Báo cáo khoa học: Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens potx

Structure and mechanism of the ThDP-dependent
benzaldehyde lyase from Pseudomonas fluorescens
Tanja G. Mosbacher
1
, Michael Mueller
2
and Georg E. Schulz
1
1 Institut fu
¨
r Organische Chemie und Biochemie, Albert-Ludwigs-Universita
¨
t, Freiburg im Breisgau, Germany
2 Institut fu
¨
r Pharmazeutische Wissenschaften, Albert-Ludwigs-Universita
¨
t, Freiburg im Breisgau, Germany
Thiamine diphosphate (ThDP)-dependent enzymes
participate in numerous biosynthetic pathways and
catalyse a broad range of reactions mainly involving
the cleavage and the formation of C–C-bonds. For
instance, they catalyse nonoxidative and oxidative de-
carboxylations of 2-ketoacids, produce 2-hydroxy-
ketones, and transfer activated aldehydes to a variety
of acceptors. However, they can also form C–N, C–O
and C–S bonds [1,2]. The ThDP-dependent benzalde-
hyde lyase (BAL, EC 4.1.2.38, suggested systematic
name: 2-hydroxy-1,2-diphenylethanone benzaldehyde-
lyase, i.e. benzoin benzaldehyde-lyase) catalyses the
reversible ligation of two aromatic aldehydes to yield

an (R)-2-hydroxyketone (Fig. 1). BAL was discovered
by Gonzales and Vicuna who isolated it from the
strain Pseudomonas fluorescens Biovar I, which was
Keywords
acyloin condensation; carbon–carbon
ligation; crystal structure; seleno-methionine
MAD
Correspondence
G. E. Schulz, Institut fu
¨
r Organische Chemie
und Biochemie, Albertstr. 21, Freiburg im
Breisgau, Germany 79104
Tel: +49 761 203 6058
Fax: +49 761 203 6161
Email:
Note
After submission of this manuscript, we
received a preprint of the following paper
reporting that the mutation of His29 to alan-
ine reduces the BAL activity to 5%. Kneen
MM, Pogozheva ID, Kenyon GL & McLeish
MJ (2005) Exploring the active site of benz-
aldehyde lyase by modeling and mutagen-
esis. Biochim Biophys Acta: Proteins and
Proteomics, doi:10.1016/j.bbapap2005.
08.025
(Received 5 August 2005, revised 22
September 2005, accepted 29 September
2005)

doi:10.1111/j.1742-4658.2005.04998.x
Pseudomonas fluorescens is able to grow on R-benzoin as the sole carbon
and energy source because it harbours the enzyme benzaldehyde lyase that
cleaves the acyloin linkage using thiamine diphosphate (ThDP) as a cofac-
tor. In the reverse reaction, this lyase catalyses the carboligation of two
aldehydes with high substrate and stereospecificity. The enzyme structure
was determined by X-ray diffraction at 2.6 A
˚
resolution. A structure-based
comparison with other proteins showed that benzaldehyde lyase belongs to
a group of closely related ThDP-dependent enzymes. The ThDP cofactors
of these enzymes are fixed at their two ends in separate domains, suspend-
ing a comparatively mobile thiazolium ring between them. While the resi-
dues binding the two ends of ThDP are well conserved, the lining of the
active centre pocket around the thiazolium moiety varies greatly within the
group. Accounting for the known reaction chemistry, the natural substrate
R-benzoin was modelled unambiguously into the active centre of the repor-
ted benzaldehyde lyase. Due to its substrate spectrum and stereospecificity,
the enzyme extends the synthetic potential for carboligations appreciably.
Abbreviations
AHAS, acetohydroxy acid synthase; ALS, acetolactate synthase; BAL, benzaldehyde lyase; BFD, benzoylformate decarboxylase; CEAS,
carboxyethylarginine synthase; IPDC, indolepyruvate decarboxylase; MAD, multiwavelength anomalous diffraction; PDC, pyruvate
decarboxylase; POX, pyruvate oxidase; SeMet, seleno-
L-methionine; ThDP, thiamine diphosphate.
FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6067
found in wood scraps in a cellulose factory [3]. They
showed that this strain can grow on lignin-like
substrates, because the endogenous BAL can cleave
the acyloin linkage of R-benzoin and R-anisoin to use
these compounds as a carbon and energy source [4].

BAL is a valuable tool for chemo-enzymatic synthe-
ses because it generates various enantiomerically pure
2-hydroxyketones through aldehyde ligation or by
partial decomposition of racemic mixtures [5–8]. The
enzyme generates activated aldehydes either via direct
aldehyde addition to ThDP or via cleavage of 2-hyd-
roxyketones but is not involved in decarboxylation
reactions [9]. BAL accepts a broad spectrum of aro-
matic donor substrates, among them ortho-substituted
benzaldehydes, and processes substituted acetaldehydes
resulting in functionalized derivatives of (R)-2-hy-
droxypropiophenone [10]. The enzyme also ligates two
aliphatic aldehydes resulting in highly enantio-enriched
acyloins [5]. In all reactions, BAL shows a high stereo-
specificity for the R-configuration of the acyloin link-
age [10]. Starting from the assumption that aldehydes
which are not accepted as donor substrates may still
be acceptor substrates and vice versa, a biocatalytic
system for the asymmetric cross-carboligation of
aromatic aldehydes has been developed [11]. Taken
together, BAL broadens appreciably the substrate
spectrum of the related enzymes benzoylformate
decarboxylase (BFD) [12,13] and pyruvate decarboxy-
lase (PDC) [14–16] used for similar syntheses. Here we
report the crystal structure of BAL with bound cofac-
tor ThDP at 2.6 A
˚
resolution, suggest the geometry of
the reaction and explain the substrate specificity in
structural terms.

Results and Discussion
Structure determination and description
BAL is a homotetramer of 4 · 563 amino acid residues
corresponding to a molecular mass of 4 · 58 919 Da.
Each subunit binds one ThDP molecule using one
Mg
2+
ion. The obtained crystals belong to spacegroup
P3
1
21 with one tetrameric BAL molecule (wild-type
plus invisible C-terminal His-tags) per crystallographi-
cally asymmetric unit (Table 1). The crystal structure
was determined by the incorporation of seleno-
l-methionine (SeMet) and subsequent phasing with
multiwavelength anomalous diffraction (MAD). Met1
was cleaved off during protein production as indicated
by electrospray ionization mass spectroscopy (ESI-
MS). The complete replacement of the 12 remaining
methionines per subunit was demonstrated by ESI-MS,
which showed a single peak at a mass of 562 ± 5 Da
(expected 563 Da) higher than the mass of the wild-
type.
The SeMet diffraction data contained a good anom-
alous signal to 3.0 A
˚
resolution. Among the expected
4 · 12 selenium sites, 4 · 11 were found and used for
the initial phasing. The 4 · 1 missing SeMet positions
were located in the mobile and therefore invisible

C-terminal ends. After phase improvement, a model of
Table 1. Data collection statistics. All crystals belong to spacegroup P3
1
21. The unit cell dimensions of the SeMet-labeled crystal were a ¼
b ¼ 150.3 A
˚
and c ¼ 195.8 A
˚
. Those of the wild-type BAL crystal were a ¼ b ¼ 154.7 A
˚
,c¼ 200.7 A
˚
. The corresponding packing parame-
ters were 2.69 A
˚
3
⁄ Da and 2.92 A
˚
3
⁄ Da, respectively. Values in parentheses refer to the highest resolution shells, which comprised 2.70–
2.58 A
˚
in all data sets.
Data set
SeMet-labeled BAL
Wild-type
peak inflection remote
Wavelength [A
˚
] 0.9793 0.9801 0.9393 0.9801

Resolution [A
˚
] 20–2.6 20–2.6 20–2.6 20–2.6
Observables 598 889 598 513 429 583 324 789
Unique reflections
a
156 252 (17 815) 156 058 (17 756) 143 633 (16 819) 79 796 (10 122)
Completeness [%] 99.5 (96) 99.2 (95) 98.2 (94) 98.8 (99)
R
sym-I
[%] 8.2 (39) 7.4 (38) 7.9 (38) 11.1 (37)
Multiplicity 3.8 (3.5) 3.8 (3.5) 3.0 (2.8) 8.1 (7.9)
Average I ⁄ r
I
12.5 (3.4) 13.9 (3.8) 12.8 (3.6) 13.3 (5.5)
a
For the MAD data sets Friedel pairs are treated as independent reflections.
O
O
H
2
BAL
benzaldehyde
R-benzoin
OH
Fig. 1. Benzaldehyde lyase (BAL) catalyzes cleavage and formation
of R-benzoin. BAL is known to accept several other substituted aro-
matic or aliphatic acyl-acceptors as substrates for the formation of
an acyloin [28].
ThDP-depending benzaldehyde lyase T. G. Mosbacher et al.

6068 FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS
SeMet-labeled BAL was built and refined to 2.6 A
˚
resolution. This model served as a template for the
wild-type BAL structure, which was determined by
molecular replacement.
The structure of wild-type BAL was refined to 2.6 A
˚
resolution resulting in a model closely similar to that
of SeMet-BAL. It included residues 2–555 of each sub-
unit as well as four molecules of ThDP and four
Mg
2+
ions. The eight C-terminal residues were disor-
dered in both structures. Data collection and refine-
ment statistics are given in Tables 1 and 2. The
crystals of SeMet-labelled BAL and wild-type BAL
grew under almost identical conditions and showed the
same packing scheme but quite different unit cell axes.
Since the B-factors were lower and the refinement
results better for the wild-type crystals than for SeMet-
labelled crystals, we refer in the following to the wild-
type structure (Fig. 2).
The BAL homotetramer has an overall size of
approximately 95 · 95 · 75 A
˚
3
. No significant struc-
tural differences were found between the four crystallo-
graphically independent subunits of the tetramer

(Fig. 3). Each subunit consists of the three domains
Dom-a, Dom-b and Dom-c (Fig. 2), named as in pre-
vious annotations. All three domains consist of a cen-
tral six-stranded parallel b-sheet flanked by a varying
number of a-helices. Residues involved in binding of
the cofactor ThDP are located at the C-terminal ends
of the b-strands of Dom-c (diphosphates and Mg
2+
)
and of Dom-a¢ of a neighbouring subunit (pyrimidine
moiety). The active centre is defined by the thiazolium
ring of ThDP, which sits in a deep pocket opening to
the outer surface of the tetramer.
The four subunits A, B, C and D form the two tight
dimers A–B and C–D around the molecular axis P
(Fig. 3), in which each subunit buries a solvent-access-
ible surface area of 3270 A
˚
2
. The two tight dimers are
associated much less tightly around the molecular axes
Q and R to form a D
2
-symmetric homotetramer.
These secondary interfaces bury 1790 A
˚
2
per subunit.
The tight contact is formed by Dom-a and Dom-c of
subunit A with their counterparts in subunit B. It is

stabilized by a large number of hydrogen bonds. The
weaker contact results from an association of Dom-a
and Dom-b of subunit A with the respective domains
of subunit D. It contains only few hydrogen bonds. A
Fig. 2. Stereo ribbon plot of a BAL subunit composed of the three domains Dom-a (residues 1–183, blue), Dom-b (residues 184–363,
orange) and Dom-c (residues 364–563, green). The cofactor ThDP is shown as a ball-and-stick model and Mg
2+
as a pink sphere. The secon-
dary structures are labeled.
Table 2. Refinement statistics.
Data set
SeMet-labeled
BAL peak data set Wild-type
Resolution range [A
˚
] 20–2.6 20–2.6
Structured peptide
(all four subunits)
3–555 2–555
Water molecules 404 477
Average B-factors [A
˚
2
]
(mainchain ⁄ total)
43.3 ⁄ 43.8 34.7 ⁄ 35.6
R
cryst
[%] 21.2 19.9
R

free
[%] 24.5 22.0
R.m.s.d. bond lengths
[A
˚
] ⁄ angles [degr.]
0.012 ⁄ 1.40 0.012 ⁄ 1.46
Ramachandran angles in
favored region [%]
96.5 96.8
Ramachandran angles in
allowed region [%]
3.5 3.2
T. G. Mosbacher et al. ThDP-depending benzaldehyde lyase
FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6069
large cavity lined by the four Dom-a is located at
the centre of the tetramer. It contains a considerable
number of crystallographic water molecules and is not
connected to the active centre pocket.
To detect possible conformational changes of the
tetramer, we compared the wild-type and the SeMet-
labelled structures of BAL. A chainfold superposition
of the four central Dom-a showed a good agreement
in these domains but a radial contraction bringing the
outer Dom-b and Dom-c of SeMet-BAL up to 1.4 A
˚
closer to the centre when compared with the wild-type.
Moreover, the shrinkage of SeMet-BAL involves a
0.5-A
˚

approach of Dom-c (fixing the diphosphate of
ThDP) towards Dom-a¢ (binding the pyrimidine moi-
ety), which may affect the thiazolium ring suspended
between the two fix points. This observed contraction
reveals possible domain rearrangements and agrees
with the crystal unit cell changes stated in Table 1.
According to the crystallization conditions, the
contraction seems to be caused by an increase of the
PEG 200 concentration from 50% to 55%, removing
water from the protein.
Comparison with related proteins
To find related proteins in the Protein Data Bank, we
performed a general search using program dali [17].
This search identified a number of closely related
structures all of which were ThDP-dependent enzymes
involved in important metabolic pathways. The
Z-scores ranged from 39.6 to 29.6 indicating close rela-
tionships (Table 3). The related proteins are acetolac-
tate synthase (ALS) [18], acetohydroxy acid synthase
(AHAS) [19], indolepyruvate decarboxylase (IPDC)
[20], benzoylformate decarboxylase (BFD) [12], carb-
oxyethylarginine synthase (CEAS) [21], pyruvate oxi-
dase (POX) [22] and pyruvate decarboxylase (PDC)
[14,15]. The overall sequence identity among these
seven enzymes ranges from 19% to 29% with an aver-
age of 24%. A comparison of the relative domain posi-
tions in the D
2
-symmetric tetramer showed in general
a good equivalence with deviations around 2 A

˚
. All
enzymes are especially similar with respect to the tight
dimer formed by Dom-a and Dom-c. Given the high
dali scores of Table 3 and the drastic drop to the next
lower score, these enzymes form a separate subset
among the ThDP-dependent enzymes, which we name
‘POX group’ after the first established structure [22].
Fig. 3. Stereo ribbon plot of the D
2
-symmetric BAL tetramer with the three molecular twofold axes P, Q and R using the colors of Fig. 2.
The tetramer should be described as a dimer of dimers. The tightest interfaces are around axis P. Each tight dimer contains two active cen-
tres at its interface. ThDP is shown as a ball-and-stick model.
Table 3. Superposition of BAL with related proteins using DALI [17].
Protein
a
PDB
code FAD
Chain
length
b
Number of
aligned residues Z-score
c
ALS
d
1OZG None 565 419 39.6
AHAS 1JSC Structural 630 416 39.6
IPDC 1OVM None 554 495 34.4
BFD 1MCZ None 528 398 33.7

CEAS 1UPA None 573 439 32.0
POX 1POX Redox 585 356 31.7
PDC
e
1ZPD None 567 397 29.3
a
In all cases we used subunit A of the PDB file except for BAL
where we used subunit D.
b
BAL contains 563 residues total.
c
The
next lower Z-score was 16.7 indicating that the eight enzymes form
a separated, closely related group. The ThDP-dependent transketo-
lase [20] showed a Z-score of 13.1.
d
PDB file 1OZF of ALS yields
the same values.
e
The PDC structure is that of Zymomonas
mobilis.
ThDP-depending benzaldehyde lyase T. G. Mosbacher et al.
6070 FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS
Within this group the enzymes BFD and PDC are
best characterized with respect to their function and
therefore most relevant in organic synthesis. A struc-
ture-based sequence alignment of BAL with BFD and
PDC is shown in Fig. 4. The alignment assigns the
residue equivalences at the active centres and it pre-
sents the sequence of BAL in relation to its secondary

structures. AHAS and POX contain FAD as a further
cofactor besides ThDP which, however, plays merely a
secondary role (Table 3). The FAD of POX accepts
two electrons from the substrate and transfers them to
dioxygen, whereas the FAD of AHAS is only required
for structural integrity indicating that it is a relic of
evolutionary development.
The POX group shows very similar binding loca-
tions for ThDP which also correspond to those of
other ThDP-dependent enzymes [23]. In all enzymes
ThDP assumes a V-conformation resulting in a close
approach between the C2 atom of the thiazolium ring
and the N4¢ atom of the pyrimidine moiety. A super-
position of the cofactors is depicted in Fig. 5 revealing
a remarkable conformational similarity. The diphos-
phates are tightly bound to the polypeptide of Dom-c
using Mg
2+
as a mediator. The Mg
2+
ion is octahed-
rally coordinated to the sidechains of Asp448 and
Asn475, to the backbone carbonyl of Ser477, to the
diphosphate as well as to a water molecule (Fig. 6).
This binding motif is present in all ThDP-dependent
enzymes. In evolutionary terms the diphosphate-bind-
ing site is the most important fix point of ThDP
because it is best conserved as demonstrated by the
diphosphate-binding sequence fingerprint G-D-G-
X24-N-N that was detected long before any structure

was known [24]. At the other end of ThDP, the pyrim-
idine is well fixed in Dom-a¢ of another subunit: its
N1¢ atom forms a strong hydrogen bond to a glutamic
acid (Glu50 in BAL). A comparison of the relative
B-factors along the ThDP molecules shows that the
Fig. 4. Structure-based sequence alignment of BAL, BFD and PDC all of which are used in organic synthesis. The secondary structure of
BAL is given for reference. The underlined segments are structurally aligned with BAL within the usual 3 A
˚
cutoff criterion. Lower case indi-
cates lack of structure. The domain borders are indicated by triangles. The crystallized BAL lacked Met1 and carried a C-terminal His-tag with
the sequence
561
pfgshhhhhh which was disordered and therefore invisible in the crystal structure. The sequence of PDC continues with
562
KPVNKv (r).
T. G. Mosbacher et al. ThDP-depending benzaldehyde lyase
FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6071
thiazolium ring and the ethylene bridge have generally
the highest mobility, which corresponds to the largest
positional differences of these parts observed in Fig. 5.
Active centre and reaction geometry
While the overall polypeptide architecture as well as
the binding mode of ThDP are quite similar within the
POX group, the active centre is not well conserved. The
active centre pocket of BAL is lined by nonpolar alipha-
tic and aromatic but only few polar residues. In this
respect, BAL is most closely related to BFD [12]. In
both crystal structures of BAL a water molecule was
identified at a distance of about 3.6 A
˚

from the C2 atom
of ThDP. This water molecule forms hydrogen bonds
with Gln113 and His29, among which Gln113 is known
to play an important role in catalysis [25].
The structures of all group members show ThDP in
the V-conformation that brings the C2 atom of thiazo-
lium in close proximity to the N4¢ atom of the
pyrimidine moiety. Moreover, one of the reported
structures of ALS [18] contains an inhibitor that fixes
the C2 to N4¢ approach through a covalent bond as
shown in Fig. 5. Moreover, all group members have a
glutamic acid forming a short hydrogen bond to the
N1¢ atom of the pyrimidine ring, which was suggested
to induce the 1¢,4¢-imino tautomer [22]. The actual
presence of this tautomer was later demonstrated
[26,27]. As shown in Fig. 6, the imino group is hydro-
gen bonded to the carbonyl of Gly419 so that its lone
electron pair points to the C2 atom of ThDP. It is
therefore most likely that the catalytic cycle starts by
transferring a proton from C2 to the imine. The result-
ing C2 carbanion may then attack the carbonyl carbon
of the substrate yielding a covalent ThDP-substrate
intermediate.
During acyloin cleavage, the next step is supposedly
the deprotonation of the hydroxyl by His29 followed by
the dissociation of the first aldehyde. The remaining
activated aldehyde is then protonated and also released.
The protonation is probably performed by the water
attached to His29. During acyloin synthesis, on the
Fig. 5. Superposition of the ThDP cofactors of BAL (grey), ALS (cyan), AHAS (green), IPDC (purple), BFD (yellow), CEAS (blue), POX (pink)

and PDC (red) together with some residues important for cofactor binding and catalysis. The BAL residues are labeled and the BAL hydrogen
bonds are displayed. The yellow and red residues at the top are His70 of BFD and Asn28 of PDC, respectively. For ALS we show an inhib-
itor with a modified C2 atom (PDB file 1OZG) instead of ThDP available from 1OZF. This inhibitor contains an additional covalent bond
between the C2 and N4¢ atoms and carries an isopropyl substituent.
Fig. 6. Stereoview of ThDP-binding at BAL showing the initial (Fo-Fc)-electron density map of ThDP and Mg
2+
at the 3 r contour level. The
cofactor binds in the typical V-conformation required for catalysis. BAL residues lining the active centre pocket and interacting with the co-
factor are shown in blue and orange, corresponding to the two domains they belong to (Fig. 2). Hydrogen bonds are given as dotted lines.
ThDP-depending benzaldehyde lyase T. G. Mosbacher et al.
6072 FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS
other hand, the intermediate is an activated aldehyde
that is going to attack an acceptor aldehyde suitably
positioned in the active centre. Again, His29 is likely to
participate in the reaction by forming a hydrogen bond
to the oxygen of the acceptor aldehyde, which is eventu-
ally converted to a hydroxyl group of the condensation
product by deprotonating His29. Proton handling by
His29-Nd1 is facilitated by the contact of the Ne2 atom
to the bulk solvent (Note).
All steps seem to involve small displacements of the
thiazolium ring, which are possible because this ring is
relatively mobile (Fig. 5). It should be noted that
ThDP is suspended between Dom-c and Dom-a¢ which
in a direct comparison between the wild-type and the
SeMet structures of BAL underwent a relative dis-
placement of 0.5 A
˚
. It is therefore conceivable that
domain motions affect the positional freedom of thia-

zolium and thus catalysis. Since such domain displace-
ments may be caused by the Brownian motion, it is
further possible that the enzymes channel thermal
energy into the chemical reaction.
Substrate specificity
BAL shows a general preference for nonpolar sub-
strates [8,28] and is highly stereospecific with respect to
benzoin, cleaving only R-benzoin out of a racemic
mixture [5]. Moreover, BAL reacts with benzaldehyde
and acetaldehyde to yield (R)-2-hydroxypropiophenone
[28], in contrast to BFD, which uses the same educts
to produce the S-enantiomer [29]. In order to explore
the geometry of the reaction catalyzed by BAL, R-ben-
zoin was modeled into its active centre (Fig. 7). The
resulting model accounts for a nucleophilic attack from
the deprotonated C2 atom of the thiazolium ring
under the expected Bu
¨
rgi-Dunitzangle of 103° ±3°
onto the carbonyl carbon of R-benzoin [30]. Fulfil-
ling this restraint, the substrate is uniquely defined
with respect to general location and conformation
because all alternatives met severe steric obstacles. In
contrast to R-benzoin, any model of the S-enantio-
mer gave rise to major sterical clashes, which
explains the stereospecificity of BAL. In the resulting
R-benzoin model the hydroxyl is located at the posi-
tion of the water molecule observed in both crystal
structures of BAL (Figs 6 and 7) as well as in the
crystal structure of BFD [12]. We suggest that this

applies for all acyloin cleavage reactions of BAL.
During acyloin C–C-bond formation, on the other
hand, this water site accommodates the oxygen of
the acceptor aldehyde.
All residues lining the active centre pocket are depic-
ted in Fig. 7. A comparison with the functionally well-
established enzymes BFD and PDC shows almost no
conservation (Fig. 4). However, Ala480 and Phe484
are conserved between BAL and BFD. These residues
were therefore mutated resulting in decreased activity,
as to be expected from their location within the active
centre [31]. In BAL, the chain around Phe484 is quite
mobile with B-factors about 20 A
˚
2
higher than average
so that this side chain may close down on a bound
substrate performing an induced-fit motion. Such a
side chain displacement is supported by a comparison
with BFD, where Phe484 points into the active centre
as shown in Fig. 7.
The established structure of BAL invites further
efforts to identify the roles of the various catalytic
residues through mutational and structural studies
combined with enzyme kinetic measurements. This
knowledge together with designed mutations are likely
to expand the range of organic compounds that can be
produced enzymatically.
Fig. 7. Model of R-benzoin (green) bound in the active centre of BAL. The model allows for a nucleophilic attack of the deprotonated C2
atom of ThDP at the targeted carbonyl-group of R-benzoin under the expected Bu

¨
rgi–Dunitz angle [30] (dotted line, red). The suggested pro-
ton transfer from C2 to N4¢ is indicated (dotted line, green). All residues lining the active centre pocket are given as ball-and-stick models in
domain colors (Fig. 2). Their Ca atoms are black. The Phe484 conformation observed in the BFD crystals is yellow. The site of the water
molecule bound to BAL is marked by a halo and occupied by the substrate hydroxyl group.
T. G. Mosbacher et al. ThDP-depending benzaldehyde lyase
FEBS Journal 272 (2005) 6067–6076 ª 2005 The Authors Journal compilation ª 2005 FEBS 6073
Experimental procedures
Expression and purification
Wild-type BAL with a C-terminal His-tag (Fig. 4) was
obtained from Escherichia coli SG13009 cells following a
previously described procedure [25]. Cells were grown at
37 °C and expression of BAL was induced with isopropyl-
b-d-thiogalactopyranoside (IPTG). After cell lysis, the
supernatant was applied to a Ni-chelate column. The
enzyme was further purified on a gel permeation column
(Superdex 200, Amersham-Pharmacia, Freiburg, Germany)
using buffer A (25 mm Hepes pH 6.9, 200 mm NaCl,
2.5 mm MgCl
2
, 0.1 mm ThDP and 2 mm dithiothreitol).
BAL-containing fractions were identified by SDS ⁄ PAGE,
pooled and adjusted to a concentration of 20 mgÆmL
)1
.
The typical yield was 8 mg proteinÆg
)1
cell pellet. SeMet-
labelled BAL was obtained by introducing the expression
vector into the methionine-auxotrophic E. coli strain

B834(DE3). Cells were cultured in LeMaster medium [32]
containing 25 mgÆL
)1
seleno-l-methionine (Acros). Cultiva-
tion and purification procedures were the same as for wild-
type BAL. The yield of purified SeMet-labeled BAL was
about 6 mgÆg
)1
cell pellet. Full incorporation of SeMet was
verified by ESI-MS.
Crystallization and data collection
The purified protein (wild-type and SeMet) was dialyzed
for 12 h against buffer B (5 mm Hepes pH 6.9, 10 mm
NaCl, 2 mm MgCl
2
and 2 mm dithiothreitol). The solution
was then adjusted to a concentration of 12 mgÆmL
)1
and
crystallised by the hanging drop vapour diffusion method
at 20 °C. The drops consisted of 1.8 lL protein in buffer B,
0.2 lL of an Agarose-LM solution (3%, 37 °C; Hampton
Research, Alieso Veijo, CA, USA) and 2 lL buffer C (50%
(v ⁄ v) PEG 200 for wild-type BAL or 55% (v ⁄ v) PEG 200
for SeMet-labelled BAL, 100 mm Mes pH 6.85), which was
also used as the reservoir. The crystals appeared after about
3 days and reached maximum sizes of 300 · 80 · 80 lm
3
a
week later. All crystals belonged to spacegroup P3

1
21. They
were flash-frozen in liquid nitrogen without a further addi-
tion of a cryo-protectant. Data collection of the wild-type
crystals was carried out at beamline PX of the Swiss Light
Source (Villigen, Switzerland). MAD data were collected
from a single SeMet crystal using beamline BW7A at the
EMBL-outstation (DESY Hamburg). All data were proc-
essed and scaled with program XDS [33].
Structure determination and refinement
Using the MAD data sets, the positions of the selenium
atoms were established with solve [34]. The selenium sites
were refined and used for initial phasing with sharp [35].
Density modification and initial model building was carried
out using resolve [36]. The model was manually completed
with XFIT [37] and subsequently refined with the Anneal
and Minimize options of CNS [38] followed by a restrained
refinement with refmac [39]. Water molecules were intro-
duced using arp ⁄ warp [40]. They were confirmed wherever
the (2Fo-Fc)-map showed a density above 0.8 r and the
environment allowed the formation of hydrogen bonds.
The procedure resulted in about 0.2 water molecules per
residue. The refinement was completed with 10 cycles of
tls ⁄ refmac [41] specifying each subunit of the tetramer as
a TLS group. Non-crystallographic symmetry restraints
were used throughout the refinement. Subsequently, the
structure of wildtype BAL was established by molecular
replacement using molrep [39]. It was refined in the same
way starting from the model of SeMet-labeled BAL. Both
structures were evaluated with procheck [42] and rampage

[43]. Model building of R-benzoin in complex with BAL
was performed by manually docking the substrate into the
active centre, followed by energy minimization using the
Anneal and Minimize options of CNS. For the structure
similarity search we used dali [17]. It should be noted that
the general search with dali failed to find the enzymes als
and ipdc in the Protein Data Bank. Structural superposi-
tions were performed with lsqman [39]. Figures were
produced with povscript+ [44] and povray [http://
www.povray.org]. The coordinates and structure factors
have been deposited in the Protein Data Bank under acces-
sion codes 2AG0 and 2AG1.
Acknowledgements
We thank Martina Pohl for kindly providing us with the
gene of the benzaldehyde lyase and for helpful discus-
sions, the teams of the Swiss Light Source (Villigen ⁄ CH)
and of the EMBL outstation Hamburg for their help
with data collection and J. Wo
¨
rth for the ESI-MS meas-
urements. Further, we thank M. J. McLeish for sending
us a preprint of his paper (details in Note). The project
was supported by the Deutsche Forschungsgemeinschaft
under grants SFB-380 and SFB-388.
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