Active site mutagenesis of the putative Diels-Alderase macrophomate synthase

Article abstract of DOI:10.1039/b703177g

Although the macrophomate synthase active site is rich in potential functional groups, site-directed mutagenesis shows that only three residues are absolutely required for catalysis of oxaloacetate decarboxylation and trapping of the resulting enolate wit

Full text of DOI:10.1039/b703177g

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Active site mutagenesis of the putative Diels–Alderase macrophomate
synthase{
Jo¨rg M. Serafimov,a Hans Christian Lehmann,a Hideaki Oikawab and Donald Hilvert*a
Received (in Cambridge, UK) 2nd March 2007, Accepted 15th March 2007
First published as an Advance Article on the web 28th March 2007
DOI: 10.1039/b703177g
We produced MPS with an N-terminal hexahistidine tag in
E. coli under the control of a T7 promoter. The protein was
purified to homogeneity by affinity chromatography on a
Ni(II)-chelating NTA resin at pH 7.0 and 4 uC. This procedure,
which yields up to 260 mg of pure protein per litre of cell culture, is
more efficient than the original purification strategy,10 which
entailed three chromatography steps. In agreement with the
crystallographic data,8 analytical ultracentrifugation measurements
show that the enzyme adopts a hexameric quaternary structure in
solution. Importantly, the hexahistidine tag does not significantly
affect its catalytic properties. The decarboxylation of oxaloacetate
is catalyzed with a kcat of 12 s21 and a Km of 80 mM, in good
agreement with literature values.6 When 2-pyrone 1 is included in
the reaction mixture, macrophomate (2) is formed at a rate that is
Although the macrophomate synthase active site is rich in
potential functional groups, site-directed mutagenesis shows
that only three residues are absolutely required for catalysis of
oxaloacetate decarboxylation and trapping of the resulting
enolate with a 2-pyrone; the other residues that line the binding
pocket are surprisingly tolerant to substitution.
A
variety of antibodies1 and oligonucleotides2 have been
engineered to catalyze Diels–Alder reactions, but only three
natural enzymes with possible Diels–Alderase activity3 – lovastatin
nonaketide synthase (LNS),4 solanapyrone synthase (SPS),5 and
macrophomate synthase (MPS)6,7 – have been isolated to date.
The recent determination of the crystal structure of MPS,8 which
promotes the conversion of 2-pyrones to benzoates (Scheme 1),
provides a valuable opportunity to examine the mechanism of one
of these catalysts in greater detail. Identification of the key catalytic
residues in the MPS active site is particularly important in light of
QM/MM calculations which suggest that a two-step Michael–
aldol sequence is energetically preferred over a concerted pericyclic
reaction (Scheme 1).9 To that end we have prepared 16 active site
variants of this catalyst and characterized them kinetically.
consistent with the reported kcat value of 0.6 s21
.
The MPS active site is a spacious cavity at the interface of two
subunits (Fig. 1).8 It contains a catalytically essential magnesium
ion, which activates oxaloacetate for decarboxylation and
coordinates the resulting enolate that subsequently reacts with
2-pyrones. One side of the pocket is formed by Trp68, an extended
network of polar residues (Asp70, Glu72, His73, Arg101, His125
and Gln183), and the Mg2+-chelating amino acids Glu185 and
Asp211. The hydrophobic side chains of Phe1499, Pro1519,
Trp1529 and Tyr1699 from a different subunit define an adjacent
apolar surface below the pyruvate binding site, creating a cleft into
which the 2-pyrone can bind. We systematically mutagenized ten
Scheme 1 The MPS-catalyzed formation of macrophomate (2) from
2-pyrone 1 and oxaloacetate may proceed via a concerted Diels–Alder
reaction or a two-step Michael–aldol sequence.
aLaboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich,
Switzerland. E-mail: hilvert@org.chem.ethz.ch; Fax: +41-44-632-1486;
Tel: +41-44-632-3176
Fig. 1 View into the MPS active site with Mg2+-bound pyruvate
(yellow). The catalytic pocket is formed at the interface of two protein
subunits (brown and blue surfaces). Hydrogen bonding interactions
between the catalytic residues and pyruvate are shown as dashed lines.
This figure was prepared using PyMOL.11
bDivision of Chemistry, Graduate School of Science, Hokkaido
University, Sapporo 060-0810, Japan
{ Electronic supplementary information (ESI) available: Details on
preparation and characterization of the protein variants. See DOI:
10.1039/b703177g
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 1701–1703 | 1701

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of these amino acids using standard procedures to probe their
contribution to catalysis. Mutant genes were constructed using the
QuickChange method12 and verified by DNA sequencing. The
protein variants were then produced and purified as described for
the parent enzyme. Their structural integrity was confirmed by
ESI-MS and circular dichroism spectroscopy. All variants were
tested for oxaloacetate decarboxylase activity in the absence of
2-pyrone 1 and, independently, for their ability to form 2 in its
presence. Pyruvate, which is an alternative substrate for the
enzyme,10 was used to assay MPS activity with variants that lack
decarboxylase activity. The steady-state kinetic parameters for the
decarboxylation reaction and the relative MPS activities are
summarized in Table 1.
The same three residues that are required for the decarboxyla-
tion of oxaloacetate also strongly influence the subsequent
conversion of 2-pyrones to benzoates. Thus, no MPS activity is
detected for the Asp70Ala/Asn or Arg101Ala/Ser variants, and
only small amounts of 2 are formed when the His73Ala/Asn and
Arg101Lys variants are incubated with pyruvate and 1 (Table 1).
Arg101 is within hydrogen bonding distance of the carboxylate
group of pyruvate, and the fact that Arg101Lys, but not
Arg101Ala/Ser, retains low levels of activity supports the
suggestion8 that a long cationic residue at this position may also
be important for orienting and activating the 2-pyrone via a
hydrogen bonding interaction. Asp70, which forms a tight salt
bridge with Arg101 but is too distant to interact with substrate
directly, probably fulfils a structural role, ensuring that the
guanidinium cation is appropriately positioned for catalysis.
Similarly, although His73 could conceivably function as an
acid or base in the complex reaction sequence leading to the
final product,14 its location within the active site makes a
structural role more plausible: it mediates intersubunit
packing through extensive van der Waals contacts through its
imidazole ring and by donating a hydrogen bond to a carboxylate
group on the adjacent subunit. Disruption of this set of
interactions would be expected to perturb local active site geometry
significantly.
Our results show that three residues, Asp70, His73 and Arg101,
are absolutely essential for efficient enzyme-catalyzed decarboxyla-
tion of oxaloacetate. Even conservative replacement of aspartate
and histidine by asparagine, or arginine by lysine, reduces
decarboxylase activity more than 104-fold. In contrast, substitution
of Glu72, His125, Gln183, and the various hydrophobic residues
leads to relatively modest reductions in activity (, 9-fold changes
in kcat, and 3–90-fold decreases in kcat/Km). The large increase in
Km for some of these mutants points to disrupted oxaloacetate
binding, perhaps due to subtle changes in the coordination sphere
of the magnesium ion or to an otherwise distorted active site
geometry. Given the location of most of these residues at the
subunit interface, altered quaternary interactions may be the
ultimate origin of these effects.
Changes in MPS activity upon mutating the other active site
residues are minor (less than 6-fold reductions in rate) and
correlate roughly with the (larger) effects on the initial decarbox-
ylation step (Table 1). These results are consistent with previous
observations that pyruvate formation from oxaloacetate is more
efficient than formation of 2.6 As shown with the Glu72Gln
variant, decarboxylase activity can be reduced by as much as a
factor of 20 without impacting MPS activity. Interestingly,
generation of the reactive enolate by deprotonation of pyruvate
can be more or less sensitive to specific mutations than the
decarboxylation of oxaloacetate (Table 1). Finally, we find no
evidence to support the suggestion that Tyr169, which sits at the
entrance of the binding pocket, engages in a productive hydrogen
bond with the exocyclic C5 carbonyl group of the 2-pyrone.8 In
our hands, the Tyr69Phe variant is fully active, perhaps because of
the improved purification protocol.
Table 1 Steady state parameters for the MPS-catalyzed decarboxyla-
tion of oxaloacetate and relative rates of formation of macrophomate
(2)a
kcat/Km
[M21 s21
b
c
Mutant
kcat [s21
12.0 ¡ 0.6 80 ¡ 10
]
Km [mM]
] k
Yield
MPA
rel;ox
MPA
rel;py
wt MPS
150000
6500
, 10
, 10
1600
6300
, 10
, 10
, 10
, 10
, 10
7700
2600
37000
42000
1400
2700
100
18
—
—
9
92
—
—
—
—
—
31
21
67
110
4
100
21
—
—
13
93
2
Trp68Tyr
Asp70Ala
Asp70Asn
Glu72Ala 1.3 ¡ 0.1 800 ¡ 110
Glu72Gln 1.0 ¡ 0.1 160 ¡ 20
13 ¡ 1
—
—
2000 ¡ 120
—
—
His73Ala
—
—
—
—
—
—
—
—
—
—
His73Asn
Arg101Ala
Arg101Ser
Arg101Lys
His125Ala 7.7 ¡ 0.2 1000 ¡ 100
Pro151Ala 9.5 ¡ 1.4 3700 ¡ 900
Trp152Tyr 7.3 ¡ 0.3 200 ¡ 30
Tyr169Phe 5.0 ¡ 0.1 120 ¡ 10
Gln183Ala 4.1 ¡ 0.2 2900 ¡ 250
Gln183Asn 8.0 ¡ 0.4 3000 ¡ 330
a
3
—
—
10
15
33
97
100
6
Despite containing a large number of potentially useful
functional groups, the MPS active site is notable for its relative
insensitivity to mutation. Aside from the catalytically essential
magnesium ion, generation of the reactive pyruvate enolate and its
reaction with 2-pyrones appear to be facilitated primarily by
Arg101, which may preorganize the reactants through hydrogen
bonds and also electrostatically stabilize negative charges that
develop in the course of the reaction. The other essential amino
acids identified by mutagenesis, Asp70 and His73, more likely
serve structural roles. Although these findings do not resolve the
central question regarding the concertedness of the key C–C bond
forming step(s),6,9 it is striking that these same residues are strictly
conserved in a structurally homologous enzyme, 2-dehydro-3-
deoxygalactarate (DDG) aldolase, which catalyzes the reversible
aldol addition of pyruvate to tartronic semialdehyde.15 Based on
the relatively minor effects associated with their mutation, the
other active site residues appear to play a more indirect role in
catalysis, fine tuning the reactivity of the magnesium ion or
influencing, through steric interactions, the choice of reaction
16
11
Assays were performed in 50 mM PIPES (pH 7.0) containing 5 mM
MgCl2 at 30 uC. The disappearance of oxaloacetate was monitored
at 305 nm in the absence of 2-pyrone 1. Under these conditions, the
spontaneous rate of decarboxylation is kuncat 5 1.5 6 1025 s21 at
pH 7.0 and 25 uC,13 giving a net rate acceleration for the enzyme
(kcat/kuncat) of ca. 8 6 105. The formation of macrophomate (MPA,
2) was monitored by reverse phase HPLC as a function of time.
b
MPA
rel;ox
k
represents the relative rate of macrophomate formation in
reactions of 2-pyrone ([1] 5 1 mM) and oxaloacetate (1.0 mM) with
MPS (0.7 mM). The standard error on the relative rates is ¡ 15%.
The relative yield of macrophomate formed in reactions of pyrone
c
([1] 5 1 mM) and pyruvate (10 mM) after incubation with MPS
(20 mM)for 72 h at pH 7.0 and 30 uC. The error on the HPLC
measurements is ¡ 15%.
1702 | Chem. Commun., 2007, 1701–1703
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This work was generously supported by the ETH Zu¨rich and a
Kekule´ fellowship from the Fonds der deutschen chemischen
Industrie (JMS).
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