3-Keto-5-aminohexanoate cleavage enzyme: A common fold for an uncommon claisen-type condensation

Article abstract of DOI:10.1074/jbc.M111.253260

The exponential increase in genome sequencing output has led to the accumulation of thousands of predicted genes lacking a proper functional annotation. Among this mass of hypothetical proteins, enzymes catalyzing new reactions or using novel ways to cata

Full text of DOI:10.1074/jbc.M111.253260

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 31, pp. 27399–27405, August 5, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
3-Keto-5-aminohexanoate Cleavage Enzyme
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A COMMON FOLD FOR AN UNCOMMON CLAISEN-TYPE CONDENSATION
Received for publication, April 20, 2011, and in revised form, May 17, 2011 Published, JBC Papers in Press, June 1, 2011, DOI 10.1074/jbc.M111.253260
Marco Bellinzoni‡1, Karine Bastard§¶ʈ1, Alain Perret§¶ʈ, Anne Zaparucha§¶ʈ2, Nadia Perchat§¶ʈ, Carine Vergne§¶ʈ
Tristan Wagner‡, Raquel C. de Melo-Minardi§¶ʈ3, Franc¸ois Artiguenave§¶ʈ4, Georges N. Cohen**,
Jean Weissenbach§¶ʈ, Marcel Salanoubat§¶ʈ, and Pedro M. Alzari‡5
,
From the ‡Unite´ de Microbiologie Structurale, Institut Pasteur, and CNRS-URA2185, 25 rue du Dr. Roux, 75724 Paris Cedex 15, the
§Direction des Sciences du Vivant, Commissariat a` l’Energie Atomique (CEA), Institut de Ge´nomique, Genoscope, 2 rue Gaston
Cre´mieux, 91057 Evry, ¶CNRS-UMR8030, 2 rue Gaston Cre´mieux, 91057 Evry, the ʈUniversite´ d’Evry Val d’Essonne, boulevard
Franc¸ois Mitterrand, 91057 Evry, and the **Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France
The exponential increase in genome sequencing output has
led to the accumulation of thousands of predicted genes lacking
a proper functional annotation. Among this mass of hypotheti-
cal proteins, enzymes catalyzing new reactions or using novel
One of the most striking outputs of genome sequencing is the
high proportion of proteins without pertinent functional anno-
tation. Predicted genes have been mainly annotated on the basis
of sequence homology to already characterized proteins from
ways to catalyze already known reactions might still wait to be other genomes, resulting, on average, in a fraction as high as
30–50% of predicted gene products that, in a given genome,
have no accurate functional assignments. These hundreds of
proteins without known function within a single organism pre-
vent a deeper understanding of the cell as a biological system
because it might be expected that some of these proteins of yet
undiscovered functionalities may end up revealing significant
novelty in chemical reactions.
identified. Here, we provide a structural and biochemical char-
acterization of the 3-keto-5-aminohexanoate cleavage enzyme
(Kce), an enzymatic activity long known as being involved in the
anaerobic fermentation of lysine but whose catalytic mechanism
has remained elusive so far. Although the enzyme shows the
ubiquitous triose phosphate isomerase (TIM) barrel fold and a
Zn2؉ cation reminiscent of metal-dependent class II aldolases,
our results based on a combination of x-ray snapshots and
molecular modeling point to an unprecedented mechanism that
proceeds through deprotonation of the 3-keto-5-aminohexano-
ate substrate, nucleophilic addition onto an incoming acetyl-
CoA, intramolecular transfer of the CoA moiety, and final
retro-Claisen reaction leading to acetoacetate and 3-aminobu-
tyryl-CoA. This model also accounts for earlier observations
showing the origin of carbon atoms in the products, as well as
the absence of detection of any covalent acyl-enzyme interme-
diate. Kce is the first representative of a large family of prokary-
otic hypothetical proteins, currently annotated as the “domain
of unknown function” DUF849.
The increasing numbers of proteins of unknown function
from newly sequenced genomes have been grouped into fami-
lies and classified within the Pfam database as “domain of
unknown function” (DUF)6 (1). Besides grouping protein fam-
ilies by sequence similarities (2, 3) and gene context analyses
(4), structural genomics has emerged as a promising tool for
functional annotation, given the assumption that proteins with
a similar fold can be related in function (5). To narrow down the
possible functions of each family and to achieve a better per-
ception of the protein universe, the NIH Protein Structure Ini-
tiative (PSI; www.nigms.nih.gov/Research/FeaturedPrograms/
PSI) has made a systematic effort to produce representative
structures of most DUF families (6). Our attention was drawn
by DUF849, a family restricted to prokaryotes and found in
ϳ850 bacterial proteins belonging to several different phyla
where, according to gene predictions, it accounts in most
(albeit not all) cases for the whole protein chain. Despite the fact
that a few crystallographic structures of proteins associated
with DUF849 have been recently deposited in the Protein Data
Bank (PDB), no mechanistic or biochemical investigation is at
present available. In a previous work, we identified a gene asso-
ciated with DUF849 as coding for the 3-keto-5-aminohexano-
ate cleavage enzyme (Kce) involved in the lysine fermentation
pathway (7), an intriguing enzyme whose catalytic mechanism
has remained so far enigmatic. The reaction catalyzed by Kce
involves the reversible condensation of a six-carbon molecule,
3-keto-5-aminohexanoate (KAH), derived from L-lysine,
* This work was supported by grants from the Institut Pasteur, the CEA, the
CNRS, and the University of Evry.
ꢀ This article was selected as a Paper of the Week.
The atomic coordinates and structure factors (codes 2Y7D, 2Y7E, 2Y7F, and
2Y7G) have been deposited in the Protein Data Bank, Research Collabora-
tory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Experimental Procedures, Tables S1–S3, and Figs. S1–S3.
1 Both authors contributed equally to this work.
2 To whom correspondence may be addressed: Laboratoire de Chimie Orga-
nique et Biocatalyse, IG Genoscope and CEA-UMR8030, 2 rue Gaston
Cre´mieux, 91057 Evry cedex, France. Fax: 33-1-60872514; E-mail:
azaparuc@genoscope.cns.fr.
3 Present address: Dept. of Computer Science, Federal University of Minas
Gerais, 6627 Pampulha, Belo Horizonte, Brazil.
4 Present address: Medical Genetics Dept., School of Medical Sciences, Uni- 6 Theabbreviationsusedare:DUF, domainofunknownfunction;KAH, 3-keto-
versity of Campinas, UNICAMP, Campinas, Sa˜o Paulo 13083-000, Brazil.
5 To whom correspondence may be addressed. Fax: 33-1-45688604; E-mail:
pedro.alzari@pasteur.fr.
5-aminohexanoate; Kce, 3-keto-5-aminohexanoate cleavage enzyme; TIM,
triose phosphate isomerase; r.m.s., root mean square; MAD, multiwave-
length anomalous diffraction.
AUGUST 5, 2011•VOLUME 286•NUMBER 31
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Structure and Catalytic Mechanism of Kce
through three different reactions with acetyl-CoA to produce through the Poisson-Boltzmann pdb2pqr server (20) applying
two four-carbon species, of which one still esterified to CoA, i.e. the CHARMM force field.
acetoacetate and a 3-aminobutyryl-CoA (8–10) (see Fig. 1A).
Substrate Synthesis—(S)-KAH and (R)-KAH were synthe-
Such ␤-keto acid-degrading activity proceeding through the sized from (SS,S)-(ϩ)-methyl N-(p-toluenesulfinyl)-3-amino-
cleavage on the ␤-carbon still has no analogues in literature and 3-methylpropanoate (21) and (Rs,R)-(Ϫ)-methyl N-(p-toluene-
therefore makes the characterization of this enzyme of partic- sulfinyl)-3-amino-3-methylpropanoate, respectively. Detailed
ular interest.
experimental procedures and compound characterization data,
Here, we investigated the catalytic mechanism of Kce from including H and 13C NMR data, are available in the supple-
1
Candidatus Cloacamonas acidaminovorans by structural and mental material.
biochemical means. We determined the crystal structures of
Molecular Modeling—Acetyl-CoA and 3-aminobutyryl-
the enzyme in its native state, as well as complexed with either CoA chemical depictions were retrieved from the PubChem
one of its natural substrates ((S)-KAH) or one of the products Compound database (pubchem.ncbi.nlm.nih.gov). Interme-
(acetoacetate). Additionally, we modeled the assumed interme- diates of reaction were built with ChemDraw, whereas dock-
diates of the reaction into the binding pocket, resulting in poses ing simulations were run with Autodock Vina (22). Energy
that agree with the proposed catalytic mechanism. The ensem- minimization of intermediate I1 (the deprotonated form of
ble of our results suggests that the enzyme, although structur- KAH) and acetyl-CoA in the binding pocket proceeded using
ally similar to class II aldolases, proceeds through a Claisen-like MMTK (23) within the Chimera package (24). More details
condensation with the peculiarity that CoA moiety is trans- of the computational methodology are provided in the sup-
ferred in an intramolecular fashion to the keto group of the plemental material.
KAH moiety.
RESULTS AND DISCUSSION
EXPERIMENTAL PROCEDURES
Overall Structure—The structure of Kce was solved by mul-
Experimental procedures for DNA cloning, protein produc- tiwavelength anomalous diffraction on selenomethionyl-la-
tion, purification and crystallization, biochemical assays, and beled protein crystals diffracting at 3.2 Å resolution (supple-
site-directed mutagenesis are described in the supplemental mental Table S2). The non-isomorphous structures of the
material. Oligonucleotide sequences are provided in supple- native enzyme at higher resolution (1.6 Å), as well as the com-
mental Table S1.
plexes with substrates and products, were solved by molecular
Data Collection, Structure Solution, and Refinement—X-ray replacement; a total of four separate models were refined at
diffraction data were collected either at Synchrotron Soleil resolutions ranging from 1.75 to 1.28 Å (Table 1). In all the
(Saint-Aubin, France) or at the European Synchrotron Radia- crystal structures determined, the enzyme is assembled as a
tion Facility (ESRF) (Grenoble, France) from single crystals at homotetramer with point symmetry 222 (Fig. 1B), consistent
100 K. Diffraction frames were processed with XDS (11) fol- with the observed tetrameric state of the recombinant enzyme
lowed by data reduction with either XSCALE (11) or SCALA in solution (7) and with earlier observations on purified bacte-
within the CCP4 suite of programs (12) (see Table 1 and sup- rial homologs from other species (8–10). Each subunit, made of
plemental Table S1). The structure was solved by multiwave- 276 residues, shows the canonical (␤/␣)8 TIM barrel fold, with
length anomalous diffraction (MAD) phasing from Se-Met-la- a short additional C-terminal ␣-helix (␣9) protruding at the
beled protein crystals. A total of 27 selenium sites in the N-terminal face of the barrel and three ␤-turn extensions com-
asymmetric unit were located with the program SHELXD (13) ing out from the barrel core, inserted in between ␤2 and ␣2
on data from a redundant single anomalous dispersion dataset (residues 49–58), ␤4 and ␣4 (residues 112–119), and ␤8 and ␣8
from a Se-Met-labeled crystal (supplemental Table S2); three- (residues 234–240) (Fig. 1C). The only missing part in the
wavelength MAD phasing and density modification were then model is the loop connecting ␤3 to ␣3 (residues 85–92), due to
carried out on data collected from an isomorphous crystal with the lack of supporting electron density in two out of the four
SOLVE/RESOLVE (14). The resulting electron density map monomers, as a consequence of its high conformational flexi-
showed clear secondary structure allowing the manual tracing bility (Fig. 1B). Each protomer buries a total of ϳ2100 Å2 (18%)
of the polypeptide chain with Coot (15). This preliminary of the monomer surface in intersubunit contacts, which mainly
model was partially refined with Refmac5 (16) and then used to involve helices ␣5 and ␣6, as well as the ␤-turn ␤4a-␤4b, for one
perform molecular replacement on higher resolution data from dyad, and ␣7 and ␣8 for the other dyad. As observed in the vast
a different crystal form (space group P212121). All subsequent majority of TIM barrel enzymes, the active site is situated in
complexes with ligands were solved by molecular replacement each monomer at the C-terminal face of the central ␤-barrel
with the program Molrep (17) using the structure of the Kce and could be identified by the presence of a metal ion coordi-
monomer, without ligands, as the search model. Each model nated by His-46, His-48, and Glu-230 (Fig. 1D). This ion was
was inspected and rebuilt with Coot and refined with auto- modeled as Zn2ϩ according to its coordination geometry, and
BUSTER (18). All models were validated through the Molpro- its nature was then confirmed by the analysis of double differ-
bity server (19). Final refinement parameters are shown in ence anomalous maps calculated from data collected around
Table 1. Figures were generated and rendered with the PyMOL the zinc K-edge (supplemental Fig. S1A and supplemental
Molecular Graphics System, Version 1.3, (Schro¨dinger, LLC); Table S3). The metal ion is situated at the bottom of a crevice
electrostatic surfaces were rendered with the APBS plug-in accessible from two opposite openings, one of which is delimited
within PyMOL, supplying atomic charges and radii calculated by the mobile ␤3-␣3 loop (Fig. 1D). A structural similarity search
27400 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure and Catalytic Mechanism of Kce
TABLE 1
Crystallographic data collection and refinement statistics
Datasets were collected at the Proxima 1 beamline (Synchrotron Soleil, Saint-Aubin, France).
Kce native orthorhombic
Kce native tetragonala
Kce-KAH
Kce-acetoacetate
Data collection
Space group
Wavelength (Å)
Cell dimensions
a, b, c (Å)
P212121
0.9724
P43212
0.8856
P212121
P43212
0.9801
0.8856
74.87, 100.82, 147.43
90.0, 90.0, 90.0
19.5-1.59 (1.68-1.59)
147,835
102.19, 102.19, 101.07
90.0, 90.0, 90.0
41.6-1.28 (1.35-1.28)
133,532
98.50, 98.48, 103.18
90.0, 90.0, 90.0
34.4-1.75 (1.84-1.75)
101,452
102.10, 102.10, 100.74
90.0, 90.0, 90.0
41.6-1.40 (1.48-1.40)
104,015
␣, ␤, ␥ (°)
Resolution (Å)
Unique reflections
I/␴(I)b
16.5 (3.0)
12.4 (3.3)
16.3 (2.9)
12.5 (3.0)
b
Rmerge
0.066 (0.501)
99.3 (95.7)
0.083 (0.466)
97.6 (89.6)
0.061 (0.434)
95.2 (92.0)
0.074 (0.504)
92.6 (88.5)
Completeness (%)b
Redundancyb
6.7 (4.5)
7.0 (5.8)
3.6 (3.3)
7.2 (6.8)
Refinement
Used reflections
140,296
126,786
96,199
98,582
R
work/Rfree (%)
15.7/17.4
14.3/16.9
15.0/17.4
16.7/18.6
No. of atoms
Protein
8432
16
4309
9
8528
45
4238
24
Ligands/ions
Solvent
976
585
872
498
B-factors (Å2)
Protein
21.5
30.4
34.0
17.2
16.6
26.5
19.9
33.3
30.6
20.0
22.1
28.3
Ligands/ions
Solvent
r.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.010
1.00
0.012
1.47
0.010
0.99
0.010
1.06
PDB entry
2Y7D
2Y7E
2Y7F
2Y7G
a A brief description of the Kce native tetragonal crystalline form is provided in the supplemental material.
b Highest resolution shell is shown in parentheses.
FIGURE 1. Overview of the Kce structure and the overall reaction. A, reaction scheme. The different colors are designed to reflect the origin of carbon atoms
in the 3-aminobutyryl-CoA and acetoacetate products, respectively, as determined by 14C-tracer experiments by Barker and co-workers on homologous
enzymes (8–10). B, ribbon diagram of the Kce homotetramer. C, top view of one monomer. Additional topology elements with respect to the canonical (␤/␣)8
TIM barrel fold are depicted in dark red, whereas the Zn2ϩ ion is depicted as a gray sphere. The arrow points to the mobile loop connecting ␤3 to ␣3. D,
electrostaticsurfacerepresentationoftheactivesiteregion, withtheentrancestotheZn2ϩ creviceindicatedbytheyellowarrows. The␤3-␣3loop, whichcloses
over the active site upon KAH binding, is depicted in red and indicated by a black arrow as in C.
showed significant matches (C␣ r.m.s. deviation ϳ1.2 Å) to seven 3LOT, 3E02, 3E49, and 3C6C) and showing a conserved divalent
structures of proteins of unknown function, all composed of the metal ion and its coordinating environment. However, no
PfamdatabaseDUF849domain(PDBentries3NO5, 3CHV, 3FA5, complexes with substrates or products, nor biochemical data
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Structure and Catalytic Mechanism of Kce
the decarboxylative formation of enolate as observed in decar-
boxylating condensing enzymes of fatty acid and polyketide
synthesis (25). The ␦-amino group of KAH interacts with the
main chain carbonyl of Gly-85 and the carboxyl of Glu-14 (Fig.
2A). These interactions play a key role in the proper positioning
of the S-ligand, suggesting a S-stereopreference of the enzyme.
Indeed, the kinetic data show a moderate stereospecificity, the
relative activity with (R)-KAH being 40% of the activity
obtained with (S)-KAH (initial rates obtained with 600 ␮M KAH
at a saturating concentration of acetyl-CoA). In addition to Gly-
85, the ␤3-␣3 loop also contributes to the binding of the sub-
strate through Val-87, whose side chain makes hydrophobic
interactions with the terminal methyl group of KAH. The side
chains of Val-87, Phe-114, and Phe-119 define a small hydropho-
bic cleft in which the methyl group of KAH is inserted (Fig. 2A). A
well ordered water molecule could be placed approximately half-
way between the NH2 of the fully conserved Arg-226 and the car-
bon 2 of KAH, the predicted site of C–C cleavage in the course of
the reaction, to which it positioned at an average distance of 2.9 Å,
suggesting a possible role in catalysis (see below).
The structure of Kce in complex with the reaction product
acetoacetate, although obtained from crystals belonging to a
different space group, shows a virtually identical enzyme fold
(C␣ r.m.s. deviation ϭ 0.3 Å) when compared with the complex
with KAH. Acetoacetate also binds to the Zn2ϩ ion as a biden-
tate ligand, with the carboxylate group superimposable to that
of KAH, making the same interactions with Ser-82, Thr-106,
and Asn-108 (Fig. 2B). However, the orientation of the mole-
cule is approximately perpendicular to the six-carbon chain of
KAH because the ketonyl oxygen of acetoacetate coordinates
the metal from the Arg-226 side, with which it interacts firmly
(Fig. 2B). This oxygen atom actually displaces the water mole-
FIGURE 2. View of the Kce active site in complex with either KAH (A) or
acetoacetate (AAE) (B). In both views, the ␴A-weighted difference electron
density map (mFo Ϫ DFc), calculated before each ligand was added to the
model and contoured at the 3␴ level, is shown in the form of a green mesh.
Neighboring side chains are depicted, as well as the water molecules likely to
be involved in the reaction (Fig. 3).
about these enzymes, were available, and no mechanistic cule observed, in both the native enzyme and the KAH com-
clues on catalysis could thus be inferred from a simple struc- plex, to make a hydrogen bond with the guanidinium group of
tural comparison.
Arg-226 (see above), whereas the methyl group of acetoacetate
Complexes with 3-Keto-5-aminohexanoate and Acetoacetate— makes van der Waals contacts with Val-172. Another water
To get insights into the catalytic mechanism, we crystallized molecule now completes the Zn2ϩ coordination sphere in the
Kce in complex with either the substrate (S)-3-keto-5-amino- position otherwise occupied by the ␤-keto oxygen of KAH.
hexanoate ((S)-KAH) or the product acetoacetate in two differ-
Reaction Mechanism and Structural Models—From the two
ent space groups (Table 1). Both molecules were found to bind snapshot structures, (S)-KAH-Kce and acetoacetate-Kce, and
at full occupancy and could be modeled unambiguously in the considering the primary results on the origin of carbon atoms of
active site (Fig. 2). The binding of KAH did not promote signif- reaction products through tracer experiments (10), we suggest
icant overall structural changes (C␣ r.m.s. deviation ϳ0.9 Å) as a potential reaction mechanism, depicted in Fig. 3. We hypoth-
the main chain displacements are essentially limited to the esize that the condensation catalyzed by Kce is a Zn2ϩ-depen-
␤3-␣3 loop and the ␣3 helix (residues 82–100). In particular, dent, Claisen-like mechanism (25) that proceeds through the
the ␤3-␣3 loop, floppy in the native enzyme, is well structured formation of four intermediates: an enolate derived from (S)-
upon the bound KAH closing one of the two entrances to the KAH (here named I1), a first tetrahedral high-energy interme-
active site and leaving only one access to the Zn2ϩ crevice for diate (I2), a second tetrahedral high-energy intermediate (I3),
the second substrate acetyl-CoA (Fig. 1D). Further, the side and eventually an enolate form of acetoacetate (I4). Computa-
chains of most residues surrounding the active site pocket tional modeling of the predicted reaction intermediates shows
adopt different rotamers with respect to the unliganded compatible positioning in the active site for all of them (Fig. 4),
enzyme, reorienting toward the bound KAH. The ␤-keto acid in agreement with our hypothesis. Indeed, it has been reported
binds as a bidentate chelator of Zn2ϩ, with a carboxylate oxygen that high-energy intermediates are more likely to adopt catalyt-
as well as the keto group coordinating the divalent ion at dis- ically competent poses in molecular docking than ground-state
tances of 2.0 and 2.2 Å, respectively (Fig. 2A). The binding of the intermediates (26, 27). Moreover, this kind of Claisen-like
substrate is further stabilized by the hydrogen bonds of the mechanism is fully compatible with the catalyzed reaction
carboxylate of KAH with the hydroxyl groups of Ser-82 and being reversible, as reported earlier from other Kce homologs
Thr-106 and the amide group of Asn-108, therefore preventing (8–10).
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Structure and Catalytic Mechanism of Kce
FIGURE 3. Schematic representation of the course of the Kce reaction. I1, I2, I3, and I4 refer to the postulated reaction intermediates (see “Results and
Discussion” for details).
FIGURE 4. View of the Kce active site at different states of the reaction, as predicted by molecular docking of the reaction intermediates I1, I2, I3, and
I4 (illustrated in Fig. 3). The CoA pantetheine chain, which is orientated toward the outside of the pocket, has been omitted for clarity.
The crystal structure of the KAH-Kce complex shows a into a hydrogen bond to a nearby water molecule (d ϭ 2.9 Å),
strong salt bridge connecting Asp-231 to Arg-226 (d ϭ 2.9 Å), which, in turn, is close enough to the C2 of KAH (d ϭ 2.9 Å) to
with the guanidinium group from the latter also being engaged abstract the proton in a series of sequential acid/base exchanges
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Structure and Catalytic Mechanism of Kce
TABLE 2
Steady-state kinetic analysis of Kce and point mutants
ND, not determined.
Mutant
K
m (S)-KAH
K
m AcCoA
mM
kcat
s
k
cat/Km ((S)-KAH)
k
cat/Km (AcCoA)
Ϫ1
Ϫ1
Ϫ1
mM
mMϪ1 ꢁ s
mMϪ1 ꢁ s
S82G
1.181 Ϯ 0.209
0.921 Ϯ 0.100
0.442 Ϯ 0.061
ND
0.046 Ϯ 0.010
0.024 Ϯ 0.002
0.012 Ϯ 0.002
ND
0.13 Ϯ 0.03
0.37 Ϯ 0.01
0.32 Ϯ 0.05
ND
0.1
0.4
0.7
Ϫ
Ϫ
12.9
2.8
E143G
E143Q
R226G
D231G
Wild type
15.4
26.7
Ϫ
ND
ND
ND
Ϫ
224.2
0.208 Ϯ 0.032
0.012 Ϯ 0.003
2.69 Ϯ 0.15
initiated by Asp-231 (28). We propose that the condensation (29)), suggest that the intermediate may collapse through an
starts by the abstraction of the pro-S proton from C2 of KAH, intramolecular SCoA nucleophilic addition to the C3 ␤-keto
mediated by a catalytic water molecule activated by the Asp- group (Fig. 3). According to molecular docking, the newly
231-Arg-226 charge relay dyad. The key role of this Asp-Arg obtained tetrahedral oxyanion intermediate I3 is also expected
dyad in catalysis is strengthened by the observation that its to coordinate the Zn2ϩ ion in a similar fashion as I2 (Fig. 4).
disruption, illustrated by the Arg-226 or Asp-231 to glycine However, the sulfur atom of acetyl-CoA is here less strongly
variants, leads to a catalytically inactive enzyme (Table 2). stabilized by Tyr-145 (d ϭ 3.5 Å) and is mainly held by weak van
According to our hypothesis, the stability of the resulting neg- der Waals contact with Phe-114. This conformation is consis-
atively charged KAH enolate (I1) is ensured by interactions tent with an intramolecular retro-Claisen reaction that would
with the Zn2ϩ cation and through hydrogen bonds with Thr- release 3-aminobutyryl-CoA as the leaving group and lead to
106 and Ser-82, as observed in the crystal structure of the Kce- the formation of acetoacetate in the enolate form, bound to
KAH complex and further supported by the docking model Zn2ϩ ion by a bidentate interaction mediated by the carboxyl
(Fig. 4, I1). Steady-state kinetic data are also consistent with this and ␤-keto groups, as well as by hydrogen bonds to Ser-82 and
scheme as the replacement of Ser-82 by a glycine significantly Thr-106 (Figs. 3 and 4). It is worth noting that the predicted
increases the Km for KAH and decreases the kcat (Table 2). conformation of acetoacetate at the end of the catalytic process
Although initial attempts of docking acetyl-CoA onto the crys- coincides with the binding mode observed in the high-resolu-
tallographic structure of the KAH-Kce complex failed, suggest- tion crystal structure of the enzyme-product complex (Fig. 2B)
ing that either KAH adopts a different conformation in the and is consistent with the origin of carbon atoms in acetoace-
presence of acetyl-CoA or the active site undergoes further tate as determined by tracer experiments with 14C made by
conformational changes to accommodate both substrates, a Barker and co-workers (8–10). The protonation of the enolic
perfect match could be obtained with the two reactive entities, form of acetoacetate, possibly by the action of another water
i.e. I1 and acetyl-CoA, in the binding pocket (Fig. 4). According molecule taking part in a proton relay with the Asp-231-Arg-
to this model, the orientation of the pantetheine chain of acetyl- 226 dyad (in a reverted process with respect to the deprotona-
CoA is parallel to (S)-KAH (supplemental Fig. S2A). The car- tion of KAH), would finally release the acetoacetate (Figs. 3 and
bonyl carbon of acetyl-CoA is within van der Waals contact 4).
range to both C1 and C2 of KAH (respective distances are 3.4
In all docking models (I1, I2, I3, and I4), the adenosine moiety
and 3.1 Å) and is therefore well placed to react with the depro- from the CoA ester is situated inside or near the shallow pocket
tonated carbon C2 of KAH (Figs. 3 and 4, I1). Nucleophilic flanked by the side chains of Arg-209 and Lys-237 (supplemen-
addition of enolate I1 to the thioester carbon of the acetyl-CoA tal Fig. S2). In contrast, it should be noted that the pantetheine
would lead to a tetrahedral oxyanion intermediate I2 strongly chain is modeled differently in the different intermediates.
coordinated to the Zn2ϩ ion (Fig. 3). As suggested by docking Indeed, CoA can adopt widely varying conformations in differ-
calculations, the KAH-derived part of I2 is perfectly superim- ent enzymes (30), making its mode of binding difficult to pre-
posed on the position of the substrate in the KAH-Kce crystal- dict (31).
lographic structure (Fig. 4, I2). The oxyanion from the acetyl
Although the first attempts at modeling acetyl-CoA onto the
moiety from acetyl-CoA takes exactly the same position as the crystallographic structure of KAH-Kce suggested a role of Glu-
oxygen atom of the well ordered water molecule and is stabi- 143 in stabilizing this substrate in the active site, activity mea-
lized by a hydrogen bond with Arg-226, whereas the three oxy- surements of the Glu-143 to Gly and Gln mutants invalidated
gen atoms (i.e. the oxyanion from acetyl-CoA and the carbox- this hypothesis as none of these mutations notably altered the
ylic and the ␤-keto oxygens from KAH) form a tripod-like Km of the enzyme for acetyl-CoA. Rather, their main effect is to
structure with the peak being the Zn2ϩ ion (Fig. 3, I2). In this affect the kcat, and to a lesser extent, the Km for KAH (Table 2).
conformation, the sulfur atom of acetyl-CoA makes polar inter- Although unexpected, this effect may be explained by the inter-
actions with Tyr-145 and van der Waals contacts with Phe-114, actions that, in the apo enzyme, Glu-143 makes with Arg-226
Val-172, and Arg-226, whereas the methyl group of the acetyl (d ϭ 3.0 Å), Asn-108 (d ϭ 3.4 Å), and Glu-141 (d ϭ 2.5 Å)
moiety is stabilized through van der Waals contacts with Glu- (supplemental Fig. S3A). As crystallographic data revealed that
143 and Val-172. The short distance between the sulfur atom of Asn-108 interacts with (S)-KAH (Fig. 2A), Glu-143 variants
the CoA moiety and the C3 keto atom of the former KAH (3.2 Å may alter or suppress the hydrogen bond with the carbamide
in the docking model), as well as the angle between the two group of Asn-108, and in turn, slightly lower the stabilization of
reactive centers (ϳ100°, close to the Bu¨rgi-Dunitz trajectory KAH in the active site, leading to an increased Km. Moreover,
27404 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 31•AUGUST 5, 2011

Structure and Catalytic Mechanism of Kce
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Glu-143 is involved in the network of interactions that hold the
catalytic water molecule in position, as observed in both the apo
Kce structure and the complex with KAH (supplemental Fig.
S3), in which it adopts a different rotamer upon substrate bind-
ing and structuring of the ␤3-␣3 loop (see above). Substitutions
of this residue are thus likely to alter the network that holds the
key water molecule, decreasing the overall catalytic efficiency.
Concluding Remarks—The wide functional diversity of TIM
barrel domains, which at present account for almost 9% of the
whole PDB, is well known. The vast majority of these proteins
are enzymes, spread ubiquitously in all kingdoms of life and
most often involved in metabolic pathways (32). However,
despite the already known large chemical variety of reactions
catalyzed by this fold, new types of reactions or novel catalytic
mechanisms performed by TIM barrel enzymes could still have
escaped identification. Indeed, we provide here strong evidence
pointing to Kce as the first representative of a new subclass of
TIM barrel Zn2ϩ-dependent condensing enzymes. Similarly to
canonical class II aldolases, which share with Kce both the pres-
ence of the zinc cation and the same (␤/␣)8 barrel fold, Zn2ϩ is
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tate product, as well as to stabilize their enolic forms, but the
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carbon Claisen condensation. Our model is fully consistent
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Acknowledgments—We are grateful to C. Pelle´ and C. Lechaplais
(CEA Genoscope) for excellent technical assistance and to A. Haouz
and P. Weber (PF6, Institut Pasteur) for performing robot-driven crys-
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AUGUST 5, 2011•VOLUME 286•NUMBER 31
JOURNAL OF BIOLOGICAL CHEMISTRY 27405

Protein Structure and Folding:
3-Keto-5-aminohexanoate Cleavage
Enzyme: A COMMON FOLD FOR AN
UNCOMMON CLAISEN-TYPE
CONDENSATION
Marco Bellinzoni, Karine Bastard, Alain
Perret, Anne Zaparucha, Nadia Perchat,
Carine Vergne, Tristan Wagner, Raquel C. de
Melo-Minardi, François Artiguenave, Georges
N. Cohen, Jean Weissenbach, Marcel
Salanoubat and Pedro M. Alzari
J. Biol. Chem. 2011, 286:27399-27405.
doi: 10.1074/jbc.M111.253260 originally published online June 1, 2011
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