Structure and mechanism of an unusual malonate decarboxylase and related racemases

Article abstract of DOI:10.1002/chem.200800918

A study was conducted to investigate the structure and mechanism of the arymalonate decarboxylase (AMDase), or malonate decarboxylase and related racemases. The AMDase is an unusual malonate decarboxylase, as it does not require biotin, or any other co-factors for activity and does not involve formation of a malonyl thioester-enzyme intermediate. It was found that the sequence of the AMDase shows similarity to the Glu and Asp rasmases. The fact that the AMDase and related Asp/Glu racemases do not require Mg²⁺, or any other co-factor, which might stabilize the postulated enediolate intermediate, made them unique enzymes for conducting investigations. It was also found that the homochiral carboxylic acid products of the AMDase are potentially valuable chiral precursors for the synthesis of pharmaceuticals and other products.

Full text of DOI:10.1002/chem.200800918

COMMUNICATION
DOI: 10.1002/chem.200800918
Structure and Mechanism of an Unusual Malonate Decarboxylase and
Related Racemases
[b]
Krzysztof Okrasa,[a] Colin Levy, Bernhard Hauer,[c] Nina Baudendistel,[c]
David Leys,*[b] and Jason Micklefield*[a]
The arylmalonate decarboxylase (AMDase, E.C. 4.1.1.76)
from Bordetella bronchiseptica, catalyses the enantioselec-
tive decarboxylation of a-aryl-a-methylmalonates to give
optically pure a-arylpropionates (Figure 1).[1] The AMDase
is unusual amongst malonate decarboxylases as it does not
require biotin or any other co-factors for activity, and does
not involve formation of a malonyl thioester-enzyme inter-
mediate.[1d,e] The sequence of the AMDase shows similarity
to the Glu and Asp racemases.[2] It is suggested[3,4a] that two
Cys residues at the active site of the Glu racemase function
in general acid–base catalysis, abstracting the a-proton from
the Glu substrate to generate a planar enediolate intermedi-
ate which is re-protonated from the opposite face leading to
the racemate (Figure 1). The AMDase, on the other hand,
possess only one essential Cys residue (Cys188). This led to
the suggestion[1e] that the decarboxylation of the substrate 2-
Figure 1. A) The AMDase catalyses decarboxylation of a range of aryl-
malonates (e.g. Ar=Phenyl, 2-naphthyl or 2-thienyl where R=H or
methyl). B) Proposed mechanism for racemisation of Asp and Glu, cata-
lysed by the Asp/Glu-racemase family.
methyl-2-phenylmalonate (2), catalysed by the AMDase re-
sults in an enediolate intermediate, which is protonated on
the si-face by Cys188 to form (2R)-phenylpropionate (4;
Figure 1).
Whilst the structure of the AMDase is unknown, a
number of crystal structures of the related Asp/Glu race-
mases are available.[4] Despite this, the mechanisms pro-
posed to date for these racemases,[3,4a] and the related AM-
Dase[1e] do not account for how the common putative ene-
diolate intermediate might be stabilised. Indeed the pKa of
the a-proton of a carboxylate anion is >29, which reflects
the instability of an endiolate dianion.[5a] Whilst the negative
charge of enediolate formed by the AMDase (Figure 1) can
be delocalised into the aryl substituent, to some extent, this
effect alone could not stabilise the dianion sufficiently to ac-
count for the high efficiency of the enzymatic decarboxyla-
tion. In the case of the mechanistically related enolase su-
perfamily the enediolate intermediates formed from abstrac-
[a] Dr. K. Okrasa, Prof. Dr. J. Micklefield
School of Chemistry & Manchester Interdisciplinary Biocentre
The University of Manchester
131 Princess Street, Manchester M1 7ND (UK)
E-mail: jason.micklefield@manchester.ac.uk
[b] Dr. C. Levy, Dr. D. Leys
tion of the a-proton of carboxylate substrates are stabilised
Faculty of Life Sciences & Manchester Interdisciplinary Biocentre
The University of Manchester
131 Princess Street, Manchester M1 7ND (UK)
E-mail: david.leys@manchester.ac.uk
[5]
by coordination to Mg2+
.
The fact that the AMDase and
related Asp/Glu racemases do not require Mg2+, or any
other co-factor, which might stabilise the postulated enedio-
late intermediate, makes them particularly intriguing en-
zymes from a mechanistic point of view. In addition to this
the homochiral carboxylic acid products of the AMDase are
also potentially valuable chiral precursors for the synthesis
[c] Prof. Dr. B. Hauer, Dr. N. Baudendistel
BASF SE, GVF/E, Carl-Bosch Strasse 38
67056 Ludwigshafen (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800918.
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Table 1. Kinetic parameters for AMDase and related enzymes[a]
of pharmaceuticals and other products. Finally the AMDase
has also been shown to catalyse an intramolecular aldol-
type reaction, whilst site-directed mutagenesis has been
used to switch the enantioselectivity of the decarboxylase
and also to generate a new racemase activity.[6] This catalytic
plasticity therefore makes the AMDase an ideal candidate
for the rational redesign and directed evolution of new or
improved biocatalysts.
Protein (Microorganism)
Km [m]
kcat [sÀ1
]
kcat/Km [sÀ1 mÀ1
]
AMDase[b]
B. bronchiseptica KU1201
EAN08019.1
Mesorhizobium sp.BNC1
NP_888076.1
B. bronchiseptica RB50
ZP_00801202.1
A. metalliredigenes QYMF
BAA30082.1
P. horikoshii OT3
MurI (AAF25672)[c]
A. pyrophilus
1
2
1
2
1
2
1
2
1
2
1
2
10.7”10À3 316
2.96”104
26.9”10À3 279
1.04”104
9.6”10À3
118
1.71”104
22.2”10À3 55.8
10.0”10À3 12.4
0.25”104
0.12”104
-
-
-
2.1
-
2.2
-
77
-
123”10À3
-
0.26
-
To establish the structure and mechanism of the B. bron-
chiseptica AMDase, the enzyme was overproduced in E. coli
as a C-terminal His6-fusion protein, purified by Ni-affinity
chromatography and shown to possess similar activity to the
native enzyme[1d] (Table 1). Following this, the AMDase was
crystallised and the structure was solved to 1.5 Š using mul-
tiple isomorphous replacement. The structure reveals overall
similarity with the crystal structures of Asp/Glu racemases,[4]
and is most similar to Asp racemase from Pyrococcus hori-
koshii OT3[4b] (pdf code 1 JFL, Z score 14.6 with a r.m.s.d.
of 3.5 Š for 192 Ca atoms). The AMDase is a tightly folded
monomer that consists of two four-stranded parallel b-sheets
each surrounded by a-helices (Figure 2A). Surprisingly, a
single phosphate molecule is tightly bound near the active
site Cys188, at the end of a solvent accessible channel locat-
ed at the end of two b-strands. Notably the phosphate O1/
O2 atoms are engaged by six hydrogen bonds of two adja-
cent oxyanion holes[7] (Figure 2B), which are described as
the “dioxyanion hole” from here on in. Kinetic studies also
show that phosphate is a weak competitive inhibitor (Ki =
8 mm) for the AMDase-catalysed decarboxylation of 2-
41.7”10À3 0.09
-
-
3.1”10À3
-
0.24
-
[a] Kinetic parameters were determined spectrophotometrically, for de-
carboxylation of phenylmalonate (1) and 2-methyl-2-phenylmalonate (2),
using BTB as an indicator following methods described previously.[10]
[b] Parameters agree closely with Literature data.[1d] [c] Glutamate race-
mase activity for the MurI from Aquifex pyrophilus: Km =0.21”10À3 m,
k
cat =3.30 sÀ1, kcat/Km =1.57”104 sÀ1 mÀ1
.
methyl-2-phenylmalonate (2). This suggests that the phos-
phate ion mimics the enediolate intermediate, bound to the
active site. The importance of the dioxyanion hole was also
demonstrated through site-directed mutagenesis. Previously
mutagenesis of Thr75 and Ser76 was investigated[6c] and in
addition we have subjected Tyr126 to saturation mutagene-
sis. All of the resulting mutants have been shown to be com-
pletely inactive, which supports the idea that the dioxanion
hole is essential for catalysis.
Figure 2. A) Ribbon representation of the AMDase crystal structure with phosphate (pink-red), Cys188 (yellow-blue) and b-sheets near solvent accessi-
ble channel (green). B) The AMDase active site showing the hydrogen bonding pattern observed between the O1, O2 and O3 atoms of the phosphate
and the dioxyanion hole as well as the active site Cys188 residue. The sigmaA weighted 2FoFc electron density map is contoured at 1s and shown in blue
mesh, whereas the omit map contoured at 3s for the bound phosphate is shown as a green mesh. The Cys188 is the only residue that lies outside the al-
lowed region in the Ramachandran plot. The phosphate O1 atom is within hydrogen bonding distance of the side chain of Tyr126 (2.76 Š), and the
amide backbone and hydroxy side chain of Ser76 (2.88 and 2.56 Š, respectively). The O2 atom of the phosphate group, on the other hand, is hydrogen-
bonded to the amide backbone of Gly189 (2.77 Š) in addition to both the hydroxy group and amide backbone of Thr75 (2.65 and 2.94 Š respectively).
Also, the phosphate O3 is within hydrogen bonding distance of the Cys188 (2.97 Š), which presumably stabilises the conformation of the Cys residue. C)
Active site model of AMDase structure, with the 2-methyl-2-phenyl-1,1-enediolate intermediate bound. Residues of the smaller binding pocket sur-
rounding the methyl group are shown in yellow and the dioxyanion hole is shown in blue. Tyr126, which also donates one hydrogen bond to the enedi-
ACHTREUNG
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Malonate Decarboxylase
COMMUNICATION
Whilst the true biological substrate for the AMDase is
not known, 2-methyl-2-phenylmalonate 2 has been widely
used as the model substrate. We therefore positioned the
proposed enediolate intermediate derived from the decar-
boxylation of this substrate into the active site of the struc-
ture (Figure 2C), using the coordinates of the O1-P-O2
atoms of the bound phosphate group to position the oxygen
atoms of the enediolate into the dioxyanion hole. In the
AMDase structure, the active site cavity volume is predomi-
nantly hydrophobic in nature and extends above the O1-P-
O2 atoms. There is relatively little space available directly
above the O1 atom, while in comparison a large cavity ex-
tending into the solvent area is available directly above O2.
This cavity is ideally shaped for binding of aromatic groups,
with the Pro14 side chain positioned approximately 8 Š
away from the Gly189–Gly190 amide bond, allowing for
ideal van der Waals stacking of a flat aromatic system in be-
tween. Furthermore, the aryl side chain is orientated in such
a way that it remains co-planar with the enediolate p
system, ensuring effective delocalisation of the negative
charge which can serve to further stabilise this intermediate.
From the available active site volume, it seems the enzyme
will be able to accommodate relatively bulky ortho, meta
and para groups including a 2-naphthyl substituent, which is
in line with the previously determined substrate specificity
of this enzyme.[1] The fact that there is little space available
above O1 clearly indicates that this is most likely to accom-
modate the smaller methyl substituent. The energy-mini-
mised model of the proposed intermediate, bound in this
way (Figure 2C), positions the Cys188 thiol in close contact
(4.16 Š) to the si-face of the enolate, which is consistent
with a subsequent protonation step leading to the R-config-
ured 2-phenylpropionate prod-
located in the solvent exposed pocket it is clear that the
pro-R carboxylate of the substrate would necessarily occupy
the opposite side of the active site to the Cys188 residue
(Figure 2D, see also Supporting Information). This positions
the carboxyl group tightly within the small hydrophobic
pocket made up by Leu40, Val43, Val156 and Tyr48, suggest-
ing that hydrophobic destabilisation of the pro-R carboxyl-
ate of the malonate substrate may be a necessary driving
force for decarboxylation. Presumably the neutral carbon di-
oxide that is lost in the reaction remains bound (or trapped)
within this hydrophobic pocket, as the residual electron den-
sity is delocalised into the pro-S carboxylate of the enedi-
AHCTREoUNG late intermediate with the build-up of negative charge
being stabilised by the dioxyanion hole (Figure 3). Thus the
AMDase is able to effect efficient decarboxylation without
co-factors or prior activation as an electron-withdrawing
thioester, simply by orientating the malonate substrate such
that one carboxy group is stabilised by an extensive H-bond-
ing network, whilst the other carboxylate is bound tightly
against a small hydrophobic pocket, deprived of any stabiliz-
ing electrostatic interactions.
Despite the significant potential of AMDases for applica-
tions in biocatalysis, only one such enzyme has been isolated
and characterised to date. Accordingly four protein sequen-
ces that show moderate to high similarity (30-52% identity)
to the AMDase (Table 1, see also Supporting Information)
and that contain a Cys residue at the same relative position
as the Cys188 in the B. bronchiseptica enzyme, were selected
from the protein databases. Genes encoding these proteins
were synthesised, cloned and overexpressed in E. coli. In ad-
dition to this, the known glutamate racemase from Aquifex
pyrophilus (MurI),[4a] which has lower sequence similarity
uct 4 (Figure 3).
Previously it was suggested,
using 13C-labeling experiments,
that decarboxylation of 2-
methyl-2-phenylmalonate
(2)
catalysed by the B. bronchisep-
tica AMDase occurs through
the loss of the pro-R carboxyl-
ate with overall inversion of
configuration.[9] In a separate
series of experiments using a
more
efficient 18O-labeling
strategy, vide infra, we con-
firmed this initial stereochemi-
cal assignment to be correct.
Based on this, it is possible to
position the substrate 2-methyl-
2-phenylmalonate (2) in the
active site of the AMDase
structure. With the location of
the oxygen atoms of pro-S car-
boxylate determined by their
position relative to the dioxyan-
Figure 3. Proposed mechanism of the decarboxylation reaction catalysed by the AMDases. At the pH optimum
of the enzyme (8.0), it is predicted that the substrate 2-methyl-2-phenylmalonate will exist predominantly as
the dianion.[8] It is therefore more likely that it is the substrate pro-S-carboxylate anion rather than the proton-
ion hole and phenyl substituent ated acid that initially binds to the dioxyanion hole.
Chem. Eur. J. 2008, 14, 6609 – 6613
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J. Micklefield, D. Leys et al.
(20% identity) to the AMDase, was also overproduced and
purified. Subsequently, apparent kinetic constants for the
AMDase activity of the purified enzymes were established
(Table 1). Notably the enzyme from Mesorhizobium sp.
BNC1, which shows highest similarity (52% identity) to the
B. bronchiseptica AMDase, showed very efficient
decarboxyACHTREUNGlase activity with both phenylmalonate (1) and 2-
methyl-2-phenylmalonate (2) as substrates.
To probe the stereochemical course of the AMDase from
Mesorhizobium a stereoselective 18O-labeling approach was
developed (Scheme 1). This involved the use of porcine liver
residues at their putative active site, suggesting that their
natural function is as racemases. Furthermore, the dioxyan-
ion hole motifs remain closely conserved across all these en-
zymes, suggesting that all are likely to form enediolate inter-
mediates. Furthermore, an overlay of the structure of the
glutamate racemase (MurI) from Helicobacter pylori (PDB
code 2 JFX)[4c] and Enterococcus faecalis (PDB code
2 JFO)[4c] with the B. bronchiseptica AMDase reveals a simi-
lar dioxyanion hole structure binding the carboxylate of the
d-Glu substrate (Figure 4). Analysis of other racemase
structures[4] also shows the presence of similar dioxyanion
holes. This suggests that all these enzymes share a common
structural motif that serves to stabilise the enediolate inter-
mediates. Surprisingly, given the available structural data,[4]
the mechanistic role of those conserved residues that make
up the common dioxyanion holes has not been proposed
until now. Indeed, whilst single isolated oxyanion holes have
been described in many protein structures,[7] the presence of
two of these in the form of the dioxyanion hole predicted to
stabilise a high-energy enediolate type intermediates has not
been described explicitly before, as far as we are aware.
However the notable recent structure of diaminopimelate
(DAP) epimerase, despite possessing low sequence or over-
all structural similarity to the AMDase, exhibits some simi-
larity in its active site.[12] In DAP-epimerase, two a-helices
orientated towards the substrate carboxy group are also sug-
gested to stabilise the formation of an endiolate intermedi-
Scheme 1. The 18O-labeling strategy used to determine the stereochemi-
cal course of the decarboxylation of 2-methyl-2-phenylmalonic acid cata-
lysed by the AMDases from Mesorhizobium sp. BNC1 & B. bronchi-
ACHTREUNGseptica. PLE=porcine liver esterase.
esterase (PLE) to catalyse the hydrolysis of the diester 5,
which gave the (R)-monoester (98% ee) in agreement with
the literature.[11] The monoester was then hydrolysed with
Na18OH in H218O to give 2-methyl-2-phenylmalonic acid
with an 18O label in the pro-R carboxylate 6. Similarly, hy-
drolysis first with PLE in 98% H218O followed by saponifi-
cation with unlabeled NaOH led to the opposite enantiomer
7. The chiral malonates 6 and 7 were separately incubated
with the AMDase from Mesorhizobium and B. bronchisepti-
ca. The products of the decarboxylation were then methylat-
ed with diazomethane and analysed by GC-MS. This
showed that decarboxylation catalysed by both enzymes
occurs through the loss of the pro-R carboxylate with overall
inversion of configuration. This along with modeling studies
(see Supporting Information) suggests that the mechanisms
of the two AMDases are similar.
The other enzymes that were investigated (Table 1)
showed low to moderate decarboxylase activity with phenyl-
malonate (1), but no activity with 2-methyl-2-phenylmalo-
nate (2). Interestingly the known glutamate racemase from
Aquifex pyrophilus (MurI) is able to catalyse the decarboxy-
lation of phenylmalonate, albeit with 196-fold lower activity
than the native glutamate racemase activity. It is possible to
rationalise these results through protein sequence align-
ments (see Supporting Information). Notably, the active site
residues of B. bronchiseptica AMDase are almost entirely
conserved within the Mesorhizobium sp. BNC1 enzyme,
with a single active site Cys residue consistent with the
AMDase activity. All the other enzymes possess two Cys
Figure 4. A) The active site of the H. pylori Glu-racemase (MurI)[4c]
showing how the Glu-carboxylate interacts with the dioxyanion hole. The
structural water molecule (2.70 Š), and the amide backbone and hydroxy
side chain of Thr72 (3.10 and 2.70 Š, respectively) are all within hydro-
gen bonding distance of one oxygen atom of the carboxy group. The
other oxygen atom is hydrogen-bonded to the backbone amide of Thr182
(2.95 Š) in addition to the side chain and backbone amide groups of
Asn71 (3.28 and 2.88 Š, respectively). In the published structure of MurI,
the positions of N and O atoms of the amide side chain of Asn71 re-
versed, with the oxygen atom orientated towards the carboxy group.[4c]
However the resolution of this structure is 1.9 Š, which precludes assign-
ment of the amide O and N atoms. B) The active site of the AMDase. C)
Overlay of the AMDase and MurI structures.
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Malonate Decarboxylase
COMMUNICATION
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ate. Such dipole interactions cannot occur in the AMDase,
as there are no a-helices correctly orientated relative to the
dioxyanion hole.
In summary, the first high-resolution X-ray crystal struc-
ture of a co-factor independent aryl malonate decarboxylase
(AMDase), which reveals the mechanism of this unusual
enzyme, is presented (PDB code 3DG9).[13] Notably, a
dioxyACHTREUNG
anion hole, a hitherto unidentified structural motif,[12]
is postulated to be critical in the stabilisation of a putative
enediolate intermediate formed during decarboxylation. Se-
quence alignments and analysis of the structures of the
mechanistically related Asp/Glu racemases suggests that
these enzymes also possess dioxyanion holes that stabilise
similar intermediates. As a result the Asp/Glu racemase also
exhibit some AMDase activity. In the mechanistically relat-
ed enolase superfamily, coordination to Mg2+ serves to sta-
bilise enediolate intermediates.[5] The results presented here
therefore suggest that nature has evolved at least two dis-
tinct solutions to the same mechanistic puzzle, which has
been a subject of considerable interest in enzymology.[5] In
addition, the structural and mechanistic insight, presented
here, provide a firm basis for engineering new decarboxylas-
es, which can provide valuable homochiral carboxylic acids
from cheap and accessible malonate precursors.[14] Moreover
it may now be possible to design mechanism-based inhibi-
tors that bind to the dioxyanion holes of the Asp/Glu race-
mases, which and are important targets for the development
of new antimicrobial agents.[4c]
[7] J. D. Robertus, J. Kraut, R. A. Alden, J. J. Birktoft, Biochemistry
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3.17 and 6.06, respectively. A more electron-withdrawing substitu-
ent, such as a phenyl group, is predicted to decrease the pKa values
slightly. See: Brown, H. C.; McDaniel, D. H.; Hꢁfliger, O. in Deter-
mination of Organic Structures by Physical Method, ed. Braude
E. A.; Nachod, F. C. Academic Press, New York, 1955, p. 567.
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6256–6257.
Experimental Section
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rjee, J. Biomol. Screening 2003, 8, 559–565.
Full experimental details can be found in the Supporting Information.
[11] a) P. Dominguez de Maria, B. Kossmann, N. Potgrave, S. Buchholz,
H. Trauthwein, O. May, H. Groeger, Synlett 2005, 11, 1746–1748;
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Kikelj, Tetrahedron Asymmetry 1997, 8, 425–434.
[12] The structure of DAP epimerase shows a carboxy group of an inhib-
itor bound by a network of five hydrogen bonds at the active site.
Minor conformational changes in this structure could result in a sim-
ilar dioxyanion hole. B. Pillai, M. A. Cherney, C. M. Diaper, A. Su-
therland, J. S. Blanchard, J. C. Vederas, M. N. G. James, Proc. Natl.
Acad. Sci. USA 2006, 103, 8668–8673; A. Sutherland, J. S. Blan-
chard, J. C. Vederas, M. N. G. James, Proc. Natl. Acad. Sci. USA
2006, 103, 8668–8673.
Acknowledgements
This work is supported by BASF through the UK Centre of Excellence
in Biocatalysis (CoEBio3). D.L. is a Royal Society University Research
Fellow and an EMBO YIP. Prof. Stanley Roberts, Prof. Nicholas Turner,
and Prof. Neil Bruce are acknowledged for helpful advice and discus-
sions.
Keywords: decarboxylase · enzymes · reaction mechanisms ·
racemase · structure elucidation
[13] The atomic coordinates for Bordetella bronchiseptica AMDase have
been deposited in the Protein Data Bank (accession number
3DG9).
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Received: May 14, 2008
Published online: June 23, 2008
Chem. Eur. J. 2008, 14, 6609 – 6613
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6613