A structure-based comparison with other proteins showed that benzaldehyde lyase belongs to a group of closely related ThDP-dependent enzymes.. While the resi-dues binding the two ends of
Trang 1benzaldehyde lyase from Pseudomonas fluorescens
Tanja G Mosbacher1, Michael Mueller2and Georg E Schulz1
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: georg.schulz@ocbc.uni-freiburg.de
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 rsuspend-ing 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.
Trang 2found 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
Mg2+ion The obtained crystals belong to spacegroup P3121 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 P3121 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˚, c ¼ 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
a For the MAD data sets Friedel pairs are treated as independent reflections.
H
2
BAL
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].
Trang 3SeMet-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
Mg2+ 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 Mg2+) 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 D2-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 Mg2+as a pink sphere The secon-dary structures are labeled.
Table 2 Refinement statistics.
Data set
SeMet-labeled BAL peak data set Wild-type
Structured peptide
(all four subunits)
Average B-factors [A ˚ 2 ]
(mainchain ⁄ total)
R.m.s.d bond lengths
[A ˚ ] ⁄ angles [degr.]
0.012 ⁄ 1.40 0.012 ⁄ 1.46 Ramachandran angles in
favored region [%]
Ramachandran angles in
allowed region [%]
Trang 4large 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 D2-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 daliscores 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
Chain length b Number of aligned residues Z-score c
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.
Trang 5Within 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 Mg2+ as a mediator The Mg2+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).
Trang 6thiazolium 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 aromaalipha-tic 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 inhibinhib-itor 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.
Trang 7other 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˚2higher 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.
Trang 8Experimental 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
37C 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 MgCl2, 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)1seleno-l-methionine (Acros)
Cultiva-tion and purificaCultiva-tion procedures were the same as for
wild-type BAL The yield of purified SeMet-labeled BAL was
about 6 mgÆg)1cell 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 MgCl2and 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 20C The drops consisted of 1.8 lL protein in buffer B,
0.2 lL of an Agarose-LM solution (3%, 37C; 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 lm3
a week later All crystals belonged to spacegroup P3121 They
were flash-frozen in liquid nitrogen without a further
addi-tion of a cryo-protectant Data collecaddi-tion 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
References
1 Mu¨ller M & Sprenger GA (2004) Thiamine-dependent enzymes as catalysts of C-C bonding reactions: the role
of ‘orphan’ enzymes In Thiamine: Catalytic Mechanisms
in Normal and Disease States(Jordan, F & Patel, MS, eds), pp 77–92 Marcel Dekker, London
2 Jordan F (2003) Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions Nat Prod Rep 20, 184–201
3 Gonzalez B & Vicun˜a R (1989) Benzaldehyde lyase, a novel thiamine PPI-requiring enzyme, from Pseudomo-nas fluorescensBiovaR-I J Bacteriol 171, 2401–2405
Trang 94 Hinrichsen P, Gomez I & Vicun˜a R (1994) Cloning and
sequencing of the gene encoding benzaldehyde lyase
from Pseudomonas fluorescens BiovaR-I Gene 144, 137–
138
5 Demir S, Pohl M, Janzen E & Mu¨ller M (2001)
Enan-tioselective synthesis of hydroxy ketones through
clea-vage and formation of acyloin linkage Enzymatic
kinetic resolution via C-C bond cleavage J Chem Soc
Perkin Trans 1, 633–635
6 Demir S, S¸es¸enoglu O¨, Eren E, Hosrik B, Pohl M,
Jan-zen E, Kolter D, Feldmann R, Du¨nkelmann P & Mu¨ller
M (2002) Adv Synth Catal 344, 96–103
7 Sanches-Gonzalez M & Rosazza JPN (2003) Mixed
aro-matic acyloin condensations with recombinant
benzalde-hyde lyase: synthesis of alpha-hydroxydihydrochalcones
and related alpha-hydroxy ketones Adv Synth Catal
345, 819–824
8 Lingen B, Pohl M, Liese A, Demir AS & Mu¨ller M
(2004) Enantioselective synthesis of hydroxyketones via
benzaldehyde lyase and benzoylformate decarboxylase
catalyzed C-C bond formation In Thiamine: Catalytic
Mechanisms in Normal and Disease States(Jordan, F &
Patel, MS, eds), pp 113–130 Marcel Dekker, London
9 Pohl M, Sprenger GA & Mu¨ller M (2004) A new
per-spective on thiamine catalysis Current Opinion
Biotech-nol 15, 335–342
10 Demir AS, S¸es¸enoglu O¨, Du¨nkelmann P & Mu¨ller M
(2003) Benzaldehyde lyase-catalyzed enantioselective
carboligation of aromatic aldehydes with mono- and
dimethoxy acetaldehyde Org Lett 5, 2047–2050
11 Du¨nkelmann P, Kolte-Jung D, Nitsche A, Demir AS,
Siegert P, Lingen B, Baumann M, Pohl M & Mu¨ller M
(2002) Development of a donor-acceptor concept for
enzymatic cross-coupling reactions of aldehydes: the
first asymmetric cross-benzoin condensation J Am
Chem Soc 124, 12084–12085
12 Hasson MS, Muscate A, McLeish MJ, Polovnikova LS,
Gerlt JA, Kenyon GL, Petsko GA & Ringe D (1998)
The crystal structure of benzoylformate decarboxylase
at 1.6 A resolution: diversity of catalytic residues in
thiamin diphosphate-dependent enzymes Biochemistry
37, 9918–9930
13 Polovnikova ES, McLeish MJ, Sergienko EA, Burgner
JT, Anderson NL, Bera AK, Jordan F, Kenyon GL &
Hasson MS (2003) Structural and kinetic analysis of
cata-lysis by a thiamin diphosphate-dependent enzyme,
ben-zoylformate decarboxylase Biochemistry 42, 1820–1830
14 Arjunan P, Umland T, Dyda F, Swaminathan S, Furey
W, Sax M, Farrenkopf B, Gao Y, Zhang D & Jordan F
(1996) Crystal structure of the thiamin
diphosphate-dependent enzyme pyruvate decarboxylase from the
yeast Saccharomyces cerevisiae at 2.3 A˚ resolution
J Mol Biol 3, 590–600
15 Dobritzsch D, Ko¨nig S, Schneider G & Lu G (1998)
High resolution crystal structure of pyruvate
decarboxy-lase from Zymomonas mobilis Implications for substrate activation in pyruvate decarboxylases J Biol Chem 273, 20196–20204
16 Lu G, Dobritzsch D, Baumann S, Schneider G & Ko¨nig
S (2000) The structural basis of substrate activation in yeast pyruvate decarboxylase A crystallographic and kinetic study Eur J Biochem 267, 861–868
17 Holm L & Sander C (1993) Protein structure compari-son by alignment of distance matrices J Mol Biol 233, 123–138
18 Pang SS, Duggleby RG, Schowen RL & Guddat LW (2004) The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate J Biol Chem 279, 2242– 2253
19 Pang SS, Duggleby RG & Guddat LW (2002) Crystal structure of yeast acetohydroxyacid synthase: a target for herbicidal inhibitors J Mol Biol 317, 249–262
20 Schu¨tz A, Sandalova T, Ricagno S, Hu¨bner G, Ko¨nig S
& Schneider G (2003) Crystal structure of thiamindi-phosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid Eur J Biochem 270, 2312–2321
21 Caines ME, Elkins JM, Hewitson KS & Schofield CJ (2004) Crystal structure and mechanistic implications of N2-(2-carboxyethyl) arginine synthase, the first enzyme
in the clavulanic acid biosynthesis pathway J Biol Chem
279, 5685–5692
22 Muller YA & Schulz GE (1994) Structure of the thia-mine- and flavin-dependent enzyme pyruvate oxidase Science 259, 965–967
23 Lindqvist Y, Schneider G, Ermler U & Sundstro¨m M (1992) Three-dimensional structure of transketolase, a thiamine diphosphate-dependent enzyme, at 2.5 A˚ reso-lution EMBO J 11, 2373–2379
24 Hawkins CF, Borges A & Perham RN (1989) A com-mon structural motif in thiamine pyrophosphate-binding enzymes FEBS Lett 255, 77–82
25 Siegert P, Pohl M, Kneen MM, Pogozheva ID, Kenyon
GL & McLeish MJ (2004) Exploring the substrate specificity of benzoylformate decarboxylase, pyruvate decarboxylase, and benzaldehyde lyase In Thiamine: Catalytic Mechanisms in Normal and Disease States (Jordan, F & Patel, MS, eds), pp 275–290 Marcel Dekker, London
26 Jordan F, Nemeria NS, Zhang S, Yan Y, Arjunan P & Furey W (2003) Dual catalytic apparatus of the thiamin diphosphate coenzyme: acid-base via the 1¢,4¢-iminopy-rimidine tautomer along with its electrophilic role J Am Chem Soc 125, 12732–12738
27 Nemeria N, Baykal A, Joseph E, Zhang S, Yan Y, Furey W & Jordan F (2004) Tetrahedral intermediates
in thiamin diphosphate-dependent decarboxylations exist as a 1¢,4¢-imino tautomeric form of the coenzyme,
Trang 10unlike the Michaelis complex or the free coenzyme.
Biochemistry 43, 6565–6575
28 Pohl M, Lingen B & Mu¨ller M (2002)
Thiamin-di-phosphate-dependent enzymes: new aspects of
asym-metric C-C bond formation Chem Eur J 8, 5288–5295
29 Iding H, Du¨nnwald T, Greiner L, Liese A, Mu¨ller M,
Siegert P, Gro¨tzinger J, Demir AS & Pohl M (2000)
Benzoylformate decarboxylase from Pseudomonas putida
as stable catalyst for the synthesis of chiral 2-hydroxy
ketones Chem Eur J 6, 1483–1495
30 Bu¨rgi HB & Dunitz JD (1983) From crystal statics to
chemical dynamics Acc Chem Res 16, 153–161
31 Janzen E (2002) Die Benzaldehydlyase aus Pseudomonas
fluoreszens Biochemische Charakterisierung und die
Untersuchung von Struktur-Funktionsbeziehungen
PhD Thesis, University of Duesseldorf
32 Hendrickson WA, Horton JR & LeMaster DM (1990)
Selenomethionyl proteins produced for analysis by
mul-tiwavelength anomalous diffraction (MAD): a vehicle
for direct determination of three-dimensional structure
EMBO J 9, 1665–1672
33 Kabsch W (1993) Automatic processing of rotation
dif-fraction data from crystals of initially unknown
sym-metry and cell constants J Appl Cryst 26, 795–800
34 Terwilliger TC & Berendzen J (1999) Automated MAD
and MIR structure solution Acta Crystallogr D55, 849–
861
35 de la Fortelle E & Bricogne G (1997)
Maximum-likeli-hood heavy-atom parameter refinement for multiple
iso-morphous replacement and multiwavelength anomalous
diffraction methods Methods Enzymol 276, 472–494
36 Terwilliger TC (2003) Automated main-chain model
building by template matching and iterative fragment
extension Acta Crystallogr D59, 38–44
37 McRee DE (1999) XtalView⁄ Xfit–A versatile program for manipulating atomic coordinates and electron den-sity J Struct Biol 125, 156–165
38 Bru¨nger AT, Adams PD, Clore GM, DeLanoWL, Gros
P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges
M, Pannu NS, Read RJ, Rice LM, Simonson T & Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure deter-mination Acta Crystallogr D54, 905–921
39 Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D50, 760–763
40 Perrakis A, Sixma TK, Wilson KS & Lamzin VS (1997) wARP: improvement and extension of crystal-lographic phases by weighted averaging of multiple refined dummy atomic models Acta Crystallogr D53, 448–455
41 Winn MD, Isupov MN & Murshudov GN (2001) Use
of TLS parameters to model anisotropic displacements
in macromolecular refinement Acta Crystallogr D57, 122–133
42 Laskowski RA, MacArthur MW, Moss DS & Thornton
JM (1993) PROCHECK – a program to check the stereo-chemical quality of protein structures J Appl Cryst 26, 283–291
43 Lovell SC, Davis IW, Arendall WB, de Bakker PIW, Word JM, Prisant MG, Richardson JS & Richardson
DC (2002) Structure validation by Calpha geometry: phi, psi and Cbeta deviation Proteins: Struct Funct Genet 50, 437–450
44 Fenn TD, Ringe D & Petsko GA (2003) POV-Script+: a program for model and data visualization using persistence of vision ray-tracing J Appl Cryst
36, 944–947