The maltodextrin transport system and metabolism in
Lactobacillus acidophilus NCFM and production of novel
a-glucosides through reverse phosphorolysis by maltose
phosphorylase
Hiroyuki Nakai
1
, Martin J. Baumann
1
, Bent O. Petersen
2
, Yvonne Westphal
3
, Henk Schols
3
,
Adiphol Dilokpimol
1
, Maher A. Hachem
1
, Sampo J. Lahtinen
4
, Jens Ø. Duus
2
and Birte Svensson
1
1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark
2 Carlsberg Laboratory, Valby, Denmark
3 Laboratory of Food Chemistry, Wageningen University, The Netherlands
4 Danisco Health & Nutrition, Kantvik, Finland
Keywords
glycoside hydrolase family 65; maltodextrin
gene cluster; reverse phosphorolysis;
a-glucosides; b-glucose 1-phosphate
Correspondence
B. Svensson, Enzyme and Protein
Chemistry, Department of Systems Biology,
Technical University of Denmark, Søltofts
Plads, Building 224, DK-2800 Kgs. Lyngby,
Denmark
Fax: +45 4588 6307
Tel: +45 4525 2740
E-mail: bis@bio.dtu.dk
(Received 12 August 2009, revised
9 October 2009, accepted 16 October 2009)
doi:10.1111/j.1742-4658.2009.07445.x
A gene cluster involved in maltodextrin transport and metabolism was
identified in the genome of Lactobacillus acidophilus NCFM, which
encoded a maltodextrin-binding protein, three maltodextrin ATP-binding
cassette transporters and five glycosidases, all under the control of a tran-
scriptional regulator of the LacI-GalR family. Enzymatic properties are
described for recombinant maltose phosphorylase (MalP) of glycoside
hydrolase family 65 (GH65), which is encoded by malP (GenBank:
AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP
catalyses phosphorolysis of maltose with inversion of the anomeric configu-
ration releasing b-glucose 1-phosphate (b-Glc 1-P) and glucose. The broad
specificity of the aglycone binding site was demonstrated by products
formed in reverse phosphorolysis using various carbohydrate acceptor
substrates and b-Glc 1-P as the donor. MalP showed strong preference for
monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose
and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy
N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides
served as acceptors. Disaccharide yields obtained from 50 mmb-Glc 1-P
and 50 mmglucose, glucosamine, N-acetyl glucosamine, mannose, xylose
or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product struc-
tures were determined by NMR and ESI-MS to be a-Glcp-(1 fi4)-Glcp
(maltose), a-Glcp-(1 fi4)-GlcNp(maltosamine), a-Glcp-(1 fi4)-GlcNAcp
(N-acetyl maltosamine), a-Glcp-(1 fi4)-Manp,a-Glcp-(1 fi4)-Xylpand
a-Glcp-(1 fi4)- l-Fucp, the three latter being novel compounds. Modelling
using L. brevis GH65 as the template and superimposition of acarbose
from a complex with Thermoanaerobacterium thermosaccharolyticum
GH15 glucoamylase suggested that loop 3 of MalP involved in substrate
recognition blocked the binding of candidate acceptors larger than
monosaccharides.
Abbreviations
ABC, ATP-binding cassette; GH, glycoside hydrolase family; HPAEC-PAD, high-performance ion-exchange chromatography equipped with a
pulsed amperometric detector; MalE, maltodextrin-binding protein; MalF and MalG, maltodextrin ABC transport permease proteins; MalL,
oligo-1,6-glucosidase; MalN, neopullulanase; MalP, Lactobacillus acidophilus NCFM maltose phosphorylase; MalR, transcriptional regulator of
the LacI-GalR family; MsmK, maltodextrin import ATP-binding protein; b-Glc 1-P, b-glucose 1-phosphate.
FEBS Journal 276 (2009) 7353–7365 ª2009 The Authors Journal compilation ª2009 FEBS 7353
Introduction
Maltose and maltodextrin metabolism has been widely
investigated using bioinformatics and mutagenesis
analysis in both Gram-negative bacteria, such as
Escherichia coli [1,2], and Gram-positive bacteria,
including Bacillus subtilis [3,4], Streptococcus pyogenes
MGAS5005 [5,6] and Lactococcus lactis [7,8]. The
maltose ⁄maltodextrin regulon in E.coli consists of 10
genes encoding four glycoside hydrolases [amylomal-
tase (EC 2.4.1.25), maltodextrin phosphorylase (EC
2.4.1.1), maltodextrin glucosidase (EC 3.2.1.20), peri-
plasmic a-amylase (EC 3.2.1.1)] involved in intra-
cellular metabolism, together with an ATP-binding
cassette (ABC) transporter containing a maltose ⁄
maltodextrin-binding protein [1,2]. In B. subtilis, malt-
ose and maltodextrin are transported by a phospho-
enol-pyruvate-dependent phosphotransferase system
mediated by a maltose-specific enzyme IICB and an
ABC transporter containing a maltodextrin-binding
protein [3,4]. Maltose was suggested to be hydrolysed
intracellularly to glucose and glucose 6-phosphate by
NAD(H)-dependent 6-phospho-a-glucosidase (EC 3.2.
1.122) [9]. In S.pyogenes MGAS5005, uptake of malt-
ose and maltodextrin is demonstrated to occur via
both a phosphoenol-pyruvate-dependent phosphotrans-
ferase system and ABC transporters containing
maltose ⁄maltodextrin-binding proteins [5,6], whereas
maltose is transported in Lactococcus lactis by an
ATP-dependent permease system and degraded by
maltose phosphorylase (EC 2.4.1.8), of which tran-
scription is controlled by a regulator belonging to the
LacI-GalR family [7,8]. Furthermore, bioinformatics
analysis on L.casei predicted that maltose is taken up
via a putative ABC transporter and degraded intracel-
lularly by a maltose phosphorylase [10].
Maltose phosphorylases are characterized from
several bacteria [11–14] and found to catalyse phos-
phorolysis of maltose with inversion of the anomeric
configuration at the C-1 atom to give b-glucose
1-phosphate (b-Glc 1-P) and glucose. Sequence analy-
sis [13–15] has assigned maltose phosphorylase to gly-
coside hydrolase family 65 (GH65), together with
trehalose phosphorylase (EC 2.4.1.64) [15,16], kojibiose
phosphorylase (EC 2.4.1.230) [17] and a,a-trehalase
(EC 3.2.1.28) [18] (http://www.cazy.org). The three-
dimensional structure of L.brevis maltose phos-
phorylase of GH65 was determined [19] and shows
similarities with the (a⁄a)
6
barrel fold of GH15 glu-
coamylase (EC 3.2.1.3) [20], and the GH94 cellobiose
phosphorylase (EC 2.4.1.20) [21] and chitobiose phos-
phorylase (EC 2.4.1.–) [22]. GH15 and GH65 together
constitute glycoside hydrolase clan L [23]. The reaction
mechanism of GH65 enzymes has been proposed to be
similar to an inverting mechanism using a general
acid ⁄base-catalysed direct displacement (Fig. 1), as
found for the GH94 phosphorylases (previously classi-
fied into glycoside transferase family 36) [22], where
direct nucleophilic attack by phosphate on the glyco-
sidic bond of the substrate is assisted by proton dona-
tion from the general acid catalyst to the glycosidic
oxygen. The inverting phosphorolysis catalysed by
maltose phosphorylase is reversible [24], which confers
the enzyme with the capacity to effectively synthesize
maltose in the reverse reaction with b-Glc 1-P as
the donor and glucose as the acceptor substrates
accommodated at subsites )1 and +1, respectively.
However, the substrate aglycone specificity remains to
be investigated.
In the present study, malP encoding a putative
GH65 maltose phosphorylase (MalP), was identified
using bioinformatics in a gene cluster of the genome of
Lactobacillus acidophilus NCFM, a well-documented
and commercially available probiotic bacterium for
which the full genome has previously been sequenced
and annotated [25]. MalP was predicted in the present
work to be involved in maltose ⁄maltodextrin metabo-
lism and its enzymatic properties were investigated
Fig. 1. Schematic reaction mechanism of reversible phosphorolysis. The general acid catalyst of MalP was predicted to be Glu484 based on
sequence alignment with L. brevis maltose phosphorylase [19]. R indicates a carbohydrate residue.
Maltose phosphorylase and reverse phosphorolysis H. Nakai et al.
7354 FEBS Journal 276 (2009) 7353–7365 ª2009 The Authors Journal compilation ª2009 FEBS
using the recombinant enzyme produced in E.coli.
Furthermore, the detailed specificity of the aglycone
substrate binding site in the reverse phosphorolysis
reaction was explored using b-Glc 1-P as the donor
and various potential carbohydrate acceptor sub-
strates. By applying this strategy, MalP was demon-
strated to efficiently catalyse the synthesis of several
a-glucosidic disaccharides, some of which have not
been reported previously.
Results and Discussion
Prediction of the enzymatic function of MalP
The amino acid sequence of MalP, deduced from malP
(GenBank AAV43670.1), showed similarity to maltose
phosphorylase, trehalose phosphorylase and kojibiose
phosphorylase, which all belong to GH65 (Table 1).
MalP has the highest (52–69%) sequence identity to
maltose phosphorylases from different Lactobacillus
species for which the enzymatic function has already
been identified experimentally [10,19]. Because no sig-
nal peptide was found using psort-b [26] and signalp
[27], MalP is suggested to play a role in maltose
metabolism in the cytoplasm.
malP belongs to a gene cluster in the genome of
L.acidophilus NCFM [25], which has been identified in
the present study and proposed to be involved in
maltodextrin transport and metabolism, as described
below (Fig. 2A). According to a blast search (Table 2),
the gene cluster contains a maltodextrin import
ATP-binding protein (MsmK, AAV43667.1), a maltod-
extrin-binding protein (MalE, AAV43666.1) and two
maltodextrin ABC transport system permeases (MalF,
AAV43665.1; MalG, AAV43664.1). This putative ABC
transporter for uptake of maltodextrin showed 40–44%
sequence identity to those of B.subtilis [3,4] and
S.pneumoniae [28,29], whereas the putative MalE of
L. acidophilus NCFM has 44% and 42% sequence
identity to the B. subtilis cyclodextrin-binding protein
and maltodextrin-binding protein (MdxE), respectively.
Cyclodextrin-binding protein has been reported to
exhibit specificity towards a-, b-, c-cyclodextrin and
maltodextrins (DP 4–5) with highest affinity for c-cyclo-
dextrin (K
d
= 110 lm), whereas MdxE was specific for
maltodextrins (DP 3–7) with highest affinity for malto-
hexaose (K
d
=3lm). This comparison suggests that
maltodextrins and cyclodextrins are taken up by L.
acidophilus NCFM via the ABC transporter system
consisting of MsmK, MalE, MalF and MalG (Fig. 2B).
Furthermore, three genes encoding a putative neopullu-
lanase ⁄maltogenic a-amylase (MalN, AAV43671.1; EC
3.2.1.135), oligo-1,6-glucosidase (MalL, AAV43672.1;
EC 3.2.1.10), and acetate kinase (AckA, AAV43673.1;
EC 2.7.2.1), respectively, are localized downstream of
malP (Fig. 2A, Table 2). The two glycoside hydrolases
(MalN and MalL) may be involved in the metabolism
of maltodextrin and cyclodextrin (Fig. 2B). Moreover,
apgmB encoding b-phosphoglucomutase (PgmB,
AAV43669.1; EC 5.4.2.2) is situated upstream of
malP (Fig. 2A, Table 2). The present sequence analysis
suggests a maltodextrin metabolic pathway in L.
acidophilus NCFM, as outlined in Fig. 2B. The maltod-
extrin transported by the ABC transporter system
(MsmK, MalE, MalF and MalG) is converted intracel-
lularly by three glycosidases (MalN, MalL and MalP)
to glucose and b-Glc 1-P, which can enter glycolysis
through conversion to glucose 6-phosphate by
b-phosphoglucomutase. The acetate kinase (AckA) can
produce pyruvic acid in the glycolysis.
Noticeably, malR encoding a transcriptional regula-
tor of the LacI-GalR family (MalR, AAV43674.1) is
situated downstream of the gene cluster (Fig. 2A,
Table 2) and is also present in the maltose metabo-
Table 1. Amino acid sequence identity of MalP predicted from L. acidophilus NCFM with functionally characterized GH65 enzymes. The
similarity of the amino acid sequences was investigated by using the BLASTP program (Swiss-Prot ⁄TrEMBL database).
Swiss-Prot ⁄TrEMBL
accession no. Score Identity (%) Gap (%) References
Maltose phosphorylase
Lactobacillus casei BL23 B3WCX8 1056 69 0 [10]
Lactobacillus brevis Q03TE9 893 59 1 [19]
Bacillus sp. RK-1 Q84IX5 820 53 1 [15]
Lactobacillus sanfranciscensis O87772 818 52 3 [13]
Paenibacillus sp. SH-55 Q50LH0 710 47 2 [14]
Trehalose phosphorylase
Thermoanaerobacter brockii ATCC 35047 Q8L164 338 33 3 [16]
Geobacillus stearothermophilus SK-1 Q8GRC3 338 29 5 [15]
Kojibiose phosphorylase
Thermoanaerobacter brockii ATCC 35047 Q8L163 319 26 5 [17]
H. Nakai et al.Maltose phosphorylase and reverse phosphorolysis
FEBS Journal 276 (2009) 7353–7365 ª2009 The Authors Journal compilation ª2009 FEBS 7355
lism operon of Lactococcus lactis [8] and the malt-
ose ⁄maltodextrin regulon of S.pneumoniae [28,29].
DNA binding of MalR in S.pneumoniae was drasti-
cally reduced in the presence of maltose, suggesting
that MalR and maltose function as the repressor and
the inducer, respectively, for the transcription of
genes involved in the uptake and the metabolism of
malto-oligosccharides [28]. The putative MalR recog-
nition site was seen at a gene locus adjacent to
sequences of )10 and )35 promoters, localized down-
stream of ackA (Fig. 2C), showing high sequence
identity to the MalR recognition site of S.pneumonia
[28] and other consensus sequences reported for tran-
scription regulators of the LacI-GalR family [30].
Therefore, it is suggested that transcription of the
gene cluster involved in maltodextrin metabolism is
regulated by MalR and maltose is produced from
maltodextrin transported by hydrolysis catalysed by
MalN and MalL.
Production and purification of MalP
Recombinant MalP was produced in E.coli
BL21(DE3) as a His-tag fusion protein and purified
by nickel chelating chromatography in a yield of
58 mg from 4 L culture. MalP migrated in
SDS ⁄PAGE as a single band with an estimated size of
88 kDa (Fig. S1), consistent with the theoretical mass
of 88 401.47 Da. However, the molecular mass was
estimated by gel filtration to be 176 kDa, indicating
that MalP is a dimer in solution. This was previously
found for L. brevis maltose phosphorylase [12], in
which the dimer was stabilized by two salt bridges and
20 hydrogen bonds, mainly from the loop between a5
and a6 [19], which is well conserved among GH65
maltose phosphorylases.
Enzymatic properties of MalP
MalP catalysed phosphorolysis of maltose with an
apparent pH optimum of 6.2 under the described con-
ditions (Fig. 3A). In dilute solution (9 nm), MalP was
stable (> 95% residual activity) in the range of pH
3.0-8.2 at 4 C for 24 h (Fig. 3B). MalP showed maxi-
mum activity at 45 C (Fig. 3C) and retained activity
during 15 min incubation at temperatures up to 45 C
at the optimum pH 6.2 (Fig. 3D). MalP was strictly
specific for maltose with a specific activity of 52
UÆmg
)1
and did not phosphorolyse the disaccharides
isomaltose (a-1,6), nigerose (a-1,3), kojibiose (a-1,2)
and trehalose (a-1,1).
Substrate recognition by MalP
Recently, substrate recognition by the GH65 trehalose
and kojibiose phosphorylases was suggested to involve
A
BC
Fig. 2. Maltodextrin utilization in L. acidophi-
lus NCFM. (A) Gene loci of the gene cluster
involved in maltodextrin transport and
metabolism. The numbers (nt) indicate the
locations of the first base of malG and the
last base of malR on the L.acidophilus
NCFM chromosome [25]. (B) Predicted
transport and metabolism of maltodextrin in
L. acidophilus NCFM. (C) Putative promoter
and regulator recognition region of the gene
cluster. Promoters ()35 and )10 elements),
ribosome binding site (RBS), stop codon for
malR, start codon for ackA are underlined.
The putative MalR binding consensus
sequence is shown as a gray box.
Maltose phosphorylase and reverse phosphorolysis H. Nakai et al.
7356 FEBS Journal 276 (2009) 7353–7365 ª2009 The Authors Journal compilation ª2009 FEBS
the sequence from a3toa6 of the catalytic (a⁄a)
6
bar-
rel domain [43]. The well-conserved stretch between a3
and a4 was unique for GH65 maltose phosphorylases
(Fig. 4A) and forms the rim of the active site pocket,
as seen in the structure of L.brevis maltose phosphor-
ylase (PDB: 1H54) [19], where it probably participates
in substrate recognition.
In an attempt to understand why malto-oligosac-
charides of DP 3–6 were not substrates for MalP,
the structure of MalP was modelled using the struc-
ture of the 59% sequence identical L.brevis maltose
phosphorylase [19] as a template. However, because
the structure of a ligand complex was not
available in GH65, the structure of the inhibitory
pseudomaltotetrasaccharide acarbose in a complex
with Thermoanaerobacterium thermosaccharolyticum
glucoamylase of GH15 [44] was superimposed onto
the MalP modelled structure. In this MalP ⁄acarbose
complex, four amino acid residues [Trp355, Asp356,
Glu484 (general acid catalyst), Gln589], which are
well conserved in GH65 disaccharide phoshorylases
including trehalose phosphorylase [15,16] and kojibi-
ose phosphorylase [17], were predicted to be involved
in the recognition of the glucose unit at subsite )1
(see Fig. 4C). Noticeably, the loop His413-Glu421
between a3 and a4 (Fig. 4A) of the (a⁄a)
6
barrel
domain appeared to block binding of malto-oligo-
saccharides longer than maltose (Fig. 4B), thus con-
forming with the strict specificity of MalP towards
maltose.
Table 2. Amino acid sequence similarities deduced from genes involved in maltodextrin metabolism from L. acidophilus NCFM to related
proteins of known function. The similarities of the amino acid sequences were analysed using the BLASTP program (Swiss-Prot ⁄TrEMBL data-
base).
Swiss-Prot ⁄
TrEMBL
accession no. Score
Identity
(%)
Similarity
(%)
Gap
(%) References
MalR
Bacillus subtilis catabolite control protein A P25144 115 27 48 6 [31]
Streptococcus pneumoniae HTH-type
transcriptional regulator MalR
P0A4T2 103 27 45 12 [28,29]
AckA
Thermotoga maritima acetate kinase AckA Q9WYB1 820 49 67 2 [32]
MalL
Bacillus cereus oligo-1,6-glucosidase MalL P21332 372 34 55 3 [33,34]
Bacillus thermoglucosidasius oligo-1,6-glucosidase P29094 370 37 56 4 [35]
MalN
Geobacillus stearothermophilus neopullulanase P38940 484 43 61 3 [36,37]
Baciillus sphaericus cyclomaltodextrinase Q08341 440 41 58 2 [38]
PgmB
Lactococcus lactis b-phosphoglucomutase PgmB P71447 153 36 58 1 [39]
Thermotoga maritima phosphorylated
carbohydrates phosphatase
Q9X0Y1 76 26 51 8 [40]
MsmK
Escherichia coli UTI89 maltose ⁄maltodextrin import
ATP-binding protein MalK
Q1R3Q1 338 44 60 3 [41]
Escherichia coli K12 maltose ⁄maltodextrin import
ATP-binding protein MalK
P68187 290 44 60 3 [1]
MalE
Bacillus subtilis cyclodextrin-binding protein CycB O07009 134 26 44 9 [3]
Bacillus subtilis maltodextrin-binding protein MdxE O06989 107 25 42 9 [4]
MalF
Bacillus subtilis maltodextrin-binding protein MdxF O06990 185 29 46 5 [4]
Escherichia coli maltose transport system permease
protein MalF
P02916 96 24 45 3 [1]
MalG
Escherichia coli K12 inner membrane ABC transporter
permease protein YcjP
P77716 128 30 52 3 [42]
Escherichia coli K12 maltose transport system
permease protein MalG
P68183 116 30 51 5 [1,42]
H. Nakai et al.Maltose phosphorylase and reverse phosphorolysis
FEBS Journal 276 (2009) 7353–7365 ª2009 The Authors Journal compilation ª2009 FEBS 7357