LmbE proteins from Bacillus cereus are de-N-acetylases
with broad substrate specificity and are highly similar
to proteins in Bacillus anthracis
Alexandra Deli
1
, Dimitrios Koutsioulis
2
, Vasiliki E. Fadouloglou
2,
*, Panagiota Spiliotopoulou
1
,
Stavroula Balomenou
1
, Sofia Arnaouteli
1
, Maria Tzanodaskalaki
2
, Konstantinos Mavromatis
3
,
Michalis Kokkinidis
1,2
and Vassilis Bouriotis
1,2
1 Department of Biology, Enzyme Biotechnology Group, University of Crete, Greece
2 Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece
3 Department of Energy ⁄Joint Genome Institute, Genome Biology Program, Walnut Creek, CA, USA
Keywords
Bacillus anthracis; de-N-acetylase;
glucosamine; LmbE; mutational analysis
Correspondence
V. Bouriotis, Department of Biology,
Enzyme Biotechnology Group, University of
Crete, PO Box 2208, Vasilika Vouton
714 09, Heraklion, Crete, Greece
Fax: +30 2810 394055
Tel: +30 2810 394375
E-mail: bouriotis@imbb.forth.gr
*Present address
Department of Biochemistry, University of
Cambridge, UK
(Received 22 October 2009, revised 15
March 2010, accepted 20 April 2010)
doi:10.1111/j.1742-4658.2010.07691.x
The genomes of Bacillus cereus and its closest relative Bacillus anthracis
each contain two LmbE protein family homologs: BC1534 (BA1557) and
BC3461 (BA3524). Only a few members of this family have been biochemi-
cally characterized including N-acetylglucosaminylphosphatidyl inositol
(GlcNAc-PI), 1-d-myo-inosityl-2-acetamido-2-deoxy-a-d-glucopyranoside
(GlcNAc-Ins), N,N¢-diacetylchitobiose (GlcNAc
2
) and lipoglycopeptide
antibiotic de-N-acetylases. All these enzymes share a common feature in
that they de-N-acetylate the N-acetyl-d-glucosamine (GlcNAc) moiety of
their substrates. The bc1534 gene has previously been cloned and expressed
in Escherichia coli. The recombinant enzyme was purified and its 3D struc-
ture determined. In this study, the bc3461 gene from B. cereus ATCC14579
was cloned and expressed in E. coli. The recombinant enzymes BC1534 (EC
3.5.1.-) and BC3461 were biochemically characterized. The enzymes have
different molecular masses, pH and temperature optima and broad sub-
strate specificity, de-N-acetylating GlcNAc and N-acetylchito-oligomers
(GlcNAc
2
, GlcNAc
3
and GlcNAc
4
), as well as GlcNAc-1P, N-acetyl-d-glu-
cosamine-1 phosphate; GlcNAc-6P, N-acetyl-d-glucosamine-6 phosphate;
GalNAc, N-acetyl-d-galactosamine; ManNAc, N-acetyl-d-mannosamine;
UDP-GlcNAc, uridine 5¢-diphosphate N-acetyl-d-glucosamine. However,
the enzymes were not active on radiolabeled glycol chitin, peptidoglycan
from B. cereus,N-acetyl-d-glucosaminyl-(b-1,4)-N-acetylmuramyl-l-alanyl-
d-isoglutamine (GMDP) or N-acetyl-d-GlcN-Na1-6-d-myo-inositol-1-HPO
4
-
octadecyl (GlcNAc-I-P-C
18
). Kinetic analysis of the activity of BC1534 and
BC3461 on GlcNAc and GlcNAc
2
revealed that GlcNAc
2
is the favored
substrate for both native enzymes. Based on the recently determined crystal
structure of BC1534, a mutational analysis identified functional key resi-
dues, highlighting their importance for the catalytic mechanism and the sub-
strate specificity of the enzyme. The catalytic efficiencies of BC1534 variants
were significantly decreased compared to the native enzyme. An alignment-
based tree places both de-N-acetylases in functional categories that are dif-
ferent from those of other LmbE proteins.
Abbreviations
GalNAc, N-acetyl-D-galactosamine; GlcNAc, N-acetyl-D-glucosamine; GlcNAc
2
,N,N¢-diacetylchitobiose; GlcNAc-1P, N-acetyl-D-glucosamine-1
phosphate; GlcNAc-6P, N-acetyl-D-glucosamine-6 phosphate; GlcNAc-Ins, 1-D-myo-inosityl-2-acetamido-2-deoxy-a-D-glucopyranoside;
GlcNAc-PI, N-acetylglucosaminylphosphatidyl inositol; GlcNAc-I-P-C
18
,N-acetyl-D-GlcN-a1-6-D-myo-inositol-1-HPO
4
-octadecyl; GMDP,
N-acetyl-D-glucosaminyl-(b-1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine; ManNAc, N-acetyl-D-mannosamine; UDP-GlcNAc, uridine
5¢-diphosphate N-acetyl-D-glucosamine.
2740 FEBS Journal 277 (2010) 2740–2753 ª2010 The Authors Journal compilation ª2010 FEBS
Introduction
Bacillus cereus, an opportunistic pathogen that causes
food poisoning, and Bacillus anthracis, the endospore-
forming bacterium that causes inhalational anthrax,
share a large number of homologous genes, as demon-
strated by recent genome sequencing and comparative
analysis [1,2]. Given the laboratory safety precautions
necessary for working with highly infectious agents
and the recent concerns regarding use of B. anthracis
as a potential bioweapon (class A agent, Centers for
Disease Control), the B. cereus enzymes are useful
models for studying the corresponding proteins of
B. anthracis.
The ERGO light database of the B. cereus
ATCC14579 genome (http://www.ergo-light.com/)
reveals the presence of two LmbE protein family
homologs, BC1534 and BC3461, which share 21%
identity [1]. Furthermore, they share 96% and 95%
identity with their homologs BA1557 and BA3524,
respectively, from B. anthracis. The LmbE protein
family (Fig. 1) includes N-acetylglucosaminylphosphat-
idyl inositol (GlcNAc-PI) de-N-acetylases from
mammals [3], yeast [4] and protozoa [5], 1-d-myo-inosi-
tyl-2-acetamido-2-deoxy-a-d-glucopynanoside (GlcNAc-
Ins) de-N-acetylase from Mycobacterium tuberculosis
(MshB) (EC 3.5.1.89) [6], N,N¢-diacetylchitobiose
(GlcNAc
2
) de-N-acetylase from the archaeon Thermo-
coccus kodakaraensis KOD1 (Tk-Dac) (EC 3.5.1.-) [7]
and antibiotic de-N-acetylases from Actinoplanes teich-
omyceticus (Orf2) [8] and Bacillus circulans (BtrD) [9].
The most important members of the LmbE protein
family, together with the structures of their substrates,
are shown in Fig. 2.
The crystal structure of BC1534 (previously reported
as BcZBP) has been determined at 1.8 A
˚resolution
(Fig. 3A) [10]. The structures of three other LmbE
protein family members have been similarly deter-
mined, namely TT1542 from Thermus thermophilus
[11], MshB from Mycobacterium tuberculosis [12] and
Orf2 from Actinoplanes teichomyceticus [8]. The
N-terminal part of the 234 amino acid BC1534 protein
adopts a Rossmann fold, and the C-terminal part con-
sists of two b-strands and two a-helices. In the crystal,
the protein forms a compact hexamer (Fig. 3A), in
agreement with solution data [13]. A zinc binding site
and a potential active site have been identified in each
monomer. These sites have extensive similarities to
those found in other known zinc-dependent hydrolases
with de-N-acetylase activity, i.e. MshB from Mycobac-
terium tuberculosis [12] and LpxC (UDP-(3-O-(R-3-hy-
droxymyristoyl))-N-acetylglucosamine de-N-acetylase)
from Aquifex aeolicus [14]. Despite a low degree of
structural homology, it has been suggested that these
enzymes are the products of convergent evolution due
to similar active site features.
The objective of this study was to shed light on
the role of two de-N-acetylases from B. cereus, which
are LmbE protein family homologs. Given the exten-
sive homology between B. cereus and B. anthracis, the
results of these studies could contribute to understand-
ing of the physiology of this interesting pathogenic
microbe. Two recombinant de-N-acetylases from
B. cereus ATCC14579 were biochemically character-
ized. A comparison of the substrate specificities of the
enzymes with those of other members of the LmbE
D
BC1534 RHPDHA´
RHPDHA´
VHPDHN
VHPDHD
SGHSNH
GHPDHV
RHPDHT
GHPDHR
EHPDHE
KHVDHR
AHADDVEIGMAGTIAKYTKQG
PHPDDEAYAAGGTIRLLTD
D
QG´
PHPDDEAFAAGGTIRLLT QG´
PHPDDGELGCGGTLARAKAEG
PHFDDVILSCASTLMELMNQG
PHLDDAVLSFGAGLAQAAQDG
PHPDDCAIGLGGTIKKLTDSG
AHPDDESLSNGATIAHYTSRG
AHPDDEAMFFAPTILGLARLK
AHADDVEIGMAGTIAKYTKQG
114
126
126
114
157
152
165
167
148
112
31
30
30
31
68
59
35
33
32
29
BC3461
BA1557
BA3524
PIG-L
MshB
TT1542
Orf2
BtrD
Tk-Dac
Fig. 1. Partial amino acid sequence alignment of BC1534 and BC3461 with five LmbE-like proteins of known function and three of unknown
function (BA1557, BA3524 and TT1542). BA1557, hypothetical protein from Bacillus anthracis strain Ames (NP_844007); BA3524, hypotheti-
cal protein from Bacillus anthracis strain Ames (NP_845802); PIG-L, GlcNAc-PI de-N-acetylase from Rattus norvegicus (BAA20869); MshB,
GlcNAc-Ins de-N-acetylase from Mycobacterium tuberculosis H37Rv (NP_215686); TT1542, conserved hypothetical protein from Ther-
mus thermophilus HB8 (BAC67240); Tk-Dac, N,N¢-diacetylchitobiose de-N-acetylase from Thermococcus kodakaraensis KOD1 (BAD29713);
Orf2, de-N-acetylase from Actinoplanes teichomyceticus (CAG15014); BtrD, de-N-acetylase from Bacillus circulans (BAE07068). The black
regions indicate identical residues and gray shading indicates similar amino acids.
A. Deli et al. LmbE proteins from Bacillus cereus
FEBS Journal 277 (2010) 2740–2753 ª2010 The Authors Journal compilation ª2010 FEBS 2741
protein family is presented. A mutational analysis of
BC1534 identified functional key residues, highlighting
their importance for the catalytic mechanism and the
substrate specificity of the enzyme. A computational
analysis to predict the biological role of the enzymes is
also reported.
Results
Identification of bc1534 and bc3461 from
B. cereus ATCC14579 and comparison with
B. anthracis homologs
In silico analysis of the B. cereus ATCC14579 genome
revealed the presence of the bc1534 and bc3461 genes
(NP_831313 and NP_833195, respectively). The bc1534
gene consists of 705 bp and encodes a protein of 234
amino acids, while the bc3461 gene consists of 663 bp
and encodes a protein of 220 amino acids. Neither
N-terminal signal sequences nor transmembrane helices
were found in the deduced amino acid sequences (based
on sequence similarities and the sequence prediction
programs http://www.cbs.dtu.dk/services/SignalP/ and
http://www.sbc.su.se/~miklos/DAS/ for signal peptide
and transmembrane domain prediction, respectively),
which is consistent with the fact that recombinant
BC1534 and BC3461 were detected and purified from
the cytosolic fraction of Escherichia coli cells as intra-
cellular enzymes.
According to the Pfam [15] and Cluster of Ortholo-
gous Groups [16] databases, BC1534 and BC3461 are
members of the LmbE protein family (Pfam02585 ⁄
COG2120). BC1534 is also classified as a member of
carbohydrate esterase family 14 (CE14), but BC3461
has not been assigned to any of the CE families. Two
open reading frames found in B. anthracis strain ‘Ames
ancestor’ [BA1557 (NP_844007) and BA3524
(NP_845802)] were identical in size and shared identities
of 96% and 95% to BC1534 and BC3461, respectively.
The bc1534 gene belongs to an operon that also con-
tains six genes that have various predicted functions
(Fig. 4). In silico data show that expression of the
operon is regulated by a common mechanism (the r
E
promoter at the 5¢end of the operon) [17]. Based on
the gene organization and predicted functions of the
genes that belong to this operon, there is no apparent
GlcNAc GlcNAc
2
OH
OH
O
O
CH3
CH3CH3
CH2OH
H3C
NH
HO
HO
HO
HO
HO O
O
ONH
2
NH
2
HO
NH
HO
HO
OH OH
OH
OH
OH OH
N
NH NH
OO
OH
OH
OH
OH
OH
HO
HO
HO
HO2CNH2
CH3
CH3
H3C
OH OH
OH
HO
OH
OH
OH
NH
O
O
O
O
O
O′
O′
P
HN
NN
Cl
Cl
H
N
H
N
HHN
HN
H
HO
O
O
O
O
O
O
O
OO
O
O
O
O
O
O
O
O
OO
O
OO
HO
HO
HO 3
1
HO
HO HO
H
Tk-Dac MshB
GlcNAc-Ins
BtrD
N-acetyl-D-glucosaminyl
aglycone
N-acetyl-D-glucosaminyl
pseudoaglycone
Orf2
GlcNAc-PI
PIG-L
Fig. 2. LmbE proteins substrates. All enzymes catalyze hydrolysis of the N-acetyl group (shown inside a circle or a rectangle) of the GlcNAc
moiety of their substrate(s).
LmbE proteins from Bacillus cereus A. Deli et al.
2742 FEBS Journal 277 (2010) 2740–2753 ª2010 The Authors Journal compilation ª2010 FEBS
AB
CD
EF
Fig. 3. (A) Overall structure of BC1534 hexamer formed through the association of three dimers [10] (PDB ID 2ixd). Dimerization is achieved
through interaction between the b8-strand of one monomer and the b6- and b7-strands of the other monomer. (B) Surface representation of
the enzyme and view of the active site from outside. The active site is occupied by GlcNAc
2
(shown as a ball model). The substrate has
been modeled into the active site by autodocking. The two conformations of R140, determined by the crystal structure, are also shown
(stick models). (C,D) GlcNAc
2
(stick model) has been docked into the active site of the enzyme, and the interactions or loss of interactions
with residues that occupy positions 140 and 42 are indicated. (C) Position 140. The native Arg residue (carbon atoms in yellow) and the
mutations Ala (carbon atoms in brown) and Glu (carbon atoms in gray) are shown as stick models. The distances between the substrate and
each of the three residues are shown as dashed lines. (D) Position 42. The native Ala residue (yellow) and the mutation Ser (gray) are shown
as stick models. The positions of the two native Ser residues (45 and 46) in the neighborhood of the mutation are shown as thin stick mod-
els. The distances from the substrate are shown as dashed lines. All the point mutations shown in (C) and (D) were computationally intro-
duced in the model of the native crystal structure (see Experimental procedures). (E) Cartoon representation of the active site of BC1534,
highlighting residues that have been mutated and showing their relative positions in the structure. The H110 ⁄D112 pair is shown as a stick
model, and three of the residues that form a hydrophilic pocket suggested to function as the ‘oxyanion hole’ are shown using Van der Waals
dotted spheres. (F) Cartoon representation that focuses on the entrance of the active site tunnel, showing that it is dominated by positively
charged residues represented here by Van der Waals dotted spheres. The position of K207 is also shown.
A. Deli et al. LmbE proteins from Bacillus cereus
FEBS Journal 277 (2010) 2740–2753 ª2010 The Authors Journal compilation ª2010 FEBS 2743
pathway in which all of these genes could be involved.
In contrast, bc3461 is not part of a gene cluster, indi-
cating that its expression is probably regulated in an
independent manner (Fig. 4).
Protein sequence alignment of characterized mem-
bers of this family with the B. cereus homologs (Fig. 1)
revealed two conserved sequence motifs. The first
[(A ⁄P)H(X ⁄P)DD] is located near the N-terminus, and
the second [H(X ⁄P)DH] is located towards the middle
of the protein. The crystal structure of BC1534 [10]
revealed that the underlined H and D residues in the
first motif and the last H of the second motif are zinc
binding ligands. Moreover, it has been proposed for
other members of the family that the underlined H
and the subsequent D of the second motif play a
charge relay role during catalysis [9].
Cloning, expression and purification of BC1534
The gene encoding BC1534 was isolated from B. cereus
ATCC14579 genomic DNA by PCR and cloned into
expression vector pET26b for recombinant protein
production in E. coli. BC1534 was produced as a
C-terminal hexahistidine fusion protein to facilitate puri-
fication by affinity chromatography (Ni-nitrilotriacetic
acid). SDS ⁄PAGE analysis revealed the presence of
three protein bands. The position of the main band is
in agreement with the predicted molecular mass of the
hexahistidine fusion protein (27 kDa). The N-terminal
amino acid sequence of the protein bands correspond-
ing to the higher-molecular-mass proteins seen in
SDS ⁄PAGE was determined to be MSGL, which is
identical to the predicted amino acid sequence of
BC1534 (MSGLHILAFG), suggesting the existence of
non-denatured homopolymers of BC1534 in the
SDS ⁄PAGE gel (Fig. 5A). The positions of these
bands are in agreement with the molecular mass of
purified BC1534 estimated by size-exclusion chroma-
tography [13] and glutaraldehyde cross-linking [18]
(approximately 160 kDa), indicating that the protein
exists as a hexamer in solution.
Cloning, expression and purification of BC3461
bc3461 was isolated from B. cereus ATCC14579 geno-
mic DNA by PCR and cloned into the pRSETA
expression vector for recombinant protein production
in E. coli. Purification was achieved in two steps, using
ion-exchange and size-exclusion chromatography. The
apparent molecular mass of BC3461 was calculated to
be 26 kDa as determined by SDS ⁄PAGE (Fig. 5B) and
gel-filtration chromatography, suggesting that the pro-
tein exists as a monomer in solution.
Enzymatic properties of BC1534 and BC3461
BC1534 and BC3461 were active on N-acetyl-d-gluco-
samine (GlcNAc), N-acetylchitooligomers (GlcNAc
2
,
GlcNAc
3
and GlcNAc
4
), GlcNAc-1P, GlcNAc-6P,
GalNAc and ManNAc (Table 1). The specificity of
the enzymes for various N-acetylchito-oligomers was
examined, and the kinetic parameters were determined
BC1531
BC1532
1482325
BC3459 BC3460
Putative transcriptional
regulatory protein
Dihydrodipicolinate
reducatase
Short chain
dehydrogenase
LmbE-related
protein
Hypothetical protein
Hypothetical protein
Arsenical pump
membrane protein
LmbE-related
protein
Glycosyltransferase
tRNA CCA-
pyrophosphorylase
Biotin-opemn
repress or/biotin–[acetyl-
CoA-carboxylase]
synthetase
Methylglyoxal
synthase
BC3461
BC3462
BC3463
3415277 3419625
BC1534 BC1536
BC1537mgsA BC1535
1487879
A
B
Fig. 4. Gene organization in the 5.5 kbp region that includes bc1534 (A) and the 4.3 kbp that includes bc3461 (B) on the B. cereus
ATCC14579 genome. Arrows indicate open reading frames. Genes of interest are indicated by colored arrows.
LmbE proteins from Bacillus cereus A. Deli et al.
2744 FEBS Journal 277 (2010) 2740–2753 ª2010 The Authors Journal compilation ª2010 FEBS