THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 43, Issue of October 22, pp. 45110 –45120, 2004
Printed in U.S.A.
A Reciprocal Single Mutation Affects the Metal Requirement of
3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) Synthases
from Aquifex pyrophilus and Escherichia coli*
Received for publication, April 26, 2004, and in revised form, August 11, 2004
Published, JBC Papers in Press, August 12, 2004, DOI 10.1074/jbc.M404561200
Smadar Shulami‡§, Cristina Furdui¶, Noam Adir§储, Yuval Shoham‡储**, Karen S. Anderson¶,
and Timor Baasov§储‡‡
From the ‡Department of Biotechnology and Food Engineering, the §Department of Chemistry, and the 储Institute of
Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel and the ¶Department of
Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066
The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase is metal-dependent in one class
of organisms and metal-independent in another. We have
used a rapid transient kinetic approach combined with
site-directed mutagenesis to characterize the role of the
metal ion as well as to explore the catalytic mechanisms of
the two classes of enzymes. In the metal-dependent
Aquifex pyrophilus KDO8P synthase, Cys11 was replaced
by Asn (ApC11N), and in the metal-independent Escherichia coli KDO8P synthase a reciprocal mutation, Asn26
to Cys, was prepared (EcN26C). The ApC11N mutant retained about 10% of the wild-type maximal activity in the
absence of metal ions. Addition of divalent metal ions did
not affect the catalytic activity of the mutant enzyme and
its catalytic efficiency (kcat/Km) was reduced by only ⬃12fold, implying that the ApC11N KDO8P synthase mutant
has become a bone fide metal-independent enzyme. The
isolated EcN26C mutant had similar metal content and
spectral properties as the metal-dependent wild-type
A. pyrophilus KDO8P synthase. EDTA-treated EcN26C retained about 6% of the wild-type activity, and the addition
of Mn2ⴙ or Cd2ⴙ stimulated its activity to ⬃30% of the
wild-type maximal activity. This suggests that EcN26C
KDO8P synthase mutant has properties similar to that of
metal-dependent KDO8P synthases. The combined data
indicate that the metal ion is not directly involved in the
chemistry of the KDO8P synthase catalyzed reaction, but
has an important structural role in metal-dependent enzymes in maintaining the correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site.
The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate
(KDO8P)1 synthase (EC 4.1.2.16) catalyzes the condensation
* This work was supported by U.S.-Israel Binational Science Foundation (Grant 2002-126) (to T. B. and K. S. A.), by Rubin Scientific and
Medical Fund for promotion of research at the Technion (Grant 060624), and by National Institutes of Health Grants GM61413 and
GM71805, (to K. S. A.). Additional support was provided by the Fund
for the Promotion of Research at the Technion, and by the Otto Meyerhof Center for Biotechnology, Technion, established by the Minerva
Foundation (Munich, Germany). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence may be addressed: Dept. of Biotechnology
and Food Engineering, Technion, Haifa 32000, Israel. Tel.: 972-48293072; Fax: 972-4-8320742; E-mail: [email protected].
‡‡ To whom correspondence may be addressed: Dept. of Chemistry,
Technion, Haifa 32000, Israel. Tel.: 972-4-8292590; Fax: 972-48295703; E-mail: [email protected].
1
The abbreviations used are: KDO8P, 3-deoxy-D-manno-2-octu-
reaction between D-arabinose-5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (Pi) (see Scheme 1) (1). This enzymatic reaction plays an
essential role in the assembly process of lipopolysaccharides of
most Gram-negative bacteria, and therefore represents an attractive target for the design of novel antibacterial drugs (2, 3).
While earlier studies on Escherichia coli KDO8P synthase
have established that this enzyme does not require metals (1),
it has recently been demonstrated that enzymes from the hyperthermophilic bacteria Aquifex aeolicus (4), Aquifex pyrophilus (5), and from the pathogenic bacterium Helicobacter pylori
(6), require a divalent metal cofactor for catalysis. Furthermore, phylogenetic analysis (7, 8) suggests that KDO8P synthases from other pathogenic bacteria, i.e. Chlamydia trachomatis, Chlamydia pneumoniae, and Campylobacter jejuni, may
also be metal-dependent enzymes. This is reminiscent of another class of enzymes, aldolases, which catalyze a similar
aldol-type C-C bond formation (9). Class I aldolases, which are
primarily found in animals and higher plants, do not require a
metal cofactor for catalysis. In contrast, Class II aldolases,
found predominantly in prokaryotes, use a divalent metal cofactor that functions as a Lewis acid. Thus, in addition to
aldolases, KDO8P synthase represents another example of an
enzyme that is metal-independent in one class of organisms
(Class I) but metal-dependent in another (Class II). Therefore,
KDO8P synthase represents a distinctive target for the development of selective, narrow-spectrum antibiotics.
The catalytic mechanism of E. coli KDO8P synthase has
been studied extensively and earlier studies have established
that the reaction occurs through the unusual cleavage of the
C-O bond of PEP (10, 11). KDO8P synthesis was shown to be a
sequential process in which the kinetically preferred order of
binding involves the PEP preceding the binding of acyclic A5P,
and the release of inorganic phosphate preceding the dissociation of the product KDO8P (12). The condensation step was
shown to be stereospecific, with the si face of PEP attaching to
the re face of the carbonyl group of A5P (see Scheme 1) (13, 14).
More recent studies using rapid chemical quench techniques
(15), together with the synthesis and evaluation of an acyclic
bisubstrate inhibitor (16), supported the reaction pathway that
involves the formation of an inherently unstable, acyclic
bisphosphate intermediate 1 (Scheme 1). Moreover, the identification of 1 as a true reaction intermediate was provided
losonate-8-phosphate; PEP, phosphoenolpyruvate; A5P, D-arabinose-5phosphate; DAHP synthase, 3-deoxy-D-arabino-2-heptulosonate-7phosphate synthase; ApC11N, C11N mutant of A. pyrophilus KDO8P
synthase; EcN26C, N26C mutant of E. coli KDO8P synthase; TEAB,
triethylammonium bicarbonate; ICP-MS, inductively coupled plasmamass spectrometry; HPLC, high pressure liquid chromatography.
45110
This paper is available on line at http://www.jbc.org
KDO8P Synthases from A. pyrophilus and E. coli
SCHEME 1. Proposed mechanism for KDO8P synthase-catalyzed reaction.
recently (17) using time-resolved electrospray ionization mass
spectrometry (ESI-MS) experiments that directly monitor the
enzymatic reaction with its natural substrates on a very short,
millisecond time scale. This study has established the time-dependent formation and decay of the enzyme bound intermediate 1 on a time scale consistent with substrate decay and
product formation, as expected for a true reaction intermediate.
In addition to the mechanistic studies described above, several crystal structures of the E. coli enzyme have recently been
reported (18, 19), including the structures of KDO8P synthase
in its binary complexes with a mechanism-based inhibitor and
with the substrate PEP (20). Based on the position of PEP at
the active site, it was suggested (20) that the condensation step
between PEP and A5P should proceed in a stepwise fashion in
which the initial formation of a transient oxocarbenium intermediate 1a, or an early transition state having oxocarbenium
character, must be followed by the capture of water at the
cationic C2 position to complete the formation of acyclic intermediate 1 (Scheme 2, path a). This mechanism is consistent
with earlier observations that examined intramolecular models
of KDO8P synthase-catalyzed reaction (21).
Unlike the reaction of E. coli KDO8P synthase, the catalytic
mechanism of the metal-dependent KDO8P synthases has been
studied to a lesser extent (22, 23). Based on the three-dimensional structures of the A. aeolicus enzyme in complex with
exogenous Cd2⫹ and various combinations of substrates and/or
inhibitors, it was proposed that a water molecule activated by
a metal ion initiates the condensation reaction by attacking the
C2 of PEP followed by subsequent coupling of the carbanionic
C3 with the carbonyl of A5P which leads to the formation of the
same acyclic intermediate 1 (Scheme 2, path b). Since this
mechanism includes the opposite sequence of condensation
steps to that suggested for metal-independent enzymes
(Scheme 2, path a), it seems that the major difference between
the mechanisms of Class I and Class II enzymes is in the
sequence of the elementary steps that leads to the formation of
the same intermediate 1.
The three-dimensional structure of the A. aeolicus enzyme in
complex with exogenous Cd2⫹ also identified the amino acid
residues involved in metal binding: Cys11, His185, Glu222, and
Asp233, along with the metal-coordinated water molecule (23).
To further delineate the role of these residues in metal binding
and/or catalysis, the H185G mutant of A. aeolicus enzyme was
recently prepared and structures of Cd2⫹-H185G enzyme in its
substrate-free form and in complex with PEP, and PEP plus
A5P were determined (24). Interestingly, unlike the wild-type
enzyme, no metal coordinated catalytic water was found in
either, the structure of the substrate-free Cd2⫹-H185G enzyme
or the structures of Cd2⫹-H185G bound with PEP or with PEP
plus A5P. Instead, in the later structures the carboxylate moiety of PEP binds directly to the metal ion and replaces the
water and the His185 as the ligands. Despite the lack of catalytic water, the Cd2⫹-H185G enzyme retains 8% of the wildtype activity, raising the question about the exact role of the
metal ion in the metal-dependent enzymes.
We have recently cloned the kdsA gene from the hyperthermophilic bacterium A. pyrophilus, expressed the gene in E. coli
45111
and conducted an initial biochemical characterization of the
recombinant enzyme (5). Sequence alignment revealed that the
four amino acid residues that were identified in A. aeolicus
KDO8P synthase as ligands of the metal ion are located at
identical positions in the A. pyrophilus enzyme. Therefore, it is
likely that these residues are also involved in metal binding in
the A. pyrophilus KDO8P synthase. It is noteworthy that three
out of the four metal binding residues, His, Glu, and Asp in
metal-dependent KDO8P synthases, are completely conserved
in all KDO8P synthases currently sequenced (7), implying that
they play an essential role in both metal-dependent and metalindependent KDO8P synthase enzymes. Furthermore, the Cys
residue is conserved only in metal-dependent enzymes (7),
whereas in metal-independent enzymes, a conserved Asn replaces Cys. Based on these observations, we hypothesized (5),
that a single amino acid replacement, Cys to Asn, could eliminate the requirement for metals in metal-dependent enzymes,
and similarly, the reciprocal replacement in the metal-independent E. coli KDO8P synthase (Asn26 to Cys) could result in
metal-dependent activity.
In the present work, we describe the characterization of a
C11N mutant of A. pyrophilus KDO8P synthase (ApC11N) and
an N26C mutant of E. coli KDO8P synthase (EcN26C) with
respect to metal binding and catalysis. The specific questions
these studies were designed to address are as follows: (i) What
is the role of the divalent metal ion in metal-dependent enzyme? Does the metal ion play a direct catalytic or structural
role in catalysis? (ii) Can a single amino acid mutation be
sufficient to convert the enzyme from a metal-dependent to a
metal-independent and conversely? (iii) How do these changes
affect the overall reaction pathway? The present study provides
the first demonstration that a reciprocal single mutation has
the potential to convert any of metal-dependent class KDO8P
synthase to a metal-independent variant and vice versa. Some
important mechanistic implications of these observations
are discussed.
EXPERIMENTAL PROCEDURES
Chemicals and Reagents—A5P was prepared enzymatically according to the procedure of Whitesides and co-workers (25). The potassium
salt of PEP was prepared in large quantities as previously described
(26). Metal salts used in this study were obtained from Aldrich and all
other chemicals were purchased from Aldrich or from Sigma and were
used without further purification. Solutions and buffers were passed
through a column of Chelex 100 (Na⫹ from 100 –200 mesh; Bio-Rad), to
remove all traces of metal ion contamination. [1-14C]pyruvate was obtained from American Radiolabeled Chemicals, and the HPLC Mono Q
5/5 anion exchange column was from Amersham Biosciences.
[1-14C]PEP Synthesis—Radiolabeled PEP was enzymatically synthesized from [1-14C]pyruvate (American Radiolabeled Chemicals) by coupling pyruvate phosphate dikinase (PPDK) obtained as a generous gift
from Dr. Dunaway-Mariano, to inorganic pyrophosphatase reaction.
After purification by Q-Sepharose anion-exchange chromatography using a linear gradient (20 mM to 1 M) of triethylammonium bicarbonate
(TEAB) buffer, the fractions containing PEP were pooled and lyophilized. Final stock solutions (5.6 mM) contained 29,300 dpm/nmol.
Bacterial Strains and Plasmids—E. coli strain XL-1 Blue (Stratagene La Jolla, CA) was used for general cloning. E. coli strain
BL21(DE3) (Promega, Madison, WI) was used for protein production
with the pET9d expression vector (Novagen, Madison, WI).
DNA Manipulations—Plasmid DNA was purified with the WizardR
DNA Clean-Up system (Promega). DNA was transformed by electroporation using GeneZapper (IBI, New Haven, CT) for strains XL-1 Blue
and BL21(DE3). DNA sequencing was performed at the DNA sequencing unit of the Weizmann Institute (Rehovot, Israel). DNA sequences
were analyzed using MacVectorTM 7.0 (Oxford Molecular Ltd.) and by
the software package of the Genetics Computer Group (GCG, version 9,
University of Wisconsin, Madison, WI).
Cloning and Mutagenesis—The kdsA gene (GenBankTM accession
number AY135660) from A. pyrophilus was cloned in the pET9d expression vector (Novagen, Madison, WI) as previously described (5). The
kdsA gene from E. coli was amplified from the pJU1 vector (12), using
45112
KDO8P Synthases from A. pyrophilus and E. coli
SCHEME 2. Proposed elementary
steps for the formation of intermediate 1.
two PCR primers that allowed the in-frame cloning in the pET vector.
The N-terminal primer (5⬘-GGAATATCATGAAACAAAAAGTCGTTAGC-3⬘) was made to contain an ATG transnational start codon inside a
BspHI restriction site (TCATGA). The C-terminal primer (5⬘-GCGATAGATCTTACTTGCTGGTATCCAGTTC-3⬘) contained a stop codon
(TAG) and a BglII restriction site (AGATCT) at the end of the gene.
Following PCR amplification the gene was cloned into the T7 expression
vector pET9d (linearized with NcoI and BamHI), resulting in plasmid
pET9d-kdsA.
Site-directed mutagenesis was performed using the QuikChange
site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutagenic
primers for C11N of KDO8P synthase from A. pyrophilus were as follows (the mutated nucleotides are shown in bold): 5⬘-GACTCGCTCTCGATCGCATTGGGTCCCGC-3⬘ and 5⬘-GCGGGACCCAATGCGATCGAGAGCGAGTC-3⬘. The primers for N26C of KDO8P synthase from
E. coli were as follows (the mutated nucleotides are shown in bold):
5⬘-GATTCCAACACGCACATGCCGCCAAACAGTAC-3⬘ and 5⬘-GTACTGTTTGGCGGCAT GTGCGTGTTGGAATC-3⬘. The mutated genes
were sequenced to confirm that only the desired mutation was inserted.
The mutated genes were overexpressed and purified as described below.
Purification of the kdsA Gene Products—The recombinant kdsA
genes from both E. coli (1) and A. pyrophilus bacteria (5) were expressed
using similar procedures. Briefly, the expression was carried out by
growing overnight cultures of E. coli BL21(DE3) carrying pET9d-kdsA
in Luria-Bertani (LB) broth (27) with kanamycin (25 ␮g ml⫺1), without
induction. Purification procedure of the kdsA gene from E. coli was
similar to that of the thermostable enzyme but with the following
modifications: all manipulations were carried out at 4 °C and without
the heat treatment step. The overnight cultures (2 liters) were harvested, resuspended in 15 ml of 50 mM NaCl and 20 mM Tris-HCl buffer
pH 7.5, and disrupted by two passages through a FrenchR Press (Spectronic Instruments, Inc., Rochester, NY) at room temperature. Cell
extracts were centrifuged, and the resulting supernatant was applied to
an anion-exchange column (HiPrep 16/10Q FF, Amersham Biosciences)
equilibrated with 20 mM Tris-HCl, pH 7.5. The column was first washed
with five column volumes of equilibration buffer and then eluted with
20 column volumes of a linear gradient of 0.05–1.0 M NaCl in 20 mM
Tris-HCl, pH 7.5. The enzymes eluted as distinct peaks, collected, and
found to be homogeneous as determined by SDS-PAGE. Aliquots (1 ml)
of the purified enzymes were stored at ⫺80 °C. In the purification of the
wild-type and mutant A. pyrophilus KDO8P synthases a heat-treatment step was included (65 °C, 30 min) as previously described (5).
Metal Content Analysis for the As-isolated and EDTA-treated KDO8P
Synthase—The procedure described below applies to both the E. coli
and A. pyrophilus KDO8P synthase and to their respective mutants
N26C and C11N. Native KDO8P synthase enzymes as isolated were
concentrated by ultrafiltration using FUGISEP (Wokingham Berkshire, England) to 6 –7 mg/ml. Enzymes were treated with 10 mM EDTA
for 3 h at room temperature, and then dialyzed against 500 ml of buffer
50 mM Tris-HCl, pH 7.5 at 4 °C with two buffer changes, one with 2 mM
EDTA and the second with 1 mM EDTA for about 4 h. Overnight dialysis
was carried out against 1.5 liters of buffer with 1 ␮M EDTA. Residual
EDTA was removed by three sequential 3-h periods of dialysis at 4 °C
against 50 mM buffer Tris-HCl, pH 7.5 (without EDTA) containing
Chelex 100 (25 g/liter, Na⫹ form, 100 –200 mesh, Bio-Rad). Additional
samples of the native enzymes were prepared without EDTA treatment.
The concentration of metals: Mn2⫹, Cd2⫹, Zn2⫹, Cu2⫹, Fe2⫹, Ni2⫹,
Co2⫹, Mg2⫹, and Ca2⫹, in both EDTA-treated and EDTA-untreated
enzymes (40 ␮M), was determined by high resolution Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) on a PerkinElmer Optima
3000DV. Samples of the dialysate buffer (from the last change) were
also analyzed for metal content, and the observed concentrations were
subtracted from those obtained in the enzymes samples.
Cu2⫹ Reconstitution of the EcN26C—An aliquot of the EDTA-treated
enzyme (20 ␮M) was diluted in 500 ␮l of metal-free buffer (50 mM
Tris-HCl), pH 7.5, and various concentrations of CuSO4 were added.
After 2 min of incubation at 35 °C, the absorbance spectra were recorded on a Biochrom 4060 spectrophotometer.
Steady-state Kinetic Analysis—The KDO8P synthase activity was
determined either by a discontinuous colorimetric assay or a continuous
spectrophotometric assay. All experiments were carried out in 100 mM
HEPES pH 7.5 at 60 °C for A. pyrophilus wild-type and C11N KDO8P
synthase and at 25 or 37 °C for E. coli wild-type and N26C KDO8P
synthase in 50 mM Tris, pH 7.5. The dependence of A. pyrophilus
wild-type and C11N-KDO8P synthase on metal chelator concentration
and the dependence of E. coli KDO8P synthase wild-type and N26C
mutant on metal ion concentration were followed using a discontinuous
assay as described in the next paragraph. All of the other steady-state
kinetic experiments were followed spectrophotometrically at 232 nm by
monitoring the consumption of PEP (⑀232 ⫽ 2.84 mM⫺1 cm⫺1).
Effect of Metal Chelators and Divalent Metal Ions on KDO8P Synthase Activity—A discontinuous assay was used to study the effects of
metal chelators and divalent metal ions on enzymatic activities. In the
discontinuous assay, the amount of the KDO8P product was determined by the thiobarbituric acid assay as specified by Ray (1). One unit
of activity is defined as the amount of enzyme required to produce 1
␮mol of KDO8P per minute. The standard discontinuous assay was
conducted in a final volume of 200 ␮l of 100 mM HEPES or 50 mM Tris
pH 7.5, 2 mM PEP, 2 mM A5P, and appropriately diluted enzyme. After
incubation, the reaction was quenched by adding trichloroacetic acid to
a final concentration of 5%. To ensure initial velocity conditions, the
enzyme concentration and the incubation time were chosen so that the
substrate conversion was less than 5%, and the product formation was
linear over the time course the measurements were made.
(i) Catalytic Activity of A. pyrophilus Wild-type and C11N KDO8P
Synthase—The specific activities of wild-type and mutant KDO8P synthase from A. pyrophilus were determined at 60 °C in 100 mM HEPES
buffer, pH 7.5. Aliquots (10 ␮l) of the wild-type enzyme (0.3 ␮M) or its
C11N mutant (50 ␮M) were first preincubated with EDTA (10 ␮l, 4 mM)
for 15 min at room temperature. In the case of wild-type enzyme, a
buffer solution (160 ␮l) containing 0.48 mM Cd2⫹ was added to the
enzyme followed by 20 ␮l of 20 mM PEP. In the case of C11N mutant,
Cd2⫹ was omitted from the reaction buffer. The resulting mixture was
incubated for 10 min at 60 °C, and the reaction was initiated by adding
20 ␮l of A5P (20 mM). After incubation at 60 °C, the reaction was
quenched by adding trichloroacetic acid to a final concentration of 5%.
The effect of chelators on the activity of A. pyrophilus wild-type KDO8P
synthase and the C11N mutant, was determined as follows: 10 ␮l of
enzyme (2 ␮M) was incubated with 10 ␮l of EDTA or 1,10-phenanthroline at a final concentration of 10 –500 ␮M for 15 min at room temperature. The assays were performed at 60 °C with the same final concentration of the chelator. The effect of various divalent metals was
measured by preincubating the enzyme with 4 mM EDTA for 15 min at
room temperature, followed by addition of reaction buffer (100 mM
HEPES, pH 7.5, 160 ␮l) containing various metal concentrations, and
the resulted mixture was again incubated for an additional 10 min at
KDO8P Synthases from A. pyrophilus and E. coli
45113
TABLE I
Metal content and specific activities of the wild-type and mutant KDO8P synthases from A. pyrophilus and E. coli
Metal content analysis experiments were performed at least three times and similar data were obtained in at least two independent experiments
within average experimental error of ⱕ10%.
Source
Enzyme
Specific activity
Wild-type as purified
EDTA-treated wild type
Cd2⫹- wild type
C11N as purified
EDTA-treated C11N
Cd2⫹- C11N
Wild type as purified
N26C as purified
EDTA-treated N26C
3.2 ⫾ 0.3
0.03 ⫾ 0.01
8.0 ⫾ 0.7
0.8 ⫾ 0.1
0.8 ⫾ 0.1
1.2 ⫾ 0.1
12.0 ⫾ 0.1
0.14 ⫾ 0.01
0.73 ⫾ 0.06
Zn2⫹
Fe2⫹
0.26
0.06
NDa
0.02
0.04
ND
0.05
0.37
0.009
0.19
0.08
ND
0.22
0.02
ND
⬍0.001
0.22
0.009
units/mg
A. pyrophilus
E. coli
a
Cu2⫹
Ni2⫹
Co2⫹
Mg2⫹
⬍0.001
⬍0.001
ND
⬍0.001
⬍0.001
ND
⬍0.001
⬍0.001
⬍0.001
⬍0.001
⬍0.001
ND
⬍0.001
⬍0.001
ND
0.05
⬍0.001
⬍0.001
mol metal/mol enzyme subunit
⬍0.001
⬍0.001
ND
⬍0.001
⬍0.001
ND
⬍0.001
⬍0.001
⬍0.001
⬍0.001
⬍0.001
ND
⬍0.001
⬍0.001
ND
⬍0.001
⬍0.001
⬍0.001
ND, not determined.
60 °C. The reactions were initiated by adding 20 ␮l of buffer containing
A5P (20 mM) and PEP (20 mM).
(ii) Catalytic Activity of E. coli Wild-type and N26C KDO8P Synthase—The effect of metals on E. coli KDO8P synthase activity was
assayed as follows: 160 ␮l reaction buffer (100 mM Tris-HCl, pH 7.5), 20
␮l of various metals and 20 ␮l of the mixture of substrates, A5P (20 mM)
and PEP (20 mM) were prewarmed for 10 min at 37 °C. The reaction was
initiated by adding 20 ␮l of wild type (0.3 ␮M) or EDTA-treated N26C
mutant (6 ␮M), and the reaction was allowed to proceed under the initial
velocity conditions.
More detailed kinetic studies were carried out using a continuous
spectrophotometric assay. The reactions were performed in 100 mM
Tris, pH 7.5 at 37 and 60 °C for E. coli KDO8P synthase and
A. pyrophilus enzymes, respectively. Assay solutions contained appropriately diluted enzymes with varying concentrations (0.1 to 5 Km) of
one substrate while the other substrate was held constant (10⫻ Km).
Data were fit to Michaelis-Menten equation for metal dependence experiments or to the Hill equation when cooperative behavior was observed for substrate dependence experiments. The kcat, Km, and Hill
coefficient (nH) parameters for metal, PEP, and A5P were determined.
The values of Km and kcat were determined by non-linear regression
analysis using the program GRAFIT 5.0 or Kaleidagraph 3.52.
Pre-steady-state Experiments—Rapid quench experiments were performed using a Kintek RFQ-3 Rapid Chemical Quench (Kintek Instruments, Austin, TX) as previously described (28). The reaction was
initiated by mixing the enzyme solution (15 ␮l) with the radiolabeled
substrate (15 ␮l). In all cases, the concentrations of the enzyme and
substrates cited in the text are those after mixing and during the
reaction. The reaction was then quenched with 67 ␮l of 0.6 N KOH. 60
␮l of each sample was removed and analyzed by HPLC with on-line
UV-vis detector (Waters 486 Tunable Absorbance Detector) followed by
Flo-One radioactivity detection (Packard Instruments, Downers Grove,
IL). The analysis system was automated using a Waters 717plus (Milford, MA) autosampler. The substrates and products were separated on
an anion exchange Mono-Q HR 5/5 column (Amersham Biosciences)
using a 25 min of linear gradient from 20 mM to 1 M TEAB followed by
10 min column equilibration with 20 mM TEAB. The elution was performed with a flow rate of 1 ml/min. Before entering the radioactivity
detector the eluent was mixed with liquid scintillation mixture (Monoflow V, National Diagnostics) at a flow rate of 5 ml/min. During the
HPLC analysis a small amount of cold PEP was always injected as an
internal standard as it co-elutes with hot PEP and its elution time can
be monitored at 232 nm by absorbance detector. The retention times for
KDO8P and PEP were 12.7 min and 16.5 min, respectively.
Pre-steady-state Burst Experiment for A. pyrophilus Wild-type Cd2⫹
and Mn2⫹ Reconstituted KDO8P Synthase—20 ␮M KDO8P synthase, 60
␮M [1-14C]PEP, and 200 ␮M CdCl2 or 2 mM MnCl2 in 50 mM, Tris pH 7.5
were mixed with 500 ␮M A5P. The reaction was quenched with 0.3 M
KOH. The samples were analyzed by HPLC as described above.
Pre-steady-state Burst Experiment for ApC11N—3 ␮M KDO8P synthase, 9 ␮M [1-14C]PEP, and 2 mM Mn2⫹ in 50 mM Tris, pH 7.5 were
mixed with 1000 ␮M A5P in a rapid chemical quench apparatus (Kintek
Instruments, Inc., Austin, TX). The same procedure for quenching and
sample analysis was used as described above. Performing the reaction
at higher enzyme concentrations was hindered by the formation of
aggregates, which resulted in lower active site concentration and lower
burst rate.
Pre-steady-state Burst Experiment for EcN26C—Pre-steady-state
analysis of this mutant enzyme was complicated by a minor contami-
nant protein(s) possessing an activity that degraded the radiolabeled
PEP to pyruvate as evidenced by HPLC analysis with radioactivity
detection. This degradation of PEP occurred when the mutant enzyme
was incubated with PEP for extended periods of time required to collect
a time course for rapid chemical quench. Removal of the contaminating
activity was accomplished by further purification using anion exchange
chromatography. The specific activity after extended purification was
somewhat lower. However, the contaminating activity causing PEP
degradation was removed thus allowing further analysis using a transient kinetic strategy. (i) Pre-steady-state burst experiment in the absence of metals: 20 ␮M N26C, 60 ␮M [1-14C]PEP, and 500 ␮M EDTA were
mixed with 1 mM A5P in 50 mM Tris, pH 7.5 in a rapid-quench apparatus. The reaction was quenched with 0.3 M KOH, and the formation of
KDO8P was followed by HPLC as described above. (ii) Pre-steady-state
burst experiments in the presence of different metal concentrations: 20
␮M N26C, 60 ␮M [1-14C]PEP, 500 ␮M EDTA, and different Cd2⫹ concentrations were mixed with 1 mM A5P in 50 mM Tris pH 7.5 in a rapid
chemical quench apparatus. The total metal concentrations added into
the solution were: 0.52 mM (20 ␮M free metal), 0.8 mM (300 ␮M free
metal), 1.2 mM (800 ␮M free metal), and 1.9 mM (1.4 mM free metal). The
reaction was quenched with 0.3 M KOH, and the formation of KDO8P
was followed by HPLC as described above.
Transient Kinetic Data Analysis—The formation of KDO8P (␮M) was
plotted against time (s), and data were fitted to a single exponential
equation followed by linear Equation 1.
C ⫽ C0 ⫻ (1 ⫺ e(⫺k1t)) ⫹ C0 ⫻ k2t
(Eq. 1)
In this expression, C represents the product concentration at time t, the
amplitude C0 corresponds to active site concentration, k1 is the rate
constant for product formation (s⫺1) and k2 is the rate of product release
(s⫺1). By dividing C0 with the enzyme concentration, we determined the
percentage of active enzyme. Data were analyzed using Kaleidagraph
version 3.52, released June 17, 2002 by Synergy Software.
RESULTS
Metal Content Analysis—For both E. coli and A. pyrophilus
enzymes, metal content was analyzed by ICP-MS (Table I). The
wild-type E. coli KDO8P synthase, as isolated, contained only
0.05 ⫾ 0.01 mol of zinc and 0.05 ⫾ 0.01 mol of magnesium per
mol of enzyme. All of the other metals tested were below the
detection limit. The EcN26C mutant, as isolated, contained
⬃0.2 mol of iron and 0.4 mol of zinc per mol of enzyme. Manganese, copper, chromium, nickel, cadmium, and cobalt were
below the detection limit. This mutant enzyme exhibited a
specific activity of 0.14 units/mg. Treatment with EDTA reduced the iron and zinc content to 0.009 mol per mol of enzyme
and produced an enzyme with a specific activity of 0.73 units/
mg. A similar increase in activity was observed when the assay
of as-isolated EcN26C was conducted in the presence of micromolar levels (⬃1–10 ␮M) of EDTA (data not shown). This observation was reproducible and may indicate the presence of
unidentified trace metal ions (such as Pb2⫹) that inhibit the
enzyme. Moreover, EDTA could overcome this inhibitory effect.
The wild-type metal-dependent KDO8P synthase from
A. pyrophilus, as isolated, contained 0.19 and 0.26 mol of iron
45114
KDO8P Synthases from A. pyrophilus and E. coli
FIG. 1. Absorption spectra of EcN26C. Curve 1 is of the N26C
mutant (40 ␮M) after treatment with EDTA (10 mM) and removal of
excess EDTA by dialysis. The spectrum of the N26C mutant (253 ␮M) as
purified is shown in curve 2, and the mutant with the addition of CuSO4
(109 ␮M) is shown in curve 3. Inset shows the titration of EDTA-treated
N26C mutant (40 ␮M) with various molar equivalents (as indicated) of
CuSO4. All spectra were acquired at 35 °C in 50 mM Tris-HCl buffer,
pH 7.5.
FIG. 2. Effect of metal chelators on the activities of the wildtype and C11N mutant of A. pyrophilus KDO8P synthase. The
effect of chelators was tested by incubating wild-type enzyme with
different concentrations of EDTA (●) or 1,10-phenanthroline (E), and
by incubating the C11N mutant with EDTA (Œ) or 1,10-phenanthroline
(‚) at final concentrations of 10 –500 ␮M, for 15 min at room temperature. After preincubation, the assays were performed in 100 mM HEPES
pH 7.5, at 60 °C and the same final concentration of the chelator. The
activities are expressed as the percentage of the initial activity in the
absence of a metal chelator. All the experiments were performed at
least three times within average experimental error of about 8%.
and zinc, respectively, per mol of enzyme (5), whereas metal
analysis of the C11N variant revealed 0.22 mol of iron and 0.02
mol of zinc per mol of enzyme (Table I). Thus, it appears that
the loss of Cys11 decreases the apparent affinity of KDO8P
synthase for zinc but not for iron. Similar analysis of the
EDTA-treated C11N showed 0.02 and 0.04 mol of iron and zinc,
respectively, per mol of enzyme.
Spectral Properties of EcN26C—A concentrated solution of
the purified EcN26C had a pinkish color, likely caused by
bound iron (Table I). The iron content is also reflected by a
broad spectrum peak centered at 575 nm (⑀575 ⫽ ⬃440 M⫺1
cm⫺1) (Fig. 1). Addition of Cu2⫹ to the purified enzyme eliminates the A575 peak and generated a new absorption peak
centered at ⬃385 nm (⑀385 ⫽ ⬃1070 M⫺1 cm⫺1) (Fig. 1). Titration of EDTA-treated enzyme with Cu2⫹ resulted in the appearance of a well-defined absorbance peak at 385 nm (⑀385 ⫽
1000 M⫺1 cm⫺1), with stoichiometry of ⬃1 eq of Cu2⫹ per
enzyme subunit (Fig. 1, inset). This confirms the ability of
EcN26C to bind metal ion. The absorbance at 385 nm is consistent with a ligand-to-metal charge transfer band caused by
the formation of a Cu2⫹-thiolate complex (4, 29).
Metal Chelators Have No Effect on the Activity of
ApC11N—To examine whether a single amino acid replacement, Cys11 to Asn, could eliminate the requirement for metal
ions in A. pyrophilus KDO8P synthase, the activity of ApC11N
was determined in the presence of metal chelators. In the
presence of EDTA, the specific activity of ApC11N (0.8 units/
mg) was 10% of the maximal activity of Cd2⫹-reconstituted
wild type (8.0 units/mg, Table I). Unlike the wild-type A. pyrophilus KDO8P synthase, neither EDTA nor 1,10-phenanthroline influenced the activity of ApC11N (Fig. 2). When subjected to ICP-MS analysis, the EDTA-treated mutant and wildtype enzymes, showed almost identical metal content, less than
0.15 mol of metals per mol of enzyme (Table I). These data
indicate that the ApC11N can catalyze the reaction without
metal assistance.
Metal Requirement for the Wild-type and C11N Mutant
A. pyrophilus KDO8P Synthase—The EDTA treatment of wildtype A. pyrophilus KDO8P synthase, almost completely abolishes its activity, resulting in kcat of 0.045 ⫾ 0.011 s⫺1. By
following the dependence of the steady-state rate on metal ion
concentration and fitting the data to a Michaelis-Menten equation, it was found that the addition of Mn2⫹ to the wild-type
KDO8P synthase resulted in a 200-fold increase in the steadyMn2⫹
state rate (kcat ⫽ 9.0 s⫺1) (Fig. 3A) with apparent Km
of 10
␮M. However, when the same experiment was performed with
Cd2⫹, it was found that although the kcat was only slightly
lower than for the Mn2⫹-reconstituted enzyme (8.5 s⫺1 versus
9.0 s⫺1), there is a 16-fold difference in the Km values (0.6 ␮M
versus 10 ␮M) (Fig. 3B).
According to the x-ray crystal structure of A. aeolicus KDO8P
synthase, Cys11 is involved in direct coordination of the metal
(22); therefore replacing Cys11 with Asn in KDO8P synthase
from A. pyrophilus should affect metal binding. The consequence of C11N replacement on metal binding was evaluated
by measuring enzyme activity in the presence of metal ions.
Addition of Mn2⫹ up to 2 mM did not stimulate the reaction rate
of C11N mutant. However, addition of Cd2⫹ increased the
steady-state rate by about 3-fold (kcat ⫽ 1.5 s⫺1, Fig. 3C). The
Cd2⫹
apparent Km
obtained from fitting the data to MichaelisMenten equation was 640 ␮M.
Metal Requirement for the Wild-type and N26C Mutant
E. coli KDO8P Synthase—As mentioned above, the activity of
ApC11N was not affected by metal chelators. Our working
hypothesis assumes that the reciprocal mutation of the conserved Asn26 to Cys in the metal-independent E. coli enzyme
might result in a metal-dependent KDO8P synthase (5). To
examine this hypothesis the wild-type and the EDTA-treated
EcN26C were separately incubated with various divalent metal
ions and the respective catalytic activities were determined. Of
the metals examined, Mn2⫹ and Cd2⫹ enhanced the original
activity of EDTA-treated N26C by about 5-fold (Fig. 4), resulting in ⬃30% of the wild-type maximum activity. While the
presence of Cd2⫹ (Fig. 4A) and Mn2⫹ (Fig. 4B) resulted in the
same specific activity (⬃3.6 units/mg), Cd2⫹ exhibited ⬃100fold higher apparent affinity for the EcN26C than that of Mn2⫹
(the apparent Km values estimated from the data in Fig. 4 were
about 1 ␮M and 100 ␮M, for Cd2⫹ and Mn2⫹, respectively).
Steady-state Kinetic Parameters for Wild-type and C11N Mutant A. pyrophilus KDO8P Synthase—To compare the kinetic
KDO8P Synthases from A. pyrophilus and E. coli
45115
FIG. 3. Effects of Cd2ⴙ and Mn2ⴙ on the activities of wild-type and C11N mutant A. pyrophilus KDO8P synthase. A, activity of
Mn2⫹
wild-type in the presence of various concentrations Mn2⫹ (kcat 9.0 ⫾ 0.8 s⫺1, Km
10 ⫾ 2 ␮M); B, activity of wild type in the presence of various
Cd2⫹
Cd2⫹
concentrations of Cd2⫹ (kcat 8.5 ⫾ 0.8 s⫺1, Km
0.6 ⫾ 0.1 ␮M). C, effect of Cd2⫹ on the activity of ApC11N (kcat 1.5 ⫾ 0.1 s⫺1, Km
0.64 ⫾ 0.11
mM). All data were fitted to the Michaelis-Menten equation.
parameters of the wild-type and the C11N mutant KDO8P
synthase, we followed the dependence of the steady-state rate
on PEP and A5P concentrations. The results of these kinetic
studies are summarized in Table II. For the wild-type Mn2⫹KDO8P synthase the kcat value was 9.0 s⫺1, and the Km for PEP
and A5P were 26 ␮M and 67 ␮M, respectively (Fig. 5, A and B).
We performed the same experiment with the C11N KDO8P
synthase (Fig. 5, C and D). The kcat for the mutant enzyme was
0.42 s⫺1 and the Km for PEP and A5P were 17 and 140 ␮M,
respectively. Cooperative behavior was noted for the Mn2⫹reconstituted wild-type A. pyrophilus KDO8P synthase as well
as for the C11N mutant. The respective Hill coefficients are
presented in Table II. The catalytic rates were not affected by
the order of substrates addition.
Steady-state Kinetic Parameters for Wild-type and N26C Mutant E. coli KDO8P Synthase—Kinetic analysis of the native
and N26C mutant of E. coli KDO8P synthase were carried out
with or without reconstitution with metals and the kinetic
parameters are presented in Table II. Comparison between the
kinetic parameters of the wild-type E. coli KDO8P synthase
and its N26C mutant reveals the following: (i) EDTA-treated
N26C mutant exhibits a 17-fold lower kcat, yet the presence of
metals increases the kcat values 5-fold. (ii) In the absence of
metals, the N26C mutant has about 4 –5-fold higher Km for
both A5P and PEP as compared with the wild-type enzyme;
however, in the presence of Mn2⫹ the Km for PEP decreases,
while in the presence of Cd2⫹ the Km is equal to the Km for the
wild-type enzyme. iii) The kcat/Km for PEP in the presence of
metals was elevated ⬃20-fold, suggesting the important role
played by the metals in the PEP interaction with the enzyme.
The value for kcat/Km for A5P is also changed although only a
factor of 4 – 6.
Pre-steady-state Burst Experiments for Mn2⫹- and Cd2⫹-reconstituted Wild-type A. pyrophilus KDO8P Synthase and the
C11N Mutant—The purpose of the pre-steady-state burst experiments was to determine the rate-limiting step of the reaction, the active site concentration, and the rate constant of
product formation for the Mn2⫹- and Cd2⫹-reconstituted
KDO8P synthase. Mn2⫹- and Cd2⫹-reconstituted enzymes
have almost the same kcat and a burst in product formation was
observed for both the Mn2⫹- and Cd2⫹-KDO8P synthase. The
metal ion addition resulted in a fully active enzyme, the burst
amplitude showing close to 100% active site concentration. The
linear rate of product formation determined from the presteady-state burst experiments matched the steady-state rate
(kcat) determined from steady-state experiments with almost
no difference observed between the Mn2⫹ (8.0 s⫺1) and Cd2⫹
(9.9 s⫺1) enzyme. However, the major differences were in the
rate constants of the burst phase that strictly determine the
rate constants of chemical catalysis. This value for the Mn2⫹-
45116
KDO8P Synthases from A. pyrophilus and E. coli
DISCUSSION
FIG. 4. Effects of Cd2ⴙ and Mn2ⴙ on the activities of wild-type
E. coli KDO8P synthase and of EDTA-treated N26C mutant. The
reactions were performed under standard assay conditions (100 mM
Tris-HCl, pH 7.5, 2 mM PEP, 2 mM A5P, 37 °C) in the presence of
various concentrations of metal ions. A, activity of wild-type (E) and
EDTA-treated N26C (●) in the presence of various concentrations Cd2⫹.
B, activity of wild-type (‚) and EDTA-treated N26C (Œ) in the presence
of various concentrations of Mn2⫹. All the experiments were performed
at least three times within an average experimental error of about 8%.
KDO8P synthase is 37.0 s⫺1 whereas the Cd2⫹-KDO8P synthase is 130 s⫺1 (Fig. 6, A and B).
In conducting pre-steady-state experiments for the C11N
mutant, it was noted that the enzyme formed aggregates at
higher concentrations. It was therefore necessary to conduct
these experiments at an enzyme concentration of 3 ␮M or less.
For the C11N mutant, the active site concentration was 60%,
the burst rate constant of product formation was 0.18 s⫺1, and
the steady-state rate was 0.1 s⫺1, as illustrated in Fig. 7.
Pre-steady-state Burst Experiments for EcN26C in the Absence and Presence of Metals—A pre-steady-state burst experiment conducted with the EcN26C in the absence of metals
showed no burst in product formation, suggesting that under
these conditions the chemistry is rate-limiting for catalysis
(Fig. 8A). The steady-state rate considering 100% active site
concentration is 0.075 s⫺1. However, the presence of Cd2⫹
stimulates the rate of product formation by 2-fold increasing
the steady-state rate to 0.14 s⫺1. Increasing the concentration
of metal ions from equimolar to 70-fold excess over enzyme
concentration did not affect the active site concentrations or the
rate of chemistry. However, the major effect of increasing metal
concentration is found on the rate constant of product release.
Up to 15-fold excess Cd2⫹ stimulates the rate constant of product release by 2-fold (0.075– 0.14 s⫺1), while a higher concentration (35 or 70-fold) decreases the rate constant of product
release to 0.04 s⫺1 (Fig. 8B).
KDO8P synthase represents one of the rare examples of an
enzyme that is metal-dependent in one class of organisms and
metal-independent in another (7, 8). In this study, a metal-dependent enzyme, the A. pyrophilus KDO8P synthase, and a
metal-independent enzyme, the E. coli KDO8P synthase, were
used to define the function of the divalent metal ion, as well as
to identify similarities and/or differences between the catalytic
mechanisms of the two classes of enzymes. For this purpose,
two mutant enzymes ApC11N and EcN26C were prepared and
characterized with respect to metal effects and catalysis.
The three-dimensional structure of KDO8P synthase of
A. pyrophilus used in this study is assumed to be similar to that
of the highly homologous A. aeolicus enzyme, for which high
resolution structures have been determined by x-ray crystallography (22, 23). When superimposed, the A. aeolicus (23) and
E. coli (20) structures are almost identical in their general fold
(root mean square deviation of 1.2 Å between comparable ␣
carbons). In Fig. 9, the residues serving as ligands to the bound
metal ion (Cd2⫹) in the A. aeolicus structure (Aa, shown in
green stick representation) are shown superimposed on the
homologous residues from the E. coli enzyme (Ec, shown in
gray stick representation). Three residues are very similar in
their orientation, although no metal ion is present in the E. coli
enzyme. The EcD250 residue and the homologous AaD233 residue (Fig. 9), are located on a flexible loop and have a dissimilar
orientation in the two crystals. Crystallographic determination
of the EcN26C in the presence of metal ions will be necessary to
determine whether under these conditions the Asp250 residue
flips over to ligate the metal ion.
ApC11N: A Metal-independent KDO8P Synthase Enzyme?—As shown in Table I, the C11N mutant enzyme contains the similar amount of iron (⬃0.2 mol) as the wild-type
enzyme; however this metal does not affect the activity of the
mutant enzyme (⬃0.8 units/mg), as it remained unchanged
before and after treatment with EDTA or 1,10-phenanthroline
(Table I and Fig. 2). Addition of exogenous divalent metal
cations such as Zn2⫹, Mg2⫹, Co2⫹, and Mn2⫹, which typically
stimulate the activity of the wild-type enzyme at different
levels (5), had no effect on the activity of C11N, and only Cd2⫹
(0.6 mM) stimulated its activity by 1.5-fold (1.2 units/mg, Table
I). These results indicate that the replacement of Cys11 by Asn
in A. pyrophilus KDO8P synthase eliminates the requirement
of a divalent metal cation for catalysis, resulting in an active
metal-independent variant.
Additional support to this conclusion was gained from detailed kinetic characterization of both the wild-type and C11N
A. pyrophilus KDO8P synthases and their comparison to the
kinetics of other Class II and Class I KDO8P synthases. Significantly, the apparent affinity to Cd2⫹ ions by the C11N
mutant (Km ⫽ 640 ␮M) is reduced by about three orders of
magnitude versus that of the wild-type enzyme (Km ⫽ 0.6 ␮M)
(Fig. 3). In contrast, the catalytic efficiency (kcat/Km) of the
EDTA-treated C11N mutant enzyme toward the PEP substrate
is reduced by only about 12-fold, versus that of the Cd2⫹reconstituted wild-type enzyme (Table II). Interestingly, very
similar (8-fold) reduction in catalytic efficiency toward the PEP
substrate of the native H. pylori enzyme (Class II enzyme)
versus E. coli enzyme (Class I enzyme) was recently reported
(30). This difference was further reduced to 4.5-fold when comparing H. pylori Cd2⫹-KDO8P synthase and E. coli KDO8P
synthase. As seen from the data in Table II, the A. pyrophilus
Cd2⫹-KDO8P synthase is also about 3-fold less efficient than
the wild-type E. coli enzyme. It is noteworthy that similar to
the H. pylori KDO8P synthase (30), the Mn2⫹-reconstituted
wild-type A. pyrophilus and C11N mutant displayed coopera-
KDO8P Synthases from A. pyrophilus and E. coli
45117
TABLE II
Kinetic parameters of the wild-type and mutant KDO8P synthases from A. pyrophilus and E. coli
All samples were assayed in triplicate, and analogous results were obtained in 2– 4 different experiments.
Source
Enzyme
kcat
s
⫹2
A. pyrophilus
E. coli
a
Cd -wild type
Mn⫹2-wild type
EDTA-treated C11N
Wild-type
EDTA-treated N26C
Mn⫹2-N26C
Cd⫹2-N26C
⫺1
6.0 ⫾ 0.8
9.0 ⫾ 0.8
0.42 ⫾ 0.03
6.1 ⫾ 0.6
0.36 ⫾ 0.04
1.9 ⫾ 0.1
1.9 ⫾ 0.1
A5P
Km
␮M
18 ⫾ 2
67 ⫾ 6 (1.6)a
140 ⫾ 12 (1.66)
20 ⫾ 2
75 ⫾ 11
70 ⫾ 9
110 ⫾ 14
PEP
Km
␮M
16 ⫾ 2
26 ⫾ 3 (2)
17 ⫾ 2 (2.5)
6.0 ⫾ 0.8
32 ⫾ 4
19 ⫾ 2
5.8 ⫾ 1.3
A5P
kcat/Km
␮M
⫺1
s
⫺1
0.33
0.13
0.003
0.3
0.005
0.03
0.02
PEP
kcat/Km
␮M⫺1 s⫺1
0.37
0.34
0.03
1.0
0.01
0.1
0.33
Hill coefficient numbers (nH) are given in parentheses.
FIG. 5. Steady-state kinetic parameters for wild-type A. pyrophilus KDO8P synthase (A and B). Data were fitted to Hill equation. For the
wild-type Mn2⫹-KDO8P synthase the kcat was 9.0 ⫾ 0.8 s⫺1 and the Km for PEP and A5P were 26 ⫾ 3 ␮M (nH 2) and 67 ⫾ 6 ␮M (nH 1.6), respectively.
Steady-state kinetic parameters for C11N A. pyrophilus KDO8P synthase (C and D). The steady-state kinetic parameters determined by fitting the
data to Hill equation were: kcat 0.42 ⫾ 0.03 s⫺1 and the Km for PEP and A5P were 17 ⫾ 2 ␮M (nH 2.5) and 140 ⫾ 12 ␮M (nH 1.66), respectively.
tive behavior. Moreover, it was observed that the metal may
influence stability since the C11N mutant enzyme aggregates
and is less active at higher protein concentrations (⬎3 ␮M).
Transient kinetic analysis of the wild-type A. pyrophilus
enzyme revealed that the overall rate-limiting step for the
reaction was product release and that the rate constant of
chemical catalysis was influenced by the type of metal with
Cd2⫹-KDO8P synthase being 3-fold faster than Mn2⫹-KDO8P
synthase (Fig. 6). Product release was rate-limiting for the
C11N mutant as well although the rate constant of chemical
catalysis was slower (Fig. 7).
Is the EcN26C a Metal-requiring Enzyme?—Several lines of
evidence obtained in this study substantiate that N26C mutant
of E. coli KDO8P synthase has properties similar to that of
metal-dependent Class II KDO8P synthase. First, the observed
spectral properties of the N26C mutant enzyme are very similar to those of Class II enzymes. Thus, unlike the wild-type
E. coli enzyme which is colorless even at concentrations of ⬎20
mg/ml, the N26C mutant as isolated displayed a characteristic
pinkish color at concentrations above 4 mg/ml as reflected by a
broad absorption band centered at 575 nm (curve 2 in Fig. 1),
similar to that observed for the wild-type A. aeolicus (4) and
A. pyrophilus (5) enzymes. This spectral property is suggestive
of the presence of protein thiolate associated ferric ion (4, 5) in
the isolated N26C enzyme, which was further confirmed by
metal analysis (Table I). The elimination of the 575 nm absorp-
45118
KDO8P Synthases from A. pyrophilus and E. coli
FIG. 7. Pre-steady-state burst experiments for ApC11N. For
C11N KDO8P synthase, 3 ␮M mutant enzyme, 9 ␮M [14C]PEP and 2 mM
Mn2⫹ in 50 mM Tris, pH 7.5 were mixed with 1000 ␮M A5P in a rapid
chemical quench apparatus. The data were fit to a pre-steady-state
burst equation and the parameters were: the active site concentration
60%, burst rate constant 0.18 ⫾ 0.04 s⫺1, and steady-state rate 0.11 ⫾
0.02 s⫺1.
FIG. 6. Pre-steady-state burst experiments for A. pyrophilus
Mn2ⴙ- and Cd2ⴙ-KDO8P synthase. A, Mn2⫹-KDO8P synthase. 20
␮M KDO8P synthase, 60 ␮M [14C-1]PEP and 2 mM MnCl2 in 50 mM Tris,
pH 7.5 were mixed with 500 ␮M A5P. Data were fitted to a pre-steadystate burst equation and the following kinetic parameters were determined: active site concentration 100%, steady-state rate 8.0 ⫾ 0.2 s⫺1
and rate constant for product formation 37 ⫾ 6 s⫺1. B, Cd2⫹-KDO8P
synthase. 20 ␮M KDO8P synthase, 60 ␮M [14C-1]PEP and 200 ␮M CdCl2
in 50 mM Tris, pH 7.5 were mixed with 500 ␮M A5P. Similar with
Mn2⫹-KDO8P synthase the data were fitted to a pre-steady-state burst
equation with the following kinetic parameters: active site concentration 100%, steady-state rate 9.9 ⫾ 0.6 s⫺1, and rate constant for product
formation 130 ⫾ 21 s⫺1.
tion band upon addition of excess CuSO4 and concomitant
appearance of the new peak at 385 nm suggests displacement
of the enzyme bound ferric ion by the copper (4, 29). Titration
of the EDTA-treated N26C with CuSO4 generates the same
385-nm band with the stoichiometry of one Cu2⫹ ion per enzyme subunit (Fig. 1).
Second, the performance of N26C in terms of its apparent
affinity to divalent metal ions and treatment with metal chelators is also similar to that of Class II enzymes. For example, the
isolated N26C displays similar content of zinc and iron as the
wild-type A. pyrophilus enzyme (Table I), and treatment with
EDTA removes these metals from the enzyme, as confirmed by
metal analysis (Table I) and by disappearance of the characteristic 575 nm absorption band (Fig. 1). Furthermore, while
the EDTA-treated N26C enzyme retains ⬃6% of the wild-type
activity, addition of Cd2⫹ or Mn2⫹ stimulate its activity up to
⬃30% of the wild-type activity (Fig. 4), indicating significant
role of metal ions on its catalytic performance.
Third, kinetic characterization of N26C strengthens the
above conclusion. As seen from Table II, while the EDTAtreated N26C is 100-fold less efficient (with respect to PEP)
than the wild-type E. coli KDO8P synthase, this difference was
reduced to 10-fold and 3-fold in the Mn2⫹- and Cd2⫹-reconstituted N26C enzymes, respectively. These differences are due to
PEP
variations in both the kcat and the Km
. The kcat of the EDTAtreated N26C was determined to be 17-fold lower than the
wild-type E. coli enzyme. However, the kcat values of both the
Cd2⫹-N26C and Mn2⫹-N26C were the same, about 3-fold lower
than that of the wild-type enzyme. In general, the apparent
affinity of the mutant enzyme for both substrates, PEP and
A5P, is reduced by a similar extent (either in EDTA-treated or
metal-reconstituted form) and only Cd2⫹-N26C has the same
PEP
Km
value as the wild-type enzyme. Importantly, the catalytic
efficiency of Cd2⫹-N26C toward PEP is the same (0.33 ␮M⫺1
s⫺1) as that of the A. pyrophilus Cd2⫹-KDO8P synthase (0.37
␮M⫺1 s⫺1).
Transient kinetic analysis of the N26C mutant revealed that
the overall rate-limiting step is influenced by the presence of
metal since it was shown that the rate-limiting step was chemical catalysis in the EDTA-treated enzyme before metal reconstitution. The Cd2⫹-reconstituted N26C enzyme showed a characteristic burst of product indicating that product release was
now the overall rate-limiting step. The enzyme/metal stoichiometry also affected the relative rates for chemical catalysis
and product release.
In summary, these results indicate that the EcN26C has
properties similar to that of metal-dependent Class II KDO8P
synthase and that the engineered Cys26 residue in N26C fulfills
the role of the native, metal-ligated cysteine in Class
II enzymes.
Mechanistic Implications—The results described above show
that reciprocal single mutations have the potential to convert
metal-dependent class KDO8P synthase to a metal-independent variant and vice versa, as demonstrated here for the
A. pyrophilus and E. coli enzymes. However, perhaps the most
important questions to be considered are the mechanistic implications of these results on KDO8P synthase catalysis.
The demonstration that ApC11N can act in the absence of
metal ion and with significant efficiency implies that the metal
ion is not directly involved in the chemistry of the metal-dependent KDO8P synthase-catalyzed reaction. Thus, the suggested role of the divalent metal ion in catalysis (22, 23), that of
providing a highly activated hydroxide ion that initiates the
condensation reaction between PEP and A5P (Scheme 2, path
b), seems less likely. If this was indeed the case, then such a
step would be an essential elementary step for catalysis to
occur, and in the absence of metal ion there should be absolutely no catalysis. In addition, it is also of note that the
KDO8P Synthases from A. pyrophilus and E. coli
45119
FIG. 9. Comparison of active sites for the structures of Class I
and Class II enzymes. The coordinates of the E. coli KDO8P synthase
structure (PDB code 1G7U, gray) and the A. aoelicus KDO8P synthase
(PDB code 1FWS, green) were superimposed using Insight II (Accelrys).
Only the four residues serving as ligands to the Cd⫹2 ion in the
A. aeolicus structure (Aa) and the homologous residues from E. coli (Ec)
are shown for clarity. Amino acid side chain oxygens, nitrogens, and
sulfur are shown in red, blue, and yellow, respectively.
FIG. 8. Pre-steady-state burst experiments for EcN26C. A, in
the absence of metals, 20 ␮M N26C, 60 ␮M [14C-1]PEP, and 500 ␮M
EDTA were mixed with 1 mM A5P in 50 mM Tris, pH 7.5 in a rapidquench apparatus. No burst in product formation was observed (r2
0.98). Steady-state rate considering 100% active site concentration was
0.075 ⫾ 0.011 s⫺1䡠s⫺1. B, in the presence of different metal concentrations, 20 ␮M N26C, 60 ␮M [14C-1]PEP, 500 ␮M EDTA, and different Cd2⫹
concentrations were mixed with 1 mM A5P in 50 mM Tris, pH 7.5 in a
rapid-quench apparatus. The free metal concentrations were: 0.02 mM
(●), 0.3 mM (䡺), 0.7 mM (〫), and 1.4 mM (⫻). No burst in product
formation was observed for 0.02 and 0.3 mM Cd2⫹ (0.14 ⫾ 0.03 s⫺1)
while there was a burst in product formation at higher metal concentration (60% active site concentration, rate constant for product formation 0.14 ⫾ 0.02 s⫺1 and steady-state rate of 0.041 ⫾ 0.004 s⫺1).
stepwise formation of a transient C3 carbanionic species at C3
of PEP (1b, Scheme 2, path b), might be less plausible since
such a species is highly basic (pKa ⬎ 30), has no resonance
stabilization, and as such, can rapidly decompose by abstracting a proton from water or from the surrounding residues.
The other feasible role of divalent metal ion in catalysis of
Class II KDO8P synthases, activation of the A5P by direct
coordination to the carbonyl oxygen and stabilization of incipient negative charge density on this oxygen during the condensation step, is also unsuitable since it is inconsistent with the
recent x-ray data of A. aeolicus KDO8P synthase (22, 23). In all
reported structures, that include enzyme complexes with various combinations of the natural substrates (A5P, Cd2⫹ in the
presence or absence of PEP), the metal ion is 6 –7 Å away from
the carbonyl oxygen, which is too far to directly affect this stage
of the catalytic cycle. Thus, unlike the metal-dependent Class
II aldolases in which the metal cofactor (usually Zn2⫹) functions as a Lewis acid by polarizing the carbonyl group of the
aldehyde substrate (31), in Class II KDO8P synthases it seems
that the metal ion has a different function.
The Role of Metal Ion in the Metal-requiring KDO8P Synthase Catalysis—One alternative possibility is that metal ion in
Class II KDO8P synthases plays a structural role: by maintaining the holoenzyme in the correct quaternary structure the
desired active site cavity is maintained, allowing for the correct
orientation of the substrates and/or reaction intermediate(s). A
similar role in the metal-independent Class I enzymes might be
achieved by the conserved Asn residue (4, 5). Thus, the observed ability of the EcN26C to act to some extent in a metaldependent fashion does not appear to be a consequence of a
change in the catalytic mechanism in the mutant enzyme, but
is more likely to be a direct consequence of the loss of function
associated directly with Asn26 in the wild-type enzyme and that
this function is partially fulfilled by the metal ion in the N26C
mutant. According to the recently solved crystal structure of
the E. coli KDO8P synthase in its binary complex with PEP
(32), Asn26 lies 3.8 Å away from N␦ of His202 and 5.4 Å away
from the carboxylate of PEP. However, His202 is largely involved in PEP binding due to interactions with both PEPphosphate and PEP-carboxylate, and plays a very important
role in selection of a particular PEP conformer in which the
phosphate group of PEP extends toward the si face. Indeed, the
mutation of His202 with glycine rendered the enzyme virtually
inactive (32). Thus, despite the lack of direct contact to the PEP
substrate, Asn26 plays an important structural role by orienting His202 in such a way that allows the binding and recognition of the correct conformation of the PEP substrate.
Taken together, the observed data suggest that the role of
Asn26 in E. coli enzyme is similar to that of the metal ion in the
A. pyrophilus enzyme, and that this role is primarily structural. The suggested structural role of metal ion in Class II
KDO8P synthases has also been indicated for the H. pylori
KDO8P synthase (30), and for the structurally and mechanistically closely related enzyme, 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase (DAHP synthase) (33). These
two studies showed that while apoenzymes were unstable and
undergo rapid inactivation, the addition of metal ions to form
the holoenzymes promoted their stability. Analogously, in the
case of the E. coli KDO8P synthase, it was previously demonstrated that tightly bound PEP substrate stabilizes the enzyme
during purification and storage and protects against heat inactivation (15).
Nevertheless, although at this stage of investigation it is not
clear whether the formation of new C-C and C-O bonds during
the condensation step of KDO8P synthase reaction is a synchronous or stepwise process, the observed data in this work
45120
KDO8P Synthases from A. pyrophilus and E. coli
suggest that both Class I and Class II enzymes operate by the
same stepwise fashion through the intermediacy of a transient
oxocarbenium ion at C2 of PEP (1a, Scheme 2, path a). The
intermediate 1a can be captured by bulk water to lead to the
formation of acyclic hemiketal phosphate 1, which then rapidly
decomposes to yield the products KDO8P and Pi. This mechanism is further indicated by results obtained through structural data on the E. coli KDO8P synthase in complex with the
substrate PEP and with a mechanism-based inhibitor (20),
with analogues of PEP (34), with various experiments using a
mechanism-based inhibitor, and with intramolecular models of
the KDO8P synthase-catalyzed reaction (21). Additionally, the
same oxocarbenium ion transition state of PEP has been suggested earlier to account for the enzymatic reactions of UDPGlcNAc enolpyruvoyl transferase (35) and 5-enolpyruvoylshikimate-3-phosphate synthase (36). These enzymes catalyze the
enol ether transfer from PEP to their respective cosubstrate
alcohols, and represent a different distinct class of enzymatic
reaction involving the same C-O bond cleavage of PEP and the
same stereospecific 2-si face addition of an electrophile at C3 of
PEP, which is observed for KDO8P synthase (37).
In conclusion, the results presented in this study imply that
the metal ion may not be directly involved in the chemistry of
the KDO8P synthase-catalyzed reaction, but has important
structural roles in Class II enzymes through maintaining correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site to allow the catalysis. The
observed data also supports the notion that the elementary
steps of catalysis in both Class I and Class II enzymes may
follow the same oxocarbenium ion mechanism as illustrated in
Scheme 2, path a.
REFERENCES
1. Ray, P. H. (1980) J. Bacteriol. 141, 635– 644
2. Clements, J. M., Coignard, F., Johnson, I., Chandler, S., Palan, S., Waller, A.,
Wijkmans, J., and Hunter, M. G. (2002) Antimicrob. Agents Chemother. 46,
1793–1799
3. Jackman, J. E., Fierke, C. A., Tumey, L. N., Pirrung, M., Uchiyama, T., Tahir,
S. H., Hindsgaul, O., and Raetz, C. R. (2000) J. Biol. Chem. 275,
11002–11009
4. Duewel, H. S., and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824 –22831
5. Shulami, S., Yaniv, O., Rabkin, E., Shoham, Y., and Baasov, T. (2003) Extremophiles 7, 471– 481
6. Krosky, D. J., Alm, R., Berg, M., Carmel, G., Tummino, P. J., Xu, B., and Yang,
W. (2002) Biochim. Biophys. Acta 1594, 297–306
7. Birck, M. R., and Woodard, R. W. (2001) J. Mol. Evol. 52, 205–214
8. Jensen, R. A., Xie, G., Calhoun, D. H., and Bonner, C. A. (2002) J. Mol. Evol.
54, 416 – 423
9. Wong, C.-H., and Whitesides, G. H. (1994) Enzymes in Synthetic Organic
Chemistry, Vol. 12, pp. 195–242, Elsevier Science, Ltd., Oxford
10. Hedstrom, L., and Abeles, R. (1988) Biochem. Biophys. Res. Commun. 157,
816 – 820
11. Dotson, G. D., Dua, R. K., Clemens, J. C., Wooten, E. W., and Woodard, R. W.
(1995) J. Biol. Chem. 270, 13698 –13705
12. Kohen, A., Jakob, A., and Baasov, T. (1992) Eur. J. Biochem. 208, 443– 449
13. Dotson, G. D., Nanjappan, P., Reily, M. D., and Woodard, R. W. (1993) Biochemistry 32, 12392–12397
14. Kohen, A., Berkovich, R., Belakhov, V., and Baasov, T. (1993) Biooorg. Med.
Chem. Lett. 13, 1577–1582
15. Liang, P. H., Lewis, J., Anderson, K. S., Kohen, A., D’Souza, F. W., Benenson,
Y., and Baasov, T. (1998) Biochemistry 37, 16390 –16399
16. Du, S., Faiger, H., Belakhov, V., and Baasov, T. (1999) Bioorg. Med. Chem. 7,
2671–2682
17. Li, Z., Sau, A. K., Shen, S., Whitehouse, C., Baasov, T., and Anderson, K. S.
(2003) J. Am. Chem. Soc. 125, 9938 –9939
18. Wagner, T., Kretsinger, R. H., Bauerle, R., and Tolbert, W. D. (2000) J. Mol.
Biol. 301, 233–238
19. Radaev, S., Dastidar, P., Patel, M., Woodard, R. W., and Gatti, D. L. (2000)
J. Biol. Chem. 275, 9476 –9484
20. Asojo, O., Friedman, J., Adir, N., Belakhov, V., Shoham, Y., and Baasov, T.
(2001) Biochemistry 40, 6326 – 6334
21. Du, S., Plat, D., Belakhov, V., and Baasov, T. (1997) J. Org. Chem. 62, 794 – 804
22. Wang, J., Duewel, H. S., Woodard, R. W., and Gatti, D. L. (2001) Biochemistry
40, 15676 –15683
23. Duewel, H. S., Radaev, S., Wang, J., Woodard, R. W., and Gatti, D. L. (2001)
J. Biol. Chem. 276, 8393– 8402
24. Wang, J., Duewel, H. S., Stuckey, J. A., Woodard, R. W., and Gatti, D. L. (2002)
J. Mol. Biol. 324, 205–214
25. Bednarski, M. D., Crans, D. C., DiCosimo, R., Simon, E. S., Stein, P. D.,
Whitesides, G. M., and Schneider, M. J. (1988) Tetrahedron Lett. 29,
427– 430
26. Hirschbein, B. L., Mazenod, F. P., and Whitesides, G. M. (1982) J. Org. Chem.
47, 3765–3766
27. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Appendix A.1, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
28. Johnson, K. A. (1992) Enzymes 20, 1– 61
29. Baasov, T., and Knowles, J. R. (1989) J. Bacteriol. 171, 6155– 6160
30. Sau, A. K., Li, Z., and Anderson, K. S. (2004) J. Biol. Chem. 279, 15787–15794
31. Plater, A. R., Zgiby, S. M., Thomson, G. J., Qamar, S., Wharton, C. W., and
Berry, A. (1999) J. Mol. Biol. 285, 843– 855
32. Sheflyan, G. Y., Duewel, H. S., Chen, G., and Woodard, R. W. (1999) Biochemistry 38, 14320 –14329
33. Park, O. K., and Bauerle, R. (1999) J. Bacteriol. 181, 1636 –1642
34. Benenson, Y., Belakhov, V., and Baasov, T. (1996) Bioorg. Med. Chem. Lett. 6,
2901–2906
35. Kim, D. H., Lees, W. J., Haley, T. M., and Walsh, C. T. (1995) J. Am. Chem.
Soc. 117, 1494 –1502
36. Alberg, D. G., Lauhon, C. T., Nyfeler, R., Faessler, A., and Bartlett, P. A. (1992)
J. Am. Chem. Soc. 114, 3535–3546
37. Walsh, C. T., Benson, T. E., Kim, D. H., and Lees, W. J. (1996) Chem. Biol. 3,
83–91