Báo cáo khoa học: LmbE proteins from Bacillus cereus are de-N-acetylases with broad substrate specificity and are highly similar to proteins in Bacillus anthracis pot

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:
*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 ª 2010 The Authors Journal compilation ª 2010 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 ( />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
AHPDDEAMFFAP
TILGLARLK
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 ª 2010 The Authors Journal compilation ª 2010 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 and
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
CH
3
CH
3
CH
3
CH
2
OH
H
3

C
NH
HO
HO
HO
HO
HO
O
O
O
NH
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
HO
2
C
NH
2
CH
3
CH
3
H
3
C
OH
OH
OH
HO
OH
OH
OH
NH
O
O
O
O

O
O′
O′
P
HN
N
N
Cl
Cl
H
N
H
N
H
HN
H
N
H
HO
O
O
O
O
O
O
O
O
O
O
O

O
O
O
O
O
O
O
O
O
O
O
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 ª 2010 The Authors Journal compilation ª 2010 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 ª 2010 The Authors Journal compilation ª 2010 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 ª 2010 The Authors Journal compilation ª 2010 FEBS
(Tables 1 and 2). BC1534 and BC3461 exhibited maxi-
mum activity towards GlcNAc
2
. Kinetic parameters
for GlcNAc and GlcNAc
2
were obtained from Linewe-
aver–Burk plot analysis, and the enzyme reaction rates
for these substrates appear to follow Michaelis–
Menten kinetics. The resulting k
cat
⁄ K

m
ratios (catalytic
efficiency, K
eff
) indicated that GlcNAc
2
was the
favored substrate for both BC1534 and BC3461
(Table 2). UDP-GlcNAc was also tested as a potential
substrate due to the presence of a glycosyltransferase
A
B
104
175
47.5
62
25
32.5
83
16.5
BC3461
6.5
37
97
50
29
20
BC1534
Fig. 5. SDS ⁄ PAGE of the purified LmbE-like
proteins BC1534 (A) and BC3461 (B). (A)

Lane 1, molecular weight markers; lane 2,
size-exclusion chromatography eluant.
Samples were electrophoresed on a 12%
polyacrylamide gel under denaturing
conditions. Protein bands were visualized
by staining with Coomassie Brilliant Blue
R-250.
Table 2. Kinetic parameters of BC3461 and BC1534 (wild-type and variants) towards GlcNAc and GlcNAc
2
. NA, not active.
Enzyme
Substrate
GlcNAc GlcNAc
2
k
cat
(s
)1
) K
m
(lM) K
eff
(lM
)1
Æs
)1
) k
cat
(s
)1

) K
m
(lM) K
eff
(lM
)1
Æs
)1
)
BC3461 6.4 9 0.71 11.1 2 5.55
BC1534 1.89 3 0.63 98.2 3 32.7
BC1534 R140A 189.7 2500 0.08 0.37 4 0.09
BC1534 R140E 0.27 8 0.03 0.27 5 0.05
BC1534 A42S 0.01 1.3 · 10
3
8.4 · 10
)6
0.01 1.1 · 10
3
9 · 10
)6
BC1534 D112N NA NA NA 5.97 6 0.99
BC1534 K207I NA NA NA NA NA NA
BC1534 Y194F 48.26 3.8 · 10
3
0.012 15.78 1.1 · 10
3
0.014
Table 1. Substrate specificity (percentage relative activity)of BC3461, BC1534 and mutants. Assay conditions were 25 mM HEPES ⁄ NaOH
pH 8.0, 200 m

M NaCl, 1 mM CoCl
2
for 30 min at 37 °C for BC1534 and its variants, and 25 mM MES ⁄ NaOH pH 6.5, 200 mM NaCl, 1 mM
MgCl
2
for 30 min at 20 °C for BC3461. The concentration of substrates was adjusted with respect to their content in terms of N-acetyl
residues. ND, not determined.
Substrate BC3461 BC1534 BC1534 R140A BC1534 R140E BC1534 A42S BC1534 D112N BC1534 Y194F
GlcNAc 39 53 93 80 41 0 100
GlcNAc
2
100 100 100 100 25 53 100
GlcNAc
3
92 43 27 40 31 16 32
GlcNAc
4
65 35 20 0 0 0 13
GlcNAc
5
000 0 0 0 6
GlcNAc
6
000 0 0 0 14
GlcNAc-1P 65 88 67 50 100 0 30
GlcNAc-6P 27 53 87 40 50 99 30
GalNAc 58 60 40 20 25 0 10
ManNAc 58 60 0 0 12 100 0
GlcNAc-I-P-C
18

ND 0 ND ND ND ND ND
GMDP 0 0 ND ND ND 19 22
A. Deli et al. LmbE proteins from Bacillus cereus
FEBS Journal 277 (2010) 2740–2753 ª 2010 The Authors Journal compilation ª 2010 FEBS 2745
gene (bc1535) downstream BC1534 (Fig. 4). The K
m
value (3 lm) was comparable to that for GlcNAc
2
, but
the K
eff
value (0.02 lm
)1
Æs
)1
) was significantly lower
compared to that for GlcNAc
2
.
The purified recombinant enzymes showed different
pH and temperature optima using GlcNAc
2
as sub-
strate. BC1534 exhibited a pH optimum at pH 8.0,
and optimum temperature for enzyme activity was
determined to be 37 °C. The pH and temperature
optima for BC3461 were 6.5 and 20 °C, respectively.
Moreover, a 3.5-fold increase in BC1534 activity was
observed on addition of 1 mm CoCl
2

to the assay buf-
fer, and a twofold increase in BC3461 activity was seen
when 1 mm MgCl
2
was added. BC1534 and BC3461
were inhibited by the presence of 1 mm Cu
2+
and
1mm Zn
2+
(both tested as chlorides), similar to previ-
ous reports on zinc hydrolases [19]. The enzymes were
not inhibited by acetate or EDTA even at concentra-
tions up to 50 and 20 mm, respectively. Moreover, they
were both inactive on radiolabeled glycol chitin and
peptidoglycan from B. cereus vegetative cell walls, and
BC1534 was also inactive on GlcNAc-I-P-C
18
, a syn-
thetic analogue of GlcNAc-PI.
Mutational analysis of BC1534
In order to elucidate the importance of selected resi-
dues in the catalytic mechanism, substrate affinity and
specificity, as well as to investigate the importance of
the oligomerization state on the activity of the enzyme,
six point mutations and one fragment deletion were
performed. The enzyme variants obtained were tested
against the same range of substrates as the wild-type
enzyme, and their substrate specificities and kinetic
parameters are presented in Tables 1 and 2.

BC1534 shares extensive similarities to the active
sites of two characterized zinc-dependent deacetylases,
i.e. MshB and LpxC [6,14]. Based on these similarities,
it was proposed [10] that BC1534 would utilize a simi-
lar catalytic mechanism to these enzymes. The domi-
nant features of this mechanism are an H ⁄ D charge
relay pair (H110 ⁄ D112 for BC1534, Fig. 3E) and an
‘oxyanion hole’ (mainly formed by the side chains of
Y194, N150 and D108 in BC1534, Fig. 3E). To test
this hypothesis, we mutated D112 and Y194 to resi-
dues that lack groups with a putative role in catalysis.
Thus, D112 was mutated to asparagine, which retains
all aspartate’s stereochemical and physicochemical
properties except its ability to relay protons. The
mutant protein (D112N) was completely inactive on
most of the substrates tested, i.e. GlcNAc, GlcNAc
4
,
GlcNAc
5
, GlcNAc
6
and GlcNAc-1P, but exhibited
higher relative activity against GlcNAc-6P and Man-
NAc than the native enzyme did (Table 1). Y194 was
mutated to phenylalanine, which retains all the hydro-
phobic interactions of the initial residue but lacks the
hydroxyl group that is proposed to participate in for-
mation of the ‘oxyanion hole’. The Y194F mutant
showed the broadest substrate specificity among

BC1534 variants, but its catalytic efficiency against the
preferred substrate of the native enzyme (GlcNAc
2
)
was highly reduced. In addition, it exhibited activity
against GlcNAc
5
, GlcNAc
6
and GMDP, in contrast to
the wild-type enzyme, which was inactive with these
substrates.
Based on the crystal structure of BC1534 [10] and a
molecular dynamics study [20], we proposed that the
rim and the loops surrounding the active site tunnel
could play a significant role in the substrate specificity
of the enzyme. We experimentally tested this hypothe-
sis by mutating R140, K207 and A42, three residues
located on the rim of the active site tunnel. In the crys-
tal structure, R140 adopts two discrete conformations
(Fig. 3B). One of these conformations highly restricts
the accessibility of the active site, while the other,
which is mainly stabilized by electrostatic interactions
with the adjacent E142 and backbone carbonyl groups,
keeps the entrance open. To further investigate the role
of R140, it was mutated (a) to a small hydrophobic
residue, Ala and (b) to a negatively charged Glu resi-
due. With minor exceptions, both variants exhibited
the same preferences as the native enzyme for the sub-
strates tested (Table 1). R140A showed a significant

decrease in efficiency compared to the native enzyme
for GlcNAc
2
and GlcNAc. However, this variant
exhibited 100 times higher catalytic activity (k
cat
)on
GlcNAc than the wild-type enzyme. In the case of
R140E, the K
m
values remain similar to that of the
native enzyme, but the k
cat
is significantly decreased
for both substrates.
K207 was mutated to isoleucine, a small hydropho-
bic residue. Remarkably, this variant was inactive on
all substrates tested. A42 is located in a position favor-
able for the formation of hydrogen bonds with the
substrate’s hydroxyl groups (Fig. 3D). Thus, A42 was
mutated to a serine, which is a residue of comparable
size to alanine and its side chain has the ability to
form hydrogen bonds. Unexpectedly, the produced
variant (A42S) exhibited a dramatic reduction in K
eff
value for both substrates (Table 2).
BC1534 is a hexamer (Fig. 3A) that may be consid-
ered as a trimer of dimers [10,20]. Dimerization is
mainly established via exchange of two short b-strands
between the monomers (b8-strand in Fig. 3A). Dele-

tion of the b8-strand by PCR resulted in an insoluble
and inactive protein.
LmbE proteins from Bacillus cereus A. Deli et al.
2746 FEBS Journal 277 (2010) 2740–2753 ª 2010 The Authors Journal compilation ª 2010 FEBS
Prediction of function
Although members of Pfam02585 (LmbE protein fam-
ily) share common sequence features, they do not exhi-
bit the same function [5–8]. Sequence alignment of 929
proteins belonging to Pfam02585 revealed a number of
different sequence groups (Fig. 6), presumably reflect-
ing different functions. All proteins for which the enzy-
matic function has been studied belong to different
groups. The two Bacillus proteins were placed in func-
tional categories different from other LmbE proteins.
As the sequence of the protein is not sufficient to
reveal the function of the enzyme, we sought other
lines of evidence to predict its function. Location of
enzymes in the same chromosomal neighborhood could
indicate a functional relationship and frequently helps
to predict the function of enzymes [21]. BC1534
appears to be in a chromosomal neighborhood that is
conserved among the Bacillus species (data not shown).
Microarrays [22] and deep sequencing of the transcrip-
tome [23] in B. anthracis revealed that ba1557 (the
homolog of bc1534) is in an operon surrounded by
genes homologous to bc1531-bc1537, and is expressed
during the early and mid log phases, while ba3524 (the
homolog of bc3461) is expressed in the early log phase
and during sporulation. Despite possible differences in
the life cycle of these Bacillus species, these data from

the B. anthracis transcriptome [22,23] could provide
strong evidence for similar gene expression in B. cereus.
In other members of the Firmicutes, the conservation
is restricted to the presence of a glycosyltransferase
(Pfam00534) downstream of the LmbE-related protein
(i.e. the proteins encoded by the bc1535 and bc1534
genes, respectively, in B. cereus).
Discussion
In an effort to shed light on the role of the LmbE pro-
tein family enzymes in bacteria and contribute to the
understanding of the pathobiology of B. anthracis,we
describe biochemical characterization of the recombi-
nant enzymes BC1534 and BC3461 from B. cereus.
BC1534 exhibited overall 21% identity and 31% simi-
larity with BC3461. Moreover, BC1534 and BC3461
shared 96% and 95% identity with their homologs
BA1557 and BA3524, respectively, from B. anthracis
(Fig. 1). The purified recombinant enzymes exhibited
different molecular masses (Fig. 5), pH and temperature
optima and were not inhibited by acetate or EDTA.
BC1534 and BC3461 were activated by CoCl
2
and
MgCl
2
, respectively. Both enzymes were effective in
de-N-acetylating GlcNAc and N-acetylchitooligomers
(GlcNAc
2
, GlcNAc

3
and GlcNAc
4
), as well as GlcNAc-
1P, GlcNAc-6P, GalNAc, ManNAc and UDP-GlcNAc
(Table 1). However, the enzymes were not active on
glycol chitin or peptidoglycan from B. cereus ATCC14579,
GMDP or GlcNAc-I-P-C
18
. Kinetic analysis of BC3461
and BC1534 towards the N-acetylchito-oligosaccharides
GlcNAc and GlcNAc
2
revealed that GlcNAc
2
is the
favored substrate for both enzymes. Comparison of
the K
eff
values showed that both enzymes are equally
effective on GlcNAc. BC1534 was six times more
THA1200 Thermus thermophilus HB8
BC3461 Bacillus cereus ATCC 14579
TK1764 Thermococcus kodakaraensis KOD1
BC1534 Bacillus cereus ATCC 14579
BtrD Bacillus circulans
Rv1170 Mycobacterium tuberculosis H37Rv
Fig. 6. Neighbor joining tree for 929 LmbE
proteins. Bacillus cereus proteins and pro-
teins of known function are indicated.

A. Deli et al. LmbE proteins from Bacillus cereus
FEBS Journal 277 (2010) 2740–2753 ª 2010 The Authors Journal compilation ª 2010 FEBS 2747
effective than BC3461 (Table 2) when GlcNAc
2
was
tested as substrate.
Chitin de-N-acetylases from fungi and insects [24],
chito-oligosaccharide de-N-acetylases from Rhizobium
(NodB) [25] and Vibrio parahaemolyticus [26], and Glc-
NAc peptidoglycan de-N-acetylases [27] are considered
to be catalytically similar de-N-acetylases. They are all
members of carbohydrate esterase family 4, catalyzing
the hydrolysis of N-linked acetyl groups on GlcNAc
residues. Chitin de-N-acetylase is capable of removing
N-acetyl groups from chitin chains [28]. NodB, which
is involved in nodulation signal synthesis, de-N-acety-
lates the non-reducing GlcNAc residue of N-acety-
lchito-oligosaccharides [25]. GlcNAc peptidoglycan
de-N-acetylase increases the resistance of peptidoglycan
to lysozyme via de-N-acetylation of GlcNAc residues
[27]. None of the above enzymes accepts GlcNAc as
substrate.
The biochemically characterized LmbE proteins are
members of distinct metabolic pathways. MshB is
involved in the second step of mycothiol biosynthesis
[6], whereas GlcNAc-PI de-N-acetylases play an impor-
tant role in biosynthesis of the glycosylphosphatidyli-
nositol biosynthesis in eukaryotes [3–5]. Orf2 [8] and
BtrD [9] are involved in the synthesis of lipoglycopep-
tide antibiotics, while Tk-Dac plays an essential role in

a novel chitinolytic pathway identified in archaea [7].
All members of the LmbE protein family with known
function share a common feature in that they de-N-acet-
ylate the GlcNAc moiety of their substrates (Fig. 2). It
has been reported that MshB [6], Orf2 [8] and PIG-L
(GlcNAc-PI de-N-acetylase from Rattus norvegicus) are
not active on GlcNAc [3–5]. Tk-Dac de-N-acetylates
GlcNAc monomers as well as N-acetyl-chitooligomers
[7], but does not de-N-acetylate GlcNAc-6P or ManNAc.
In contrast to other LmbE protein family members,
BC1534 and BC3461 are active on GlcNAc, GlcNAc
2
,
GlcNAc
3
, GlcNAc
4
, GlcNAc-1P, GlcNAc-6P, GalNAc,
ManNAc and UDP-GlcNAc, thus exhibiting a broader
substrate specificity compared to other LmbE protein
family members.
The exact biological role of BC1534 and BC3461
proteins remains unclear. Microarray data [29] and
deep sequencing [23] of the transcriptome showed that
the homologs in B. anthracis (BA1557 and BA3524 for
BC1534 and BC3461, respectively) are expressed in dif-
ferent phases of the cell cycle (early to mid log phase
for BC1534 and late sporulation to early log phase for
BC3461). Of GlcNAc, GlcNAc
2

and GlcNAc-6P,
which were tested for their docking properties in the
BC1534 active site, GlcNAc
2
had the lowest calculated
binding energy (data not shown), which is in agree-
ment with the kinetic parameters shown in Table 2
indicating that GlcNAc
2
is the favored substrate.
RT-PCR experiments (data not shown) revealed
similar expression profiles for BC1534 and BC3461 to
that for an exochitinase (BC3725, EC 3.2.1.14) from
B. cereus ATCC14579. This observation, in combina-
tion with the reported chitinolytic activity of B. cereus
[30] and the presence of an endochitinase and a chito-
sanase genes (bc0429 and bc2682 respectively) in its
genome, support possible involvement of these
enzymes in a chitinolytic pathway, similar to Tk-Dac.
An alignment-based tree (Fig. 6) placed both enzymes
in functional categories different from other LmbE
proteins. Analysis of the chromosome organization of
bc1534 revealed the existence of a glycosyltransferase
gene (bc1535) immediately downstream in the operon
(Fig. 4). This gene organization is common for most
Firmicutes genomes, suggesting that the two proteins
are functionally related in these organisms. Interest-
ingly, we observed that BC1534 is also active on
UDP-GlcNAc. As glycosyltransferases and UDP-
GlcNAc are situated at a biosynthetic branch point

leading to peptidoglycan formation, a possible role of
BC1534 (and BC1535, EC 2.4.1 ) in modulating pepti-
doglycan biosynthesis can be envisaged.
In order to elucidate the importance of selected resi-
dues in the catalytic mechanism, substrate affinity and
specificity, as well as to investigate the importance of
the oligomerization state on the activity of the enzyme,
six point mutations and one fragment deletion were
performed. Central features of the catalytic mechanism
are an H ⁄ D pair (H110 ⁄ D112, Fig. 3E) [10] that is
proposed to play the role of a charge relay, and a
hydrophilic pocket proposed as an ‘oxyanion hole’
(Y194, N150 and D108, Fig. 3E). We tested the valid-
ity of this hypothesis by mutating D112 to N and
Y194 to F. The variant D112N shown complete aboli-
tion of catalytic efficiency against GlcNAc, and the
k
cat
against GlcNAc
2
was decreased approximately 16
times with a subsequent decrease in K
eff
, indicating
that the hydroxyl group of D112 plays a significant
role in catalysis. The Y194F variant exhibited broad
substrate specificity similar to the native enzyme
(Table 2). However in contrast to the native enzyme, it
showed a dramatic increase (> 10
3

)inK
m
for GlcNAc
and GlcNAc
2
, and similar catalytic efficiency for both
substrates. These results suggest that the tyrosine
hydroxyl group is directly associated with the enzyme’s
affinity for the substrate.
In order to test the suggestion that the loops sur-
rounding the active site and the rim of the tunnel are
directly implicated in determining the accessibility of
the active site and the enzyme’s substrate specificity
[10,20], we mutated three residues located on the rim
LmbE proteins from Bacillus cereus A. Deli et al.
2748 FEBS Journal 277 (2010) 2740–2753 ª 2010 The Authors Journal compilation ª 2010 FEBS
(R140, K207, A42) (Fig. 3B–D,F). Automated docking
of GlcNAc
2
substrate in the R140A active site showed
that the substrate was arranged in a loose way with
fewer hydrophobic interactions and a weaker hydrogen
bond compared with the native enzyme. This different
orientation of the reaction site of the substrate could
explain the lower reported K
eff
values (Table 2) for
R140A. In the native enzyme, the rim of the active site
tunnel is dominated by positively charged residues, i.e.
R109, K154, K187, K207, K220 etc., that may drive

acetate out of the active site after the end of the reac-
tion. The positioning of a negatively charged residue
(E140) could hamper the release of the acetate from
the active site. This unfavorable behavior could
account for the lower catalytic reaction rates (k
cat
)in
the case of R140E (Table 2).
Surprisingly, the mutation K207I resulted in an inac-
tive enzyme. K207 is located on the rim of the active
site and it is quite unlikely that it has any influence on
the overall structure of the enzyme as it was found at
the edge of an a-helix (Fig. 3F). K207 is not conserved
among other LmbE family members, indicating that
this residue is a unique feature of BC1534 that could
be related to the specificity of the enzyme.
A42S exhibited lower K
eff
values, which could be
due to changes in interactions stabilizing the substrate
and ⁄ or interactions with residues involved in the cata-
lytic mechanism (R53 and ⁄ or D76) [10] (Fig. 3D).
Deletion of the short b8-strand resulted in an insoluble
and inactive enzyme, supporting a previous suggestion
[10] that the structural building block of the BC1534 is a
dimer formed via b-strand exchange (Fig. 3A).
Glucosamine (GlcN) is of importance in biomedicine
as it is used as dietary supplement for osteoarthritis
[31,32]. Currently GlcN is produced by acid hydrolysis
of chitin extracted from crab and shrimp shells [33]. A

new fermentation process utilizing E. coli cells modified
by metabolic engineering for the production of high-
quality and low-cost GlcN has recently been reported
[34]. The BC1534 R140A enzyme variant is potentially
a candidate for the enzymatic production of GlcN due
to its significantly increased k
cat
towards GlcNAc.
In conclusion, we have biochemically characterized
two LmbE proteins from B. cereus that exhibit the
broadest substrate specificity compared to other LmbE
protein family members, so far reported. BC1534 and
BC3461 appear to have distinct functional roles, as
shown by their different expression profiles, chromo-
somal organization and sequence alignments. Due to
their high similarity to their B. anthracis homologs,
clarification of their biological roles will contribute to
a better understanding of the properties of this life-
threatening bacterium.
Experimental procedures
Materials
Primers were synthesized by the Microchemistry Facility of
the Institute of Molecular Biology and Biotechnology
(Heraklion, Greece). The expression plasmid pET26b and
E. coli BL21 DE3 were obtained from Novagen (Merck,
KGaA, Darmstadt, Germany). The expression plasmid
pRSETA was purchased from Invitrogen (Carlsbad, CA,
USA). E. coli BL21 T7 Express lysY was purchased from
New England Biolabs GmbH (Frankfurt, Germany). All
chromatographic materials were obtained from GE Health-

care Bio-Sciences AB (Uppsala, Sweden). Ni-nitrilotriacetic
acid agarose, PCR and gel extraction kits were purchased
from Qiagen (Valencia, CA, USA). Plasmid purification and
RNA isolation kits were purchased from Macherey-Nagel
GmbH & Co. KG (Duren, Germany). The RT-PCR kit was
obtained from Finnzymes Oy (Espoo, Finland). Substrates
(including glycol chitosan) and common biochemicals were
purchased from Sigma-Aldrich Ltd (St Louis, MO, USA).
Restriction enzymes and DNA-modifying enzymes were
purchased from MINOTECH Biotechnology (Heraklion,
Greece) and New England Biolabs GmbH. The instruments
Fluostar Galaxy and Mastercycler Gradient (for PCR and
RT-PCR) were purchased from BMG Labtechnologies
GmbH (Offenburg, Germany) and Eppendorf Netheler-
Hinz GmbH (Hamburg, Germany), respectively.
Construction of expression plasmids
bc1534 and bc3461 genes were isolated from B. cereus
ATCC14579 genomic DNA. The primers used for bc1534
were BC1534-For (5¢-GGAATTC
CATATGATGAGTGG-
ATTACATATATTA-3¢; NdeI restriction site underlined)
and BC1534-Rev (5¢-CCG
CTCGAGTTTACATCCCCCT-
AATAAATC-3¢; XhoI restriction site underlined). Plasmid
pET26b was digested with NdeI and XhoI, and bc1534 was
ligated into the corresponding sites, resulting in plasmid
pET26b-bc1534. This plasmid construction was used for
the production of BC1534 protein with a histidine tag at its
C-terminus. The primers used for bc3461 were BC3461-For
(5¢-ATGGAGAGACATGTACTTGTT-3¢) and BC3461-

Rev (5¢-CCG
CTCGAGCTACT CCCAT TTATA AGTCCA- 3¢;
XhoI restriction site underlined). Initial digestion of plasmid
pRSETA with NdeI was followed by incubation with the
Klenow fragment of DNA polymerase I, and finally diges-
tion with XhoI. The bc3461 gene was ligated into the corre-
sponding sites of the plasmid.
Production and purification of BC4361, BC1534
and BC1534 variants
To over-express bc1534, the plasmid pET26b-bc1534 was
used for transformation of E. coli BL21 DE3. The resultant
A. Deli et al. LmbE proteins from Bacillus cereus
FEBS Journal 277 (2010) 2740–2753 ª 2010 The Authors Journal compilation ª 2010 FEBS 2749
transformant was cultured in LB medium at 30 °C. When
an attenuance at 600 nm of 0.6 was reached, isopropyl-b-d-
thiogalactopyranoside (final concentration 1 mm) was
added to the culture. After 12–16 h incubation at 30 °C,
the cells were harvested by centrifugation (5000 g, 15 min,
4 °C), and resuspended in 25 mm Tris ⁄ Cl pH 8.0, 300 mm
NaCl, 1 mm phenylmethylsulfonyl fluoride. After sonica-
tion, the soluble fraction was collected by centrifugation
(11 000 g, 30 min, 4 °C) and loaded onto a Ni-nitrilotriace-
tic acid agarose column (5 mL) equilibrated with 25 mm
Tris ⁄ Cl pH 8.0, 300 mm NaCl. Proteins were eluted using a
gradient of imidazole (0–300 mm). Active fractions were
pooled and dialyzed overnight against 25 mm Tris ⁄ Cl pH
8.0, 200 mm NaCl, and subsequently applied to a Sephacryl
S200 HR column (380 mL) previously equilibrated with
25 mm Tris ⁄ Cl pH 8.0, 200 mm NaCl. Fractions containing
enzyme activity were pooled and concentrated. Expression

and purification for all BC1534 variants were the same as
for the native enzyme.
For over-expression of bc3461, the plasmid pRSETA-
bc3461 was used for transformation of E. coli Bl21 T7
Express lysY. The resultant transformant was cultured in
LB medium at 28 °C. When an attenuance at 600 nm of
0.35–0.4 was reached, isopropyl-b-d-thiogalactopyranoside
(final concentration 0.01 mm) was added to the culture.
After 2 h of incubation at 28 °C, the cells were harvested by
centrifugation (5000 g, 30 min, 4 °C), and resuspended in
50 mm Tris ⁄ Cl pH 7.6, 200 mm NaCl, 1 mm dithiothreitol.
After sonication, the pellet was collected by centrifugation
(11 000 g, 30 min, 4
°C), and resuspended in 20 mm Tris ⁄ Cl
pH 7.6. The centrifugation was repeated (11 000 g, 30 min,
4 °C), and the pellet was resuspended in 20 mm Tris ⁄ Cl pH
7.6, 2% Triton X-100, and subsequently incubated for 3 h
at 4 °C under mild stirring. The soluble fraction was col-
lected by centrifugation (11 000 g, 30 min, 4 °C), diluted five
times against 20 m m Tris ⁄ Cl pH 7.6, and subsequently
loaded onto a Source 15Q HR column (21 mL) equilibrated
with 20 mm Tris ⁄ Cl pH 7.6. Proteins were eluted using a
gradient of NaCl (0–1 m). Active fractions were pooled,
concentrated (3 mL) and subsequently applied to a Super-
dex 75 HR column (1770 mL) previously equilibrated with
20 mm Tris ⁄ Cl pH 7.6, 200 mm NaCl. Fractions containing
enzyme activity were pooled and concentrated.
Preparation of radiolabeled substrates
Preparation of cell-wall peptidoglycan from vegetative
B. cereus was performed as previously described [35]. Label-

ing of glycol chitin and peptidoglycan was performed using
[
3
H]acetic anhydride as previously described [36].
Enzyme assays
Standard enzyme assays were performed in a mixture con-
taining 25 mm HEPES ⁄ NaOH pH 8.0 (for BC1534) or
25 mm MES ⁄ NaOH pH 6.5 (for BC3461) with 1 mm CoCl
2
(for BC1534) or 1 mm MgCl
2
(for BC3461). The concentra-
tion of each substrate was adjusted so that there was a
constant content of N-acetyl residues (2.26 mm). Incubation
time was 30 min at 37 °C (for BC1534) or 20 °C (for
BC3461). Cu
2+
and Zn
2+
were tested as chlorides at con-
centration of 1 mm, under standard enzyme assay condi-
tions for wild type enzymes BC1534 and BC3461.
The kinetic properties of BC1534 and BC3461 towards
GlcNAc and GlcNAc
2
were determined as described below.
Assays were performed under the reaction conditions
mentioned above including various concentrations of
N-acetyl-chitooligosaccharides. The rates of the reactions
catalyzed by wild-type BC1534 and BC3461 enzymes and

BC1534 variants as a function of substrate concentration
were determined using the graphing and statistical software
package hyper ( />software.html). Values for K
m
and V
max
were calculated by
fitting initial velocities at various substrate concentrations to
the appropriate forms of Michaelis–Menten equation. The
k
cat
values were calculated from V
max
using molecular
masses of 27 214 and 24 919 Da for BC1534 and BC3461,
respectively. The reported values are the mean of three
measurements.
Fluorescamine-based de-N-acetylase activity
assay
Purified BC1534 and BC3461 were tested for de-N-acetylase
activity using a 96-well plate assay as previously reported
[37]. Standard reaction mixtures consisting of 0.052 lm
BC1534 or 0.352 l m BC3461 in a final volume of 50 lL
were incubated for 30 min at 37 °C (for BC1534) or 20 °C
(for BC3461). The reactions were performed in COSTAR
96-well flat-bottomed microtiter plates (Corning Incorpo-
rated, New York, USA), and terminated by addition of
50 lL 0.4 m borate buffer (pH 8.5). Free amines were
labeled with 20 lL fluorescamine (6.96 mm) in dimethylfor-
mamide for 10 min at room temperature. The labeling reac-

tion was terminated by the addition of 150 lL
dimethylformamide: H
2
O (1 : 1). Fluorescence was quanti-
fied using a Fluostar Galaxy microplate fluorescence reader
with excitation and emission wavelengths of 390 and
460 nm, respectively. A calibration curve using glucosamine
showed that the free amine labeling reaction was linear up
to a concentration of 2.4 mm glucosamine. All measure-
ments are shown are the mean of three replicates.
RT-PCR
B. cereus ATCC14579 was cultivated in LB medium (half-
strength). Total RNA was isolated using a Nucleospin
RNA II kit (Macherey-Nagel). For RT-PCRs, 100 ng of
RNA was used. The reactions were performed using a
RobusT II RT-PCR kit (Finnzymes). Primer pairs were
LmbE proteins from Bacillus cereus A. Deli et al.
2750 FEBS Journal 277 (2010) 2740–2753 ª 2010 The Authors Journal compilation ª 2010 FEBS
BC1534-For and BC1534-Rev for bc1534, BC3461-
For and BC3461-Rev for bc3461, BC1535-For (5¢-AT
GAAATTAAAAATAGGTATTACAT-3¢) and BC1535-
Rev (5¢-TTAAATCTTTCCATTTTTGTCATC-3¢)forbc1535,
BC3725-For (5¢-ATGTTAAACAAGTTCAAATTTTTTT-3¢)
and BC3725-Rev (5¢-TTATTTTTGCAAGGAAAGACC
AT-3¢) for bc3725.
Construction of BC1534 variants
Mutants were constructed using a two-step ⁄ four-primer
overlap ⁄ extension PCR method [38]. The amplified product
was sub-cloned into the pET26b vector. The sequences of
the primer pairs were R140A-For (5¢-TTCACCACAT

GCT
GTGGAGTCTTTTTAT-3¢) and R140A-Rev (5¢-ATAAA
AAGACTCCAC
AGCATGTGGTGAA-3¢) for point muta-
tion R140A; R140E-For (5¢-TTCACCACAT
GAGGT
GGAGTCTTTTTAT-3¢) and R140E-Rev (5¢-ATAAAAA
GACTCCAC
CTCATGTGGTGAA-3¢) for point mutation
R140E; A42S-For (5¢-TTAACAGAA
TCTGATCTTTC
TTCAAAT-3¢) and A42S-Rev (5¢-TTTGAAGAA
AGA
TCAGATTCTGTTAAA-3¢) for point mutation A42S;
D112N-For (5¢-GCCATCCA
AATCATGCAAATTG-3¢)
and D112N-Rev (5¢-CAATTTGCATG
ATTTGGATG
GC-3¢) for point mutation D112N; K207I-For (5¢-AA
GATGTTTGGA
ATAGAAGTTCGA-3¢) and K207I-Rev
(5¢-ACTCCAACTTC
TATTCCAAACATC-3¢) for point
mutation K207I; Y194F-For (5¢-TAACAGAAGGT
TTC
GTTGAAACT-3¢) and Y194F-Rev (5¢-CAGTTTCAAC
GAAACCTTCTGTTA-3¢) for point mutation Y194F.
Engineered codons are underlined. Deletion of the gene
fragment encoding the b8-strand was achieved by PCR.
pET26b-bc1534 was used as the template, and the primers

used were BC1534-For and delb8-Rev (5¢-CCG
CTC
GAGACTCATAAATCCCTCGGCAT-3¢; XhoI restriction
site underlined).
Autodocking
Automated docking simulations were performed using
AutoDock4 [39–41] using the crystal structure of BC1534
(2ixd.pdb) to generate atomic energy grids. Point mutations
were introduced at positions 140 and 42 using Xtal-
View ⁄ Xfit [42] and ⁄ or MIFit (Rigaku Americas Corp.). All
water and acetate atoms were removed. Hydrogen atoms
were added to the crystal structure, and the substrates and
atomic partial charges were assigned to the protein and car-
bohydrate atoms. Atomic interaction energy grids were
determined using probes corresponding to each atomic type
found in the ligand at 0.375 A
˚
grid positions in a cubic box
of 30 A side length centered on the active site. After dock-
ing, all structures generated for a single compound were
assigned to clusters with an rmsd among the structures of
the same cluster no greater than 1. For each ligand, the glo-
bal minimum structure is discussed here. The figure 3 was
prepared using the programs pymol [43] and GIMP (http://
www.gimp.org/).
Alignment-based tree
Alignments of LmbE proteins were performed using
clustalw [44]. Alignment-based trees were constructed
using neighbor-joining and maximum-likelihood algorithms
implemented in the software package PHYLIP [45]. In all

cases, the topologies of the trees were very similar, and they
were identical with respect to the proteins of known func-
tion. Information about the gene context was obtained
using the Integrated Microbial Genomes system [46].
Transcription profiles of B.cereus genes were inferred
from transcription analysis studies on other Bacillus gen-
omes. Orthologs of B.cereus genes to genes with transcrip-
tion data from other Bacillus species were identified using
the Integrated Microbial Genomes [46], and the corre-
sponding transcription profiles were mapped to the query
genes. The transcriptional expression data were clustered
using the program cluster (v3.0) [47] using the Pearson
correlation similarity metric.
Acknowledgements
We thank Michael D. Urbaniak (Division of Biologi-
cal Chemistry and Drug Discovery, College of Life
Sciences, University of Dundee, Dundee DD1 5EH,
Scotland, UK) for testing BC1534 enzyme on GlcNAc-
I-P-C
18
and Georges Feller (Laboratory of Biochemis-
try, Center for Protein Engineering, University of
Lie
`
ge, Lie
`
ge, Belgium) for the N-terminal analysis of
selected protein samples. This work was supported by
research grant PENED 2003 03ED737 from the Gen-
eral Secretariat for Research and Technology of

Greece.
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