytic residues. BoGH43A was
also observed to have a wider active site pocket than
BoGH43B, which possibly explains the higher activity of
BoGH43A toward XyG oligosaccharide substrates.
Although the reason for the presence of two GH43 is
still unclear, the structural differences between active sites
may reflect tuning to as-yet unspecified side chains.172
• The α-xylosidase BoGH31A consists of a central (β/α)8
(TIM) barrel fold that is typical of GH31 catalytic
modules. Notably, a β-sandwich domain and a PA14
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domain at the N-terminus extend the active-site and
support the recognition of larger XyG oligosaccharide
substrates.172 Specifically, BoGH31 shows higher activity
toward XyG hepta- (“XXXG”, that is, Xyl3Glc4) and
nonasaccharides (“XLLG”, that is, Gal2Xyl3Glc4) than p-
nitrophenyl-α-xyloside, suggesting multiple substrate-
binding subsites.108
• The β-glucosidases BoGH3A and BoGH3B remove
glucose from nonreducing ends of XyG oligosaccharides
and cello-oligosaccharides. Although the β-glucosidase
BoGH3A resisted crystallization, the solution of the
BoGH3B tertiary structure in complex with product
(glucose) revealed three domains, including an N-
terminal (TIM) barrel-like domain, a central α/β
sandwich domain, and a C-terminal fibronectin type-III
(FN-III)-like domain.172 As for the GH43 paralogs in the
XyGUL, the reason for the prima facie catalytic
redundancy of this GH3 pair is currently unclear.
As described above for other β-glucan utilization loci,
functional analysis of the B. ovatus XyGUL enabled the
confident identification of homologous XyGULs in other
Bacteroidetes. A perfect concordance was observed between
the presence of a XyGUL and growth on xyloglucan in 70 strains
(representing 6 individual species) of over 290 Bacteroidetes
(primarily Bacteroides) strains surveyed. Thus, homologous
XyGULs serve as molecular markers to reveal XyG metabolic
capability in human metagenomes, in which it was found to be
essentially ubiquitous across global populations.108 Of note, the
GH43 α-L-arabinofuranosidases of the B. ovatus XyGUL suggest
specific adaptation to (arabinogalacto)xyloglucans found in
solanaceous fruits such as tomato, peppers, and eggplant. In
contrast, the presence of GH95 α-L-fucosidases in homologous
XyGULs extends the substrate range to include (fucogalacto)-
xyloglucans found generally in dicots, including most vegeta-
bles.169 Presently, Firmicutes systems for xyloglucan utilization
have not been extensively studied,104 but we anticipate that
future functional analysis work in this area will enable more
informed analyses of (meta)genomes.
■ CONCLUSION
The metabolism of β-glucans by the human gut microbiota is a
key contributor to our nutrition and health. The molecular
systems that address β-glucans in the HGM are as complex and
varied as the structures of the polysaccharides themselves. As
such, the fine structural analysis of dietary glycans and the
biochemical analysis of the proteins and enzymes responsible for
their metabolism are intrinsically linked.173 Although our focus
in this review has been on the metabolism of β-glucans in the
human diet, it should be understood that the molecular systems
described here will, in many cases, have parallels in diverse
monogastric animals and other complex ecosystems. For
example, homologous β1,3-GULs have been identified and
biochemically characterized in marine Bacteroidetes.62,174 And
while the currently characterized β-glucan-specific PULs serve as
exemplar representatives, further functional characterization of
homologues will undoubtedly be warranted, given the micro-
heterogenity observed in PUL content across
taxa.66,108,132,133,169 In the future, we also look forward to the
genetic, biochemical, and structural characterization of potential
β-glucan utilization systems in Firmicutes, Actinobacteria, and
other members of the HGM, which are currently unexplored at
this level of resolution.21 As our collective knowledge of such
2097
Reviews
systems grows, so too does our ability to use PULs as molecular
markers to predict the individual and combined metabolic
capacity of members of the HGM.87 We anticipate that this will
greatly enable microbiome-based “personalized medicine” to
improve human nutrition and prevent or ameliorate disease.
■ AUTHOR INFORMATION
Corresponding Author
Harry Brumer − Michael Smith Laboratories, Department of
Chemistry, Department of Biochemistry and Molecular
Biology, and Department of Botany, University of British
Columbia, Vancouver, British Columbia V6T 1Z4, Canada;
orcid.org/0000-0002-0101-862X; Email: brumer@
msl.ubc.ca
Authors
Benedikt Golisch − Michael Smith Laboratories and
Department of Chemistry, University of British Columbia,
Vancouver, British Columbia V6T 1Z4, Canada
Zhenhuan Lei − Michael Smith Laboratories and Department
of Chemistry, University of British Columbia, Vancouver,
British Columbia V6T 1Z4, Canada
Kazune Tamura − Michael Smith Laboratories and
Department of Biochemistry and Molecular Biology, University
of British Columbia, Vancouver, British Columbia V6T 1Z4,
Canada
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschembio.1c00563
Author Contributions
⊥
B.G. and Z.L. made equal contributions. H.B. and K.T.
conceived the overall structure of this review, which was drafted
by B.G. and Z.L. and revised with input from all of the authors.
Funding
We thank the Canadian Institutes of Health Research (CIHR,
Operating Grant MOP-142472) and the Natural Sciences and
Engineering Research Council of Canada (NSERC, Discovery
Grant RGPIN-2018-03892) for funding. B.G. and K.T. are
recipients of Four-Year Doctoral Fellowships from the
University of British Columbia.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank previous members of the Brumer lab and our
collaborators for their invaluable contributions to our collective
work on β-glucan metabolism in the microbiota.
■ KEYWORDS
Dietary fiber: The components of plant-based foods that are
not broken down by human digestive enzymes.
Nonstarch polysaccharides (NSP): Complex carbohydrates
with diverse structures that comprise the major component of
dietary fiber.
Complex carbohydrates: Oligosaccharides and polysacchar-
ides typically comprised of multiple monosaccharide residues
and linkages.
β-Glucans: A family of polysaccharides that are related by a
poly(glucosyl) backbone comprising β-anomeric linkages.
Individual β-glucans are distinguished by the regiochemistry
of the linkages joining two glucosyl residues in the chain.
https://doi.org/10.1021/acschembio.1c00563
ACS Chem. Biol. 2021, 16, 2087−2102
ACS Chemical Biology pubs.acs.org/acschemicalbiology
Human gut microbiota (HGM): The composite of all
microorganisms that residue in the human gastrointestinal
tract.
Polysaccharide utilization locus (PUL): Physically linked and
coordinately regulated bacterial genes that encode a suite of
enzymes and other proteins responsible for the uptake and
hydrolysis to monosaccharides of complex glycans.
Bacteroidetes: A phylum of Gram-negative bacteria, members
of which predominate in the HGM. PULs were originally
defined in Bacteroides species of this phylum.
Firmicutes: A phylum of Gram-positive bacteria, members of
which predominate in the HGM. The term “Gram-positive
PUL (gpPUL)” has been coined to describe the distinct
clusters of polysaccharide utilization genes in some species of
this phylum.
Carbohydrate-active enzymes (CAZymes): Protein-based
catalysts involved in the biosynthesis and biodegradation of
complex carbohydrates, comprising glycosyltransferases
(GTs), glycoside hydrolases (GHs), polysaccharide lyases
(PLs), carbohydrate esterases (CEs), and auxiliary activity
(AA) enzymes.
Surface glycan binding proteins (SGBP): Noncatalytic
carbohydrate-binding proteins located on the exterior of the
outer membrane of bacteria that facilitate the capture and
uptake of complex carbohydrates.
Glycobiology: The study of the structure, biochemistry, and
biology of carbohydrates.
■
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