Cross-functionalities of Bacillus deacetylases involved in bacillithiol biosynthesis and bacillithiol-S-conjugate detoxification pathways

Article abstract of DOI:10.1042/BJ20130415

BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the acetyl group from N-acetylglucosamine malate to generate glucosamine malate. In Bacillus anthracis, BA1557 has been identified as the N-acetylglucosamine malate deacetylase (BshB); however, a

Full text of DOI:10.1042/BJ20130415

Biochem. J. (2013) 454, 239–247 (Printed in Great Britain) doi:10.1042/BJ20130415
239
Cross-functionalities of Bacillus deacetylases involved in bacillithiol
biosynthesis and bacillithiol-S-conjugate detoxification pathways
Zhong FANG*, Alexandra A. ROBERTS†, Karissa WEIDMAN*, Sunil V. SHARMA†, Al CLAIBORNE‡, Christopher J. HAMILTON†
and Patricia C. DOS SANTOS*1
*Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, U.S.A., †School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.,
and ‡Center for Structural Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27109, U.S.A.
BshB, a key enzyme in bacillithiol biosynthesis, hydrolyses the
acetyl group from N-acetylglucosamine malate to generate gluc-
osamine malate. In Bacillus anthracis, BA1557 has been identi-
fied as the N-acetylglucosamine malate deacetylase (BshB); how-
ever, a high content of bacillithiol (∼70%) was still observed in
the B. anthracis ꢀBA1557 strain. Genomic analysis led to the pro-
posal that another deacetylase could exhibit cross-functionality in
bacillithiol biosynthesis. In the present study, BA1557, its para-
logue BA3888 and orthologous Bacillus cereus enzymes BC1534
and BC3461 have been characterized for their deacetylase activity
towards N-acetylglucosamine malate, thus providing biochemical
evidence for this proposal. In addition, the involvement of
deacetylase enzymes is also expected in bacillithiol-detoxifying
pathways through formation of S-mercapturic adducts. The
kinetic analysis of bacillithiol-S-bimane conjugate favours the in-
volvement of BA3888 as the B. anthracis bacillithiol-S-conjugate
amidase (Bca). The high degree of specificity of this group of
enzymes for its physiological substrate, along with their similar
pH–activity profile and Zn2 + -dependent catalytic acid–base
reaction provides further evidence for their cross-functionalities.
Key words: bacillithiol, bimane, BshB, deacetylase, N-
acetylglucosamine malate, zinc hydrolase.
So far, BSH-deficient mutants have been shown to display
impaired sporulation, sensitivity to acid and salt, increased
sensitivity to fosfomycin [8] and reduced viability in mouse
macrophage cell lines [9]. A detailed understanding of the
enzymes mediating BSH biosynthesis and BSH-dependent drug
resistance is central to identifying whether and how such
biological targets could be exploited for the design of new
antibiotic chemotherapies.
The three-step biosynthetic pathway for BSH (Figure 2)
shares similarities with the key steps in MSH biosynthesis.
BSH biosynthesis is initiated by a retaining glycosyltransferase
(BshA) that catalyses the glycosylation of L-malic acid to
afford GlcNAc-Mal (N-acetylglucosamine malate) [8,10]. An N-
acetylhydrolase (BshB) then liberates the free amine GlcN-Mal
(glucosamine malate) [8,10]. Gene-knockout studies in B. subtilis
have identified a bacillithiol synthase (BshC) [8], proposed to
catalyse the final conversion of GlcN-Mal into BSH, but attempts
to demonstrate the BSH synthase activity of BshC in vitro have
so far been unsuccessful.
In many bacilli (e.g. B. subtilis, B. anthracis and B. cereus),
bshA and bshB are adjacent genes located in the same operon
[8] (Table 1). Whereas BshA and BshC are essential for BSH
biosynthesis, in many cases bshB-knockout mutants display only
a partial reduction in BSH levels [8,10]. This has been attributed to
the presence of one, or more, additional bshB-like gene products,
which can also N-deacetylate GlcNAc-Mal. In B. subtilis, for
example, a ꢀbshB1 (ypjG) mutant retains almost half of the
WT (wild-type) levels of BSH, whereas the ꢀbshB1 + ꢀbshB2
(yojG) double-knockout strain showed no detectable levels of
BSH [8,10]. Although in vivo data provide a strong indication
INTRODUCTION
The cysteine-containing tripeptide glutathione (GSH) is the major
low-molecular-mass thiol in eukaryotes and many Gram-negative
bacteria (Figure 1). In addition to its major role in thiol-redox
homoeostasis [1], GSH is also widely implicated in xenobiotic
detoxification. This is facilitated by GSTs, which can catalyse S-
conjugation of GSH to a diverse range of electrophilic xenobiotics
[2]. In Gram-negative bacteria, GSH plays a sacrificial role in
electrophile detoxification as these glutathione S-conjugates are
directly exported from the cell [3].
Many Gram-positive bacteria lack GSH, but instead produce
other low-molecular-mass thiols. Mycothiol (MSH) (Figure 1)
is the dominant low-molecular-mass thiol in Actinobacteria
(formerly known as Actinomycetes) (e.g. Mycobacterium,
Corynebacterium and Streptomyces), which serves functions
analogous to those of GSH [4,5]. However, one notable
difference is that MSH-dependent drug detoxification pathways
utilize a mycothiol-S-conjugate amidase (Mca) to hydrolyse the
mycothiol S-conjugate amide linkage to liberate the CysNAc (N-
acetylcysteine) S-conjugate (mercapturic acid), which is exported
from the cell, and glucosamine inositol, which is recycled back
into the MSH biosynthetic pathway [6].
In 2009, bacillithiol (BSH) (Figure 1) was identified as
a low-molecular-mass thiol among many low-G + C Gram-
positive bacteria (Firmicutes) lacking GSH or MSH [7].
These include Bacillus spp. (e.g. B. anthracis, B. subtilis, B.
cereus, B. megaterium and B. pumilis) and some, but not all,
staphylococci (e.g. Staphylococcus aureus and Staphylococcus
saprophyticus) and streptococci (e.g. Streptococcus agalactiae).
Abbreviations used: Bca, bacillithiol-S-conjugate amidase; BS–Fos, bacillithiol–fosfomycin; BSH, bacillithiol; BSmB, bacillithiol-S-bimane; CysmB,
cysteine monobromobimane derivative; DETAPAC, diethylenetriaminepenta-acetic acid; FU, fluorescence unit(s); GlcNAc-Mal, N-acetylglucosamine
malate; GlcNAc-OBn, O-benzyl-N-acetylglucosamine; GlcNAc-OMe, O-methyl-N-acetylglucosamine; GlcN-Mal, glucosamine malate; ICP-AES,
inductively coupled plasma atomic emission spectroscopy; IMAC, immobilized metal-ion-affinity chromatography; mBBr, monobromobimane; Mca,
mycothiol-S-conjugate amidase; MSH, mycothiol; NDA, naphthalene-2,3-dialdehyde; WT, wild-type.
1
To whom correspondence should be addressed (email dossanpc@wfu.edu).
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240
Z. Fang and others
Figure 1 Low-molecular-mass thiols found in bacteria
Table 1 Functional assignment of BSH biosynthetic and detoxification
genes
Organism
BshA
BshB
Bca/BshB2
B. anthracis
B. cereus
B. subtilis
S. aureus
BA1558
BC1535
BSU22460 (ypjH)
SA2981_1414
BA1557
BC1534
BSU22470 (ypjG)
–
BA3888
BC3461
BSU19460 (yojG)
SA2981_0544
activity was observed [8,10]. Although it is anticipated that
GlcNAc-Mal is the physiological substrate of BC1534, the
participation of BC1534 and/or BC3461 in BSH biosynthesis
and/or detoxification has not been explored at all.
In the present study, we have investigated the BshB and
Bca properties of the B. anthracis N-deacetylases BA1557,
BA3888 and BA3524 as well as the orthologous B. cereus
enzymes BC1534 and BC3461 (Table 1). The results indicate
that both BA1557 and BA3888 (and the B. cereus homologues)
are competent in catalysing the N-deacetylation of GlcNAc-Mal,
whereas BA3888 showed activity against BSmB (bacillithiol-S-
bimane) adduct. Assays with GlcNAc-Mal substrate analogues
demonstrate that the malate portion of the molecule plays a role
in controlling substrate specificity. In addition, a site-directed
mutagenesis approach provides insight into the mechanistic
details of the Zn2 + -dependent catalytic acid–base reaction.
Figure 2 The proposed biosynthetic pathway and detoxification mechanism
of bacillithiol
MATERIALS AND METHODS
Materials
for cross-functionality of BshB and orthologous enzymes in
the biosynthesis of bacillithiol, biochemical validation of this
proposal has not been carried out. In Mycobacterium smegmatis,
functional redundancy was also observed for analogous N-
deacetylase in MSH biosynthesis (MshB). In this case 5–10%
of WT MSH levels are still produced in ꢀmshB mutants due to
the residing background N-deacetylase activity of Mca [11,12].
Interestingly, in S. aureus, which only contains a single bshB-
like gene, preliminary studies have indicated the presence of a
bacillithiol-S-conjugate amidase (Bca) pathway that is capable of
detoxifying the electrophilic xenobiotic monobromobimane [13].
Considering these in vivo observations, it seems plausible that
one or more of these BshB-like enzymes could also present Bca
activity in xenobiotic detoxification. In pathways involving BSH,
neither the identity of any Bca enzyme (the equivalent of Mca in
BSH-producing species) nor the kinetic analysis of enzymes able
to perform both reactions has been determined.
Before the discovery of BSH, the crystal structure of a B. cereus
Zn2 + -binding protein (BC1534) (shown in the present study to
be a BshB) was reported [14,15], which was characterized as
a potential chitin deacetylase on the basis of its observed N-
deacetylase activity with GlcNAc (Km, 3 μM; kcat, 2 s− 1) and
chitobiose (Km, 3 μM; kcat, 98 s− 1) [14,15]. Since then, initial
kinetic studies of the homologous (97% sequence identity)
B. anthracis enzyme (BA1557) have demonstrated its BshB
activity with GlcNAc-Mal, but no notable GlcNAc deacetylase
BSmB [16], GlcNAc-Mal [17], GlcNAc-OBn (O-benzyl-
N-acetylglucosamine) [18] and GlcNAc-OMe (O-methyl-N-
acetylglucosamine) [19] were chemically synthesized as
described previously. BS–Fos (bacillithiol–fosfomycin) was
enzymatically synthesized via FosB-catalysed S-conjugation of
BSH with fosfomycin [16]. GlcNAc was purchased from Acros,
and NDA (naphthalene-2,3-dialdehyde) was from Anaspec. All
other reagents were purchased from Fisher Scientific or Sigma.
Expression and purification of B. anthracis BA1557, BA3524 and
BA3888, and B. cereus BC1534 and BC3461
The cloning of BA1557 (BshB) was described by Parsonage
et al. [10]. The codon-optimized genes for BA3524 and BA3888
were synthesized by GenScript and subcloned into pET28a ( + )
(Novagen). BC1534 in pET26b ( + ) [15] and BC3461 in pET24a
( + ) were generously provided by Dr Vassilis Bouriotis and Dr
Vasiliki Fadouloglou (both at Department of Biology, University
of Crete, Heraklion, Crete, Greece). All variants of BA1557 were
prepared using the QuikChange® site-directed mutagenesis kit
(Stratagene), and confirmed by DNA sequencing. All proteins
used in the present study contained a C-terminal hexahistidine
tag.
BA1557, BC1534 and all BA1557 variants were purified using
the following general protocol. The plasmid was transformed into
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241
chemically competent Escherichia coli BL21(DE3) cells, which
were plated on LB plates with 40 μg/ml kanamycin in a 37◦C
incubator overnight. For protein expression, 3 litres of LB broth
supplemented with 40 μg/ml kanamycin were inoculated with
freshly transformed cells and incubated at 37◦C with shaking at
300 rev./min. Expression was induced upon addition of 5.8 mM
lactose when the D600 reached 0.5, and the culture was incubated
further at 15◦C with shaking at 300 rev./min overnight before
being harvested by centrifugation at 5000 g for 10 min at 4◦C.
The cells were stored at − 20◦C until further use. Cells were
resuspended with 25 mM Tris/HCl (pH 8.0), 150 mM NaCl and
10% glycerol (buffer A), and then lysed using an EmulsiFlex-
C5 high-pressure homogenizer. The cell debris was separated
by centrifugation at 12000 g for 30 min, and the supernatant
was cleared upon treatment with 1% (w/v) streptomycin sulfate
followed by centrifugation at 12000 g for 20 min. After the
treatment, the clear supernatant was loaded on to pre-equilibrated
(buffer A) Zn2 + - or Co2 + -IMAC (immobilized metal-ion-affinity
chromatography) columns (GE Healthcare). The column was
washed with buffer A until the A280 reached baseline. Proteins
associated with the resin were eluted in a step gradient of 5%,
15% and 50% of buffer B (25 mM Tris/HCl, pH 8.0, 150 mM
NaCl, 300 mM imidazole and 10% glycerol). The fractions eluted
from 50% of buffer B containing pure proteins were diluted five
times with buffer C (25 mM Tris/HCl, pH 8.0 and 10% glycerol),
and loaded on to a 5 ml Hitrap Q FF (GE Healthcare) column
pre-equilibrated with buffer C. The desired protein was eluted
with 0.5 M NaCl in the same buffer. Protein concentration was
determined by the Bradford assay using BSA as the standard
[20]. Protein aliquots were frozen with liquid nitrogen and stored
at − 80◦C.
were analysed at 5 μM in 10 mM phosphate buffer (pH 7.4) and
scanned from 250 nm to 190 nm with 0.5 nm increments. The
final spectrum of each sample is the average of ten scans.
Enzyme assays for N-deacetylation of GlcNAc-Mal, GlcNAc-OBn,
GlcNAc-OMe and GlcNAc
The deacetylation activity was assayed by quantification of the
primary amine product. In general, a 200 μl assay mixture
containing 50 mM Mops (pH 7.4) and 0.1–20 μg of enzyme was
pre-equilibrated at 37◦C for 5 min. Reactions were initiated by
the addition of substrate. Sample aliquots (20 μl) were taken at
different time points and quenched with 20 μl of acetonitrile.
The primary amine products (GlcN, GlcN-Mal, etc.) were then
derivatized by addition of 200 μl of freshly prepared NDA-mix
(0.4 M borate, pH 9, 2.5 mM KCN and 0.5 mM NDA). The
solution mixture was allowed to react in the dark for 30 min
before being read in a flat-bottom microplate (Costar) using
a Synergy H1 plate reader (Biotek) with λex 390 nm and λem
480 nm. Initial velocity was determined from the slope of a plot
of FU (fluorescence units) against time using four different time
points over a period of up to 3 h. A glucosamine standard curve
was used to convert the FU/min slopes into [product]/min; the
fluorescence intensity of the GlcN–NDA product was identical
with that of the GlcN-Mal–NDA. Initial deacetylation rates of
GlcNAc-Mal were measured over the concentration range 0–
3.5 mM. For determination of the steady-state parameters, results
representing the means of triplicate values were fitted to the
Michaelis–Menten equation using SigmaPlot 11.0.
Enzyme assays for amidase activity with BSmB, BSH and BS–Fos
Because of the poor solubility experienced with BA3888 in
pET vector expression, the gene was subcloned into the pBAD
vector. The purification of pBAD-BA3888 and pET24a-BC3461
used the same procedure. The plasmids were transformed into
E. coli BL21(DE3) cells, and cells were plated on LB agar with
100 μg/ml ampicillin or 40 μg/ml kanamycin. The inoculum was
prepared with several colonies taken from a plate and transferred
into 100 ml of LB broth with appropriate antibiotics. After 1 h of
incubation at 37◦C with shaking at 300 rev./min, the inoculum
was added to 4 litres of LB broth with antibiotics and incubated
at 37◦C with shaking at 300 rev./min. Protein expression was
induced at a D600 of 0.4 by the addition of 5.8 mM lactose or
20 mM arabinose (final concentrations). The culture was shaken
at 37◦C for another 4 h before being harvested by centrifugation.
The cells were lysed as described above, and the soluble proteins
were loaded on to a nickel-IMAC column pre-equilibrated with
buffer A. The column was washed with buffer A, and proteins
associated with the column were eluted with a step gradient (5%,
15%, 30%, 50% and 100%) of buffer B. Fractions containing
deacetylase activity were pooled, diluted 6-fold with 25 mM
Hepes (pH 8.0) and 10% glycerol (buffer D), and loaded on
to a MonoQ column (GE Healthcare) equilibrated with buffer D.
Proteins associated with the column were eluted in a 20 ml linear
gradient from 0 to 0.75 M NaCl in buffer D. Pure protein fractions
(by SDS/PAGE) containing activity were combined and aliquots
of 20 μl were then frozen in liquid nitrogen for storage at − 80◦C.
Within 10 days of the purification date, freshly thawed aliquots
were used for kinetic experiments.
Amidase activity of BSH was assayed by quantification of
the mBBr (monobromobimane) derivative of cysteine (CysmB)
produced during hydrolysis of BSmB. A representative assay
contained 1.5–20 μg of enzyme in 200 μl of 50 mM Mops
(pH 7.4) pre-warmed at 37◦C for 5 min. The reaction was initiated
by addition of BSmB. After incubation for various time intervals,
reaction aliquots (10 μl) were quenched by the addition of 20 μl of
acetonitrile followed by centrifugation (5 min). The supernatant
(20 μl) was diluted 50-fold with 5 mM HCl before analysis by
HPLC using procedures described previously [7].
The amidase activity towards BSH was assayed by
quantification of cysteine formation by derivatization of the thiol
with mBBr. The assay was performed by first equilibrating 40 μM
of the enzyme in 50 mM Hepes buffer (pH 7.4) for 10 min at 37◦C.
The reactions were initiated upon addition of substrate (0.2–
10 mM BSH) followed by incubation for 30 min at 37◦C. Aliquots
of 10 μl were collected at various time points and the reaction was
quenched by heating at 80◦C for 10 min. Reaction aliquots (4 μl)
were then derivatized with 6 μl of 100 mM mBBr for 15 min
at room temperature (25◦C) to label the cysteine product and
quenched upon addition of 0.25 μl of 5 M methanesulfonic acid
and centrifuged at 10000 g for 5 min. Finally, the supernatant
was diluted with 100 μl of 10 mM methanesulfonic acid and
50 μl of sample was analysed by HPLC as described previously
[7]. The activities were calculated from a calibration curve using
CysmB as the standard. The means of three duplicates were fitted
into the Michaelis–Menten equation to determine the steady-state
parameters.
The activity towards BS–Fos was assayed by quantifying
cysteine–fosfomycin and GlcN-Mal using the AccQ Fluor reagent
kit (Waters) to detect the primary amine products. A solution
containing 40 μM of enzyme in 50 mM Hepes (pH 7.4) was
pre-warmed at 37◦C for 5 min and the reaction was started
CD spectroscopy
CD spectra were obtained using an Aviv CD spectrometer (Model
215; AVIV Biomedical) with a 1-mm-pathlength quartz cuvette
(Hellma Analytics) and a bandwidth of 1 nm. Protein samples
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242
Z. Fang and others
upon addition of 0.5 mM BS–Fos. After 30 min, reactions were
quenched by heating the samples at 85◦C for 10 min. The
derivation of amine products with AccQ Fluor reagent was
performed as recommended by the manufacture. The samples
were assayed via HPLC as described previously [21].
Preparation of metal-free deacetylase and metal reconstitution
Figure 3 Structures of substrates included in Table 2
To prepare metal-free BA1557, Co2 + -IMAC-purified protein
(∼75 μM) was incubated with 25 mM Tris/HCl (pH 7.5),
25 mM DETAPAC (diethylenetriaminepenta-acetic acid) and
10% glycerol on ice for 30 min. The protein solution was
then dialysed three times over 2 litres of 25 mM Tris/HCl
(pH 7.5) and 10% glycerol buffer at 4◦C. The concentration of
residual metal ion was determined to be less than 5% by ICP-
AES (inductively coupled plasma atomic emission spectroscopy)
(Tededyne Leeman Labs) analysis.
Ph, phenyl.
Table 2 Substrate specificity of B. anthracis and B. cereus N-deacetylases
Allassayswereperformedinthepresenceof5 mMsubstratewith0.23–6.25 μMenzyme, except
for GlcNAc-Mal, which was assayed in the presence of 0.5 mM substrate with 0.033–1.05 μM
enzyme.
Specific activity (nmol/min per mg)
For the reconstitution with Zn2 + , apo-BA1557 (10 μM) was
incubated with different stoichiometric ratios of ZnSO4 on ice for
30 min. The solution was then dialysed against 1 litre of 25 mM
Tris/HCl (pH 8.0) and 10% glycerol at 4◦C to remove unbound
Zn2 + , and the protein metal content was measured by ICP-AES.
Substrate
BA1557
BC1534
BA3888
BC3461
+
+
+
GlcNAc
0.55 0.01
<0.05
0.87 0.12
<0.05
<0.05
0.46 0.025
−
−
−
+
+
GlcNAc-OMe
GlcNAc-OBn
GlcNAc-Mal
BSmB
4.55 0.32
0.8 0.02
−
−
+
+
+
2.00 0.04
1.90 0.02
<0.05
0.36 0.02
−
−
−
+
+
+
+
14227 192
17705 207
8430 490
10.7 0.3
−
−
−
−
+
+
0.363 + 0.058
0.226
73.7 7.9
2.58
0.4 0.05
1.1
−
−
BSH
1.3
0.7
pH-dependent activity profiles
For the pH-dependence experiments, the following buffers were
used: 50 mM Mes (pH 6.0–7.0); 50 mM Mops (pH 7.0–8.0);
50 mM bicine (pH 8.0–9.0); and 50 mM borate (pH 9.0–10.0).
The standard assay protocol was conducted by equilibrating
200 μl of buffer containing 0.4 μg of deacetylase at 37◦C.
The deacetylation reaction was initiated by the addition of
0.25 mM GlcNAc-Mal. Samples (20 μl) were taken at intervals,
and the reaction was terminated by addition of an equal volume
of acetonitrile. Steady-state kinetic parameters Km, kcat and
kcat/Km for deacetylase activity were determined by fitting initial
velocities to the Michaelis–Menten equation. Eqn (1) was fitted to
the pH rate profile, wherein V is the observed rate of the reaction,
K is the pH-independent rate constant for GlcNAc or GlcNAc-
Mal substrates, and Ka and Kb are the ionization constants of the
acid and base species respectively [22]:
and background signal associated with other primary amines
present in the reaction mixture is a recurring limitation of such
amine-specific fluorescence-based assays [25]. Nevertheless, the
high sensitivity of this method allowed us to optimize the use of
synthetic substrates (Table 2 and Figure 3), many of which were
only available in limited quantities.
+
The activity of BA1557 with GlcNAc-Mal (19 1.1 μmol/min
−
per mg), when tested using this method, was comparable
+
with that obtained with the AccQ tag (21.5 0.2 μmol/min
−
+
per mg) or fluorescamine procedures (19.8 1.0 μmol/min per
−
mg). BA3888 also displayed comparable substrate kinetics with
those of GlcNAc-Mal, thereby demonstrating the functional
redundancy of BA1557 in the second step of BSH biosynthesis
(Table 3 and Supplementary Figure S1A at http://www.biochemj.
org/bj/454/bj4540239add.htm). The kinetic analysis of both
enzymes fits well with the in vivo data, which demonstrated a
70% reduction in BSH levels in the B. anthracis ꢀbshB strain
[10]. As expected, the orthologous BC1534 (97% sequence
identity) displays very similar kinetic behaviour in the BshB
reaction (Table 3 and Supplementary Figure S1A), supporting
the proposed role for this enzyme in the biosynthesis of BSH.
The catalytic efficiencies (kcat/Km) of BA1557 and BC1534 with
GlcNAc were four orders of magnitude lower than that determined
against GlcNAc-Mal, whereas BA3888 showed no detectable
activity (Table 3 and Supplementary Figure S1A). The calculated
kcat/Km values for BA1557 and BC1534 with GlcNAc were
substantially (six orders of magnitude) lower than the values
reported previously for the B. cereus enzyme [15], but similar
to the values reported for B. anthracis [10].
Additional N-deacetylase candidates, BA3524 and BC3461,
were investigated in the BshB reaction. The two enzymes are 95%
identical in sequence and are likely to perform similar intracellular
functions (Supplementary Figure S2B at http://www.biochemj.
org/bj/454/bj4540239add.htm). Phylogenetic analysis shows that
they constitute a separate branch of N-acetylhydrolases, different
from those including BA1557 or BA3888 (Supplementary Figure
S3 at http://www.biochemj.org/bj/454/bj4540239add.htm). In
our hands, BC3461 showed limited activity with GlcNAc-Mal
1
V/K =
(1)
1 + 10−pH/Ka + Kb/10−pH
RESULTS
Cross-functionality of deacetylases in BshB catalysis
Previous work described the ability of BA1557 to catalyse the
hydrolysis of GlcNAc-Mal using an assay that involved HPLC
analysis of GlcN-Mal product formation after derivatization
with the amine-specific fluorophore (AccQ tag) [10]. In the
present study, BA1557 and other BshB-like enzymes have been
extensively characterized through the application of a faster assay
procedure that bypasses the need to quantify derivatized reaction
products by HPLC separation. This was achieved by developing
a fluorescence microplate assay to detect GlcN-Mal formation
following derivatization of the secondary amine with NDA [23].
This method is more cost-effective and faster than the commercial
AccQ tag kit [10], and is ∼50-fold more sensitive than methods
involving a direct fluorescamine-based procedure [24]. Despite
the aforementioned benefits of this method, NDA-derivatization
of primary amines is not specific to the deacetylation product
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Kinetic analysis of BshB and Bca enzymes
243
Table 3 Kinetic parameters of B. anthracis and B. cereus N-deacetylases
ND, not determined.
Enzyme
BA1557
Substrate
Km (mM)
kcat (s− 1
)
kcat/Km (s− 1·M− 1
)
Reference
+
+
+
GlcNAc-Mal
GlcNAc-Mal
GlcNAc
GlcNAc
BSH
BSmB
GlcNAc-Mal
GlcNAc
BSH
BSmB
GlcNAc-Mal
GlcNAc
GlcNAc
GlcNAc-Mal
GlcNAc
GlcNAc
0.19 0.03
0.16
8.24 0.46
42
43300 7300
The present study
[9]
The present study
−
−
−
262000
− 3
+
+
+
57.1 8.5
(3.3 0.14)×10
0.058 0.01
−
−
−
ND
∼8×10− 5
[9]
− 4
− 4
+
+
+
0.48 0.05
(7.0 0.15)×10
1.4 0.18
The present study
The present study
The present study
The present study
The present study
The present study
The present study
The present study
[14]
−
−
−
+
+
+
0.32 0.05
(2.0 0.10)×10
0.7 0.13
−
−
−
+
+
+
BA3888
0.32 0.09
6.48 0.66
20300 6000
−
−
−
–*
–*
–*
− 3
+
>5†
(1.1 0.14)×10
−
+
>5†
0.031 0.0033
−
+
+
+
BC1534
BC3461
0.20 0.01
10.80 0.22
54000 2900
0.12 0.014
−
−
−
− 3
+
+
+
16.9 1.8
(2.0 0.1)×10
−
−
−
0.003
1.89
6.3×105
+
+
+
0.21 0.03
–*
0.068 0.003
320 4.8
The present study
The present study
[14]
−
−
−
–*
–*
0.009
6.4
7.1×105
*Activity was not detected when using 1–10 mM substrate.
†Rate against substrate concentration was still in the linear region at the maximum concentration assayed (5 mM); the reaction rate under these conditions was used to calculate turnover rate.
(Table 3), whereas BA3524 displayed no detectable activity.
Far-UV CD spectra and ICP-AES analyses of these proteins
indicated comparable secondary structure, and Zn2 + content with
those of BA1557, BA3888 and BC1534 (Supplementary Figure
S4 at http://www.biochemj.org/bj/454/bj4540239add.htm). This
observation indicates that the lack of activity seems not to be
attributed to protein misfolding or lack of metal cofactor resulting
from heterologous E. coli expression.
Zn2 + -dependent deacetylase activity
The Zn2 + -binding site identified in the structure of BC1534
(BcZBP) displayed a similar arrangement to that found in
the active sites of other Zn2 + -dependent deacetylases [14].
Like the MshB structure, the BC1534 active site displays
two histidine residues and one aspartate residue (His12, His113
Figure 4 Active-site region of B. cereus BC1534 (PDB code 2IXD)
and Asp15) providing a facial triad Zn2 + co-ordination [14,26]
The Zn2 + ion is co-ordinated with His12, Asp15 and His113
.
(Figure 4). All three metal-ion-co-ordinating residues are strictly
conserved in the MshB and Mca sequences, in addition to
the BshB and Bca candidate enzymes from B. anthracis
and other BSH-producing species (Supplementary Figure S2).
BA1557 can also be activated upon stoichiometric addition of
other metal cations such as Ni2 + , Co2 + and Fe3 + (Supplementary
Table S1 at http://www.biochemj.org/bj/454/bj4540239add.htm).
These results indicate that BA1557 shares similar profiles for
non-specific metal-dependent deacetylation, comparable with the
characterized cambialistic behaviours of MshB [28], LpxC [29]
and the histone deacetylases [30].
Hexahistidine-tagged recombinant BA1557, purified by Zn2 +
-
2 +
+
affinity chromatography contains 2.55 0.04 Zn cations per
−
monomer. Treatment with 20 mM EDTA or EGTA had a minor
effect on either metal content or enzyme activity (less than
20% decrease upon 1 h of incubation), whereas treatment with
the stronger metal-chelating reagent DETAPAC removes nearly
98% of the bound Zn2 + and almost completely inactivates the
enzyme. This indicates that Zn2 + is a tight-binding metal ion
cofactor in this enzyme. The apo form of the enzyme displays an
identical far-UV CD spectrum, indicating that neither DETAPAC
treatment nor Zn2 + displacement causes major changes to
secondary structure (Figure 5, inset). Zn2 + titration of the
apo form completely reconstitutes the activity of the enzyme,
correlating with a stoichiometry of one Zn2 + /mol of enzyme
(Figure 5). Further addition of >1 molar equivalent of Zn2 + causes
inhibition. This Zn2 + activation/inhibition profile is very similar
to that observed for UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc
General acid–base catalysis
The pH–activity profile for the BshB reaction with BA1557
presents a bell-shaped curve indicative of general acid–base
catalysis [22]. The maximum catalytic activity at subsaturating
concentrations of GlcNAc-Mal was reached at pH 7.8. The
pH–activity curve fit is consistent with two ionization states
with associated pKa values of 6.5 and 8.5. Interestingly,
BA3888 and BC1534 displayed nearly identical pH–activity bell-
shaped curves and similar ionization constants (Figure 6A and
Table 4). The pH profile of Zn2 + - or Co2 + -reconstituted BA1557
displayed similar ionization events (Supplementary Figure S5 at
http://www.biochemj.org/bj/454/bj4540239add.htm). Likewise,
deacetylase (LpxC). It has been proposed that LpxC has two Zn2 +
binding sites, one necessary for catalytic activity and a second
site with allosteric inhibitory properties [27]. In addition to Zn2 +
-
,
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244
Z. Fang and others
acid and general base respectively. Both residues are strictly
conserved in all members of this family of enzymes including
the BshB, MshB and Mca sequences (Supplementary Figure S2).
ND1 of His110 and OD2 of Asp14 are located on opposite sides of
the acetate group that interacts with Zn2 + , approximately 4 Å
(1 Å = 0.1 nm) from the metal centre. Individual alanine
substitutions of His110 and Asp14 in BA1557 resulted in decreased
activity levels of 4-fold and 3000-fold respectively (Figure 6B and
Table 5). The pH–activity profile of H110A BA1557 retained a
bell-shaped curve, ruling out the possible participation of this
residue as the acid or base in this mechanism. Interestingly,
in the structure of BC1534, OD2 of Asp112 is located within
hydrogen-bonding distance from both NE2 of His110 and NE2
of His12, which is a known ligand for the Zn2 + (Figure 4).
In BC1534, the D112A substitution completely eliminates the
GlcNAc deacetylase activity of this enzyme [15]. Unexpectedly,
the D14A BA1557 variant retained the ionization event associated
with pKa1, but not that for pKa2, suggesting its role as a general
acid in the reaction. Although the identity of the general base
remains unknown, the pH–activity profile of the D14A variant
eliminates the possibility of any dual involvement of this residue,
in both ionization events of this reaction mechanism.
Figure 5 Deacetylation activity of apo-BA1557 reconstituted with different
stoichiometric ratios of Zn2 +
The preparation and reconstitution of apo-BA1557 were described in the Materials and methods
section. The assays were conducted with 0.2 μg of enzyme (0.035 μM) in the presence
of 0.25 mM GlcNAc-Mal. The inset panel shows the far-UV CD spectrum of holo-BA1557
(continuous line) and apo-BA1557 (broken line).
Gatekeepers of substrate binding and hydrolysis
The very low catalytic efficiency of the enzyme with GlcNAc
compared with GlcNAc-Mal prompted the investigation of
substrate analogues where the malate aglycone was replaced by
an uncharged methyl or benzyl motif. Table 2 shows that all
substrates lacking malate at this position were at least three orders
of magnitude less effective as substrates than GlcNAc-Mal. An
in silico auto-docking approach was used to explore potential
binding modes of GlcNAc-Mal with BC1534 [31]. The results
from these docking experiments pointed to two arginine residues,
Arg53 and Arg109, as providing potential electrostatic interactions
with the substrate (Supplementary Figure S6 at http://www.
biochemj.org/bj/454/bj4540239add.htm). These residues are
located on opposite sides of the active site, ∼10 Å from each
other and ∼5 Å from the C1-position of bound acetate, whereas
the distances between the Zn2 + to the ω-N of Arg53 and Arg109
were 5.72 Å and 8.03 Å respectively. Interestingly, structural and
functional analyses of malate dehydrogenases have shown that
two arginine residues (∼12 Å apart) are involved in substrate
binding providing electrostatic interactions with both carboxy
groups of the L-malate substrate [32].
Figure 6 pH–activity profiles of deacetylase enzymes
(A) Relative activity (V/K) of BA1557 (᭜), BC1534 (᭢), BA3888 (᭿) and BC3461 (᭡, inset).
(B)Relativeactivity(V/K)ofBA1557WT(᭜), H110A(᭹)andD14A(᭡). Assaysweremeasured
with 0.25 mM GlcNAc-Mal under the pH range 5.97–9.56. All of the pH curves exhibit a bell
shape except for D14A which loses the basic limb. The pKa values were determined by fitting
the curve into eqn (1) in the text.
Table 4 pH-dependence of different enzymes/mutants for N-deacetylation
of GlcNAc-Mal
Enzyme
pKa
pKb
Compared with WT BA1557, an R53A mutation completely
eliminated the BshB activity, whereas a more conservative
R53K substitution displayed 103-fold lower activity for GlcNAc-
Mal and no detectable activity with GlcNAc (Table 5 and
Supplementary Figure S7 at http://www.biochemj.org/bj/454/
bj4540239add.htm). Both substitutions did not impair the
secondary structure of this enzyme; the far-UV CD spectrum was
nearly identical with that of the WT (Supplementary Figure S4).
Sequence alignment of several deacetylases including MshB and
Mca indicate that Arg53 is strictly conserved. It has been suggested
that Arg68 and His144 of MshB (equivalent to Arg53 and His110 of
BC1534 and BA1557) participate in electrostatic interactions with
a sugar hydroxy group of the substrate [26]. Our results with the
Arg53 mutants provide support for the involvement of this residue
in substrate binding. Arg109, however, is only conserved among a
group of deacetylases limited to the BshB1 candidates; including
BA1557, BC1534 and YpjG (Supplementary Figure S2). The
BA1557 R109K variant showed a 6-fold decrease in kcat for
GlcNAc-Mal (Table 5 and Supplementary Figure S7). Although
+
+
BA1557 (wild-type)
BA1557 (D14A)
BA1557 (H110A)
BA3888
BC1534
BC3461
6.50 0.1
6.82 0.02
8.60 0.2
–
−
−
+
−
+
+
6.66 0.08
8.05 0.04
−
−
+
+
6.82 0.06
8.50 0.05
−
−
+
+
6.58 0.2
8.60 0.02
−
−
+
+
6.64 0.05
8.94 0.04
−
−
the pH-dependence on the GlcNAc deacetylation reaction
displayed pKa values of 6.7 and 9.3 (Supplementary Figure S5).
These results discard the participation of the malate carboxy
group(s) of the substrate or the metal cofactor on either ionization
events displayed for this reaction. The similar pH–activity profile
of BshB enzymes suggests the participation of conserved residues
in the general acid–base catalytic mechanism of the BshB
reaction.
Close inspection of the protein environment surrounding the
Zn2 + cation at the active site of BC1534 (Figure 4) initially
suggested His110 and Asp14 as possible candidates for the general
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Kinetic analysis of BshB and Bca enzymes
245
Table 5 Steady-state kinetic parameters of BA1557 variants for N-deacetylation of GlcNAc-Mal
Substrate
Enzyme
Km (mM)
kcat (s− 1
)
kcat/Km (s− 1·M− 1
)
Relative efficiency
+
+
+
GlcNAc-Mal
Wild-type
D14A
H110A
R53A
0.19 0.03
0.15 0.03
8.24 0.46
(2.8 0.14)×10
43300 7263
1
−
−
−
− 4
4.1×10− 5
+
+
+
1.8 0.37
−
−
−
+
+
+
0.69 0.08
2.19 0.22
3170 486
0.073
−
−
−
–*
–*
–*
0
R53K
0.31 0.05
(1.4 0.1)×10
45 7.9
1.0×10− 3
− 2
+
+
+
−
−
−
+
+
+
R109K
Wild-type
R53K
R109K
H110A
0.24 0.03
1.29 0.16
5380 947
0.124
−
−
−
− 3
+
+
+
GlcNAc
57.1 8.5
(3.3 0.14)×10
0.058 0.010
1
0
2.76
0
−
−
−
–†
–†
–†
− 3
+
+
+
18.6 1.8
(2.9 0. 1)×10
0.16 0.016
−
−
−
− 3
+
–‡
(0.9 0.1)×10
‡
–‡
−
◦
*Assays were performed using 20 μg of enzyme and 0–5 mM GlcNAc-Mal in 50 mM Mops (pH 7.4) at 37 C.
†Assays were performed in the presence of 20 μg of enzyme and 0–120 mM GlcNAc in 50 mM Mops (pH 7.4) at 37 C.
◦
‡No substrate saturation was observed up to 180 mM GlcNAc, calculated kcat was based on the velocity at 120 mM.
conclusion supports in vivo demonstrations that inactivation of
bshB in B. anthracis and bshB1 (ypjG) in B. subtilis decreased,
but did not eliminate, the levels of BSH [8]. Kinetic analyses
of the BshB reactions with BA1557 and BA3888 show that
they display equivalent catalytic efficiencies towards GlcNAc-
Mal. However, BA3888 showed a nearly 200-fold higher activity
with BSmB compared with BA1557 (Table 2). The reactivity
of this enzyme towards larger bacillithiol conjugates provides
experimental evidence supporting the role of BA3888 as a catalyst
for the Bca reaction. However, it is worth noting that the substrate
kinetics for BSmB with BA3888 are still poor and it remains to
be seen what the physiologically relevant bacillithiol conjugate
substrate(s) is(are) for this enzyme. With BA1557, the very weak
amidase activity observed with BSH and a Km value comparable
with its cellular concentration indicates that BSH functions as
a feedback inhibitor of this enzyme. However, such feedback
inhibition is redundant in the presence of a functional BA3888
enzyme, which can substitute for the GlcNAc-Mal activity of
BA1557.
Interestingly, in S. aureus, only one BshB-like enzyme has been
identified [8]. Among the enzymes described in the present paper,
BA3888 is the closest orthologue of S. aureus BshB (70/49%
identity/similarity) supporting the notion that a single enzyme in
S. aureus could fulfil both BshB and Bca functions, although
this remains to be proved. Amino acid sequence alignment
indicates the presence of a ten-residue insertion (GDPFFANRET
in BA3888) in Bca/BshB2 sequences, including BA3888 and
S. aureus BshB (Supplementary Figure S2A). Although the
structural location of sequence is not known, adjacent residues
in the BC1534 structure constitute a short loop surrounding the
active site. It is possible that this decameric insertion sequence
could expand the flexibility of the active site to accommodate po-
tentially larger substrates as in the case of Bca. Nevertheless, the
low reactivity of these enzymes against BSmB/BS–Fos suggests
the possible occurrence of alternative pathways for detoxification
of such adducts and/or the involvement of this group of enzymes
in serving as amidases to a specific group of bacillithiol adducts.
Compared with GlcNAc-Mal, the low activity values against
GlcNAc, GlcNAc-OMe and GlcNAc-OBn reveals the high degree
of specificity for the malate aglycone. Our results from site-
directed mutagenesis pointed to BA1557 Arg109 as one potential
gatekeeper in controlling this specificity or stabilizing a reaction
intermediate. Although this model is favoured for BshB enzymes
containing arginine residues at positions equivalent to Arg109, this
scheme may not apply to all deacetylase enzymes capable of
catalysing the BshB reaction (e.g. BA3888 and BC3461 contain
valine at the equivalent position). Nonetheless, all four enzymes
this substitution impaired the catalytic efficiency of BA1557 with
GlcNAc-Mal, the R109K variant showed a modest improvement
in reactivity against GlcNAc. This variant enzyme had a lower
Km for GlcNAc (18.6 mM) and similar kcat (2.9×10− 3 s− 1) when
compared with the WT constants of 57 mM and 3.3×10− 3 s− 1
respectively (Table 5).
Deacetylase enzymes participating in detoxification pathways
BSH is already known to be implicated in some aspect of drug
detoxification as exemplified by BST (bacillithiol-S-transferase)
(FosB) [16]. A second class of BSTs have also recently
been identified, but their target electrophilic substrates are not
yet known [13]. On the basis of what has been observed
previously in MSH metabolism, enzymes catalysing the BshB
reaction (the hydrolysis of GlcNAc-Mal to yield GlcN-Mal)
could also potentially be capable of catalysing the hydrolysis of
bacillithiol S-conjugates produced in BSH-dependent pathways.
The proposed Bca enzyme is thought to be involved in the
subsequent step, thus utilizing the products of the BST reaction
as substrates. Because of the similarities in mechanism between
the BshB and Bca reactions, we have determined the reactivity
of BA1557 and BA3888 against BSmB, BSH and BS–Fos. The
activity of BA3888 in the hydrolysis of the CysmB side chain
of BSmB was 200-fold higher than that of BA1557; however,
at 5 mM BSmB the turnover rate of BA3888 was only 1.84
min− 1 (Table 2). Kinetic analysis of BA1557 and BA3888 with
BSH also showed low rates of BSH degradation by formation of
cysteine and GlcN, indicating that these enzymes do not contribute
to the pool of reduced cysteine levels. Interestingly, the Km of
BA1557 for BSH (0.48 mM) is in the same range as cellular
BSH concentrations, suggesting that BSH could act as a feedback
inhibitor of this enzyme (Table 3). On the other hand, the BA3888
Km for BSH was not determined since no substrate saturation
was reached up to 5 mM BSH. In our hands, neither BA1557
nor BA3888 showed any detectable activity against BS–Fos. The
remarkable selectivity of this group of enzymes for a restricted
subset of substrates highlights the importance for in vivo screening
of candidate compounds in strains lacking these enzymes.
DISCUSSION
In the present study, we performed comparative kinetic analysis
of the B. anthracis and B. cereus deacetylases in performing BshB
and Bca reactions to provide biochemical evidence for the cross-
functionality of these enzymes in the biosynthesis of BSH. This
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246
Z. Fang and others
tested in the present study showed a high degree of selectivity for
GlcNAc-Mal. Controlled substrate binding and hydrolysis may
serve as a molecular strategy used by this group of enzymes to
restrict the repertoire of physiological substrates.
FUNDING
Financial support for this work has been provided by a Multidisciplinary Research Grant
from the North Carolina Biotechnology Center [grant number M2011-MRG-1116 (to A.C.
and P.D.S.)] and the Biotechnology and Biological Sciences Research Council [grant
number BB/H013504/1 (to C.J.H)].
Functional assignment of residues surrounding Zn2 + at the
active site confirmed the involvement of Asp14 with the second
ionization event in the deacetylation mechanism. The abnormally
high pKa of 8.5 is 4 units above the pKa of free aspartate.
Large shifts in aspartate pKa (>4 units) have been reported
for the active-site aspartate residues of human thioredoxin and
bacteriorhodopsin [33]. In both cases, the active-site environment
surrounding the aspartate side chain favours the protonated acid
form during turnover. Studies on the MshB pH–activity profile
showed that the rate of deacetylation was also dependent on
two ionization events (pKa1 = 7.4 and pKa2 = 10.5) [34]. The first
ionization event was attributed to Asp15 which was proposed to
be the general base on this catalytic mechanism. Whereas there is
an agreement for the role of Asp15 as a general base in the MshB
mechanism, the identity of the general acid remains controversial.
Solvent isotope effect and site-directed mutagenesis experiments
suggest the participation of His144 as the general acid [34];
however, pH-dependence studies on H144A MshB [34] and recent
structural analysis of this enzyme [35] do not support this model.
A previous proposal has evoked the dual role of Asp15 as a single
general acid and base [26,35]. In this later model, it would be
expected that the pKa associated with the deprotonation of Asp15
(pKa2) would be higher than 7.4 (pKa1). In the structure of BA1534
[14], Asp14 and His110 occupy positions equivalent to Asp15 and
His144 of MshB [26,35]. Interestingly, in the present study, we
identified the pKa associated with Asp14 of BA1557 (pKa2 of 8.6,
Table 3) to be only 1.2 pH units higher than the pKa1 of the
MshB reaction. Collectively, these results support a mechanism
for the BshB reaction involving Asp14 as a general acid during the
protonation of the nascent amino group on GlcN-Mal product.
The identity of the residue associated with the first ionization
event awaits further investigation. Results of the present study rule
out the possible participation of His110 and Asp14 in performing
this function. The pH–activity curve of BA1557 using GlcNAc
as substrate showed an identical profile eliminating the potential
involvement of the malate carboxy groups of the substrate in the
first ionization event (Supplementary Figure S5). Alternatively,
the activation of a water molecule by the active-site Zn2 + , as
observed with carbonic anhydrase, or the participation of the
Zn2 + -co-ordinating Asp15 in abstracting the proton from this
water, provide possible candidates for the general base in
this mechanism. In any event, the conservation of amino acids
within the active site imposes a controlled substrate specificity
(along with conserved kinetic behaviour) of this group of
deacetylases and opens the door for further investigation into
their roles in BSH biosynthesis and BSH-mediated xenobiotic
detoxification.
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AUTHOR CONTRIBUTION
Zhong Fang, Alexandra Roberts, Karissa Weidman and Sunil Sharma performed
experiments. Al Claiborne and Patricia Dos Santos conceived the idea, and provided
overall direction. Zhong Fang, Christopher Hamilton and Patricia Dos Santos planned
experiments, analysed data and wrote the paper. All authors read and approved the content
of the paper.
ACKNOWLEDGEMENTS
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We thank Vassilis Bouriotis and Vasiliki E. Fadouloglou for the gift of expression plasmids
containing BC1534 and BC3461.
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Received 20 March 2013/6 June 2013; accepted 11 June 2013
Published as BJ Immediate Publication 11 June 2013, doi:10.1042/BJ20130415
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Biochem. J. (2013) 454, 239–247 (Printed in Great Britain) doi:10.1042/BJ20130415
SUPPLEMENTARY ONLINE DATA
Cross-functionalities of Bacillus deacetylases involved in bacillithiol
biosynthesis and bacillithiol-S-conjugate detoxification pathways
Zhong FANG*, Alexandra A. ROBERTS†, Karissa WEIDMAN*, Sunil V. SHARMA†, Al CLAIBORNE‡, Christopher J. HAMILTON†
and Patricia C. DOS SANTOS*1
*Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, U.S.A., †School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.,
and ‡Center for Structural Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27109, U.S.A.
Figure S1 Substrate saturation curves of BA1557 (᭡), BC1534 (᭿), BA3888 (᭹) and BC3461 (᭢) with GlcNAc-Mal (A) and GlcNAc (B) as substrates
Assays were performed with GlcNAc-Mal (0–3.36 mM) and GlcNAc (0–150 mM) at fixed concentrations of deacetylases as described in the Materials and methods section of the main text.
Table S1 Activity of BA1557 upon reconstitution with different metals
Assay was performed with 0.2 μg of apo-BA1557 reconstituted with one stoichiometric ratio of
different metal ion in the presence of 0.25 mM GlcNAc-Mal.
Metal ion
Velocity (μmol/min per mg)
Relative activity
+
Apo (<5%)
Fe3 +
1.5 0.3
7.1 0.2
0.20
0.95
1.50
1.00
3.01
0.40
1.91
−
+
−
Co2 +
11.3 1.1
+
−
Zn2 +
7.5 0.4
+
−
Ni2 +
22.6 9.6
+
−
Mg2 +
Ca2 +
3.0 0.1
+
−
+
14.3 3.4
−
1
To whom correspondence should be addressed (email dossanpc@wfu.edu).
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Figure S2 Sequence alignment of deacetylases
(A) Amino acid sequence alignment of deacetylase enzymes. ClustalW alignment using Gonnet series matrix including deacetylase sequences from B. anthracis str. Ames (BA1557, BA3888 and
BA3524), B. cereus ATCC 14579 (BC1534 and BC3461), B. subtilis subsp. subtilis str. 168 (BSU22470 and BSU19460), B. pumilus SAFR-032 (BPUM1978 and BPUM_1869), B. megaterium
DSM 319 (BMD_1367 and BMD_1967), S. aureus 04-02981 (SA2981_0544), S. saprophyticus subsp. saprophyticus ATCC 15305 (SSP2151), M. smegmatis str. MC2 155 (MSMEG5261 and
MSMEG5129), Mycobacterium tuberculosis H37Rv (Rv1082 and Rv1170), Corynebacterium efficiens YS-314 (CE1052 and CE1158) and Streptomyces coelicolor A3(2) (SCO4967 and SCO5126).
Shown is the N-terminal portion of the sequence alignment with completely conserved residues shaded in dark grey with white font, identical residues shaded in grey and similar residues in light
grey. Residues co-ordinating the Zn2 + ion in the structures of BshB1 (BC1534) and MshB1 (Rv1170) are indicated with a red box. Residues investigated in the present study are indicated with a red
star. Arg109 proposed to be involved in substrate binding and hydrolysis in BshB1 sequences is highlighted in yellow. Alignment was constructed in Biology Workbench. (B) Sequence identity (grey
boxes) and similarity (white boxes) between the N-deacetylase enzymes analysed in the present study from B. anthracis str. Ames and B. cereus ATCC-14579.
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Kinetic analysis of BshB and Bca enzymes.
Figure S3 Drawgram of deacetylase enzymes included in Figure S1A
PHYLIP rooted phylogenetic tree phenogram (Drawgram) constructed in Biology Workbench
depicts the phylogenetic distribution of deacetylases. The shaded boxes indicate proposed
functional assignments for deacetylase candidate sequences.
Figure S4 Far-UV CD spectra of deacetylases BA1557, BC1534, BA3888, BC3461 and BA3524 (A) and BA1557 variants H110A, D14A, R53A, R53K and
R109K (B)
Figure S5 pH–activity profiles of deacetylase activity of BA1557
(A) Relative activity (V/K) of Co2 + -BA1557 (᭹) and Zn2 + -BA1557 (᭡) using GlcNAc-Mal as substrate. (B) Relative activity (V/K) of Zn2 + -BA1557 with GlcNAc-Mal (᭡) and GlcNAc (᭜) as
substrates. Assays were measured with 0.25 mM GlcNAc-Mal or 50 mM GlcNAc under the pH range 5.5–9.5. The line is the best fit of eqn (1) as described in the Materials and methods section of
the main text. pKa and pKb are shown in the inset.
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Figure S6 Model of the active site of BC1534 (PDB code 2IXD) in complex
with GlcNAc-Mal
TheGlcNAc-MalmoleculewasgeneratedbyGaussian09toachievethemoststableconformation.
The model is the result of a simulated annealing docking of GlcNAc-Mal to BC1534 using
Autodock Vina [31]. Arg53 and Arg109 on each site of the catalytic Zn2 + with a distance 6.17
˚
˚
and 2.39 A respectively are shown. These residues locate in a position which has ∼4.5 A from
the carboxy groups of the malate moiety.
Figure S7 Kinetic analysis of BA1557 variants
(A) BA1557 WT (᭿), 109K (᭢) and R53K (᭹). (B) BA1557 WT (᭿), H110A (᭡) and D14A (᭜). The initial rates for the deacetylation of GlcNAc-Mal (0–3.36 mM) were measured by the slopes of
four-time point during a 30 min reaction for WT and R109K and a 3 h reaction for R53K.
Received 20 March 2013/6 June 2013; accepted 11 June 2013
Published as BJ Immediate Publication 11 June 2013, doi:10.1042/BJ20130415
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