(Received for publication, October 5, 1994; and in revised form, December 22, 1994)
From the
Clavaminate synthase (CS), a key enzyme in the clavulanic acid
biosynthetic pathway, has been purified to electrophoretic homogeneity
from Streptomyces antibioticus (Tü 1718),
a species that does not produce clavulanic acid. A comparison of the
physical and kinetic properties of clavaminate synthase from S.
antibioticus (CS3) and the two isozymes from Streptomyces
clavuligerus (CS1 and CS2) has been conducted. In oxidative
reactions requiring the co-substrates O,
-ketoglutaric
acid, and catalytic Fe
, both CS1 and CS2 catalyze
three distinct transformations, the hydroxylation of
deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and
the cyclization and desaturation of proclavaminic acid to clavaminic
acid. We have demonstrated that CS3 from S. antibioticus also
catalyzes these three oxidations. The apparent molecular mass of CS3
from matrix-assisted laser desorption mass spectrometry is 35,839
± 36 Da. The enzyme is a monomer in solution as determined by
gel filtration chromatography. Analysis of the four possible
proclavaminic acid diastereomers confirmed the absolute configuration
of the substrate to be 2S,3R. Based upon N-terminal
sequence comparisons among the three proteins, CS3 possesses the higher
degree of homology with the CS1 isozyme from S. clavuligerus.
Although previously associated solely with clavulanic acid
biosynthesis, we propose these findings and recent precursor
incorporation data support the view that clavaminate synthase plays a
critical role in the biosynthesis of the clavam metabolites.
Clavulanic acid (Fig. 1, clavam 1) is a clinically
important -lactamase inhibitor produced by Streptomyces
clavuligerus(1) . Clavaminate synthase (CS)
, a
non-heme iron,
-KG-dependent oxygenase, catalyzes three distinct
transformations in the biosynthesis of 1 (Fig. 1)(2, 3) . The first of these
reactions is conventional hydroxylation of deoxyguanidinoproclavaminic
acid (2) to produce guanidinoproclavaminic acid (3) (4, 5) . Before CS can perform its second oxidative
function, an intervening enzyme, proclavaminic acid amidino hydrolase
catalyzes the hydrolysis of the guanyl moiety of 3 to produce
proclavaminic acid (4)(5) . Clavaminate synthase then
carries out the oxidative cyclization of 4 to produce the
transient intermediate dihydroclavaminic acid (5)(6, 7) . In a third oxidative process the
later is desaturated to yield clavaminic acid (6)(2, 3) . Clavaminic acid is thought to
undergo oxidative deamination (8) and an unusual stereochemical
inversion to yield the aldehyde (7), which is then reduced to
afford 1(9) .
Figure 1: Clavulanic acid biosynthetic pathway. PAH, proclavaminic acid amidino hydrolase.
Initial isolation of CS from S.
clavuligerus revealed the unexpected presence of two isozymes, CS1
and CS2(3) , whose genes (cs1 and cs2) have
been cloned, sequenced(10) , and overexpressed in Escherichia coli(11) . ()The genes are 87%
identical and the proteins differ in length by a single amino acid
residue(10) . The two isozymes have the same cofactor
requirements and possess nearly identical kinetic
properties(3) . All genes required for the biosynthesis of 1 are believed to flank cs2(
)in a 12-kilobase
cluster (12, 13) . Surprisingly, cs1 is not present in this DNA fragment raising questions as to the
role of CS1 in the biosynthesis of 1(10) .
The
clavams 8, 9, 10, and 11 are members of a
family of bicyclic -lactams structurally related to 1 (Fig. 2) and coproduced by S.
clavuligerus(14, 15) . Clavams 12(16, 17) and 13(18) are isolated
from an allied species, Streptomyces antibioticus, which does
not produce 1. We have demonstrated that an advanced
biosynthetic intermediate of clavulanic acid (1), proclavaminic
acid (4), is also a precursor of clavams 8, (19) 12, and 13(20) . The common
intermediacy of 4 suggests that a shared biosynthetic pathway
exists to the clavams and 1, at least until the formation of 4. In view of the ``enantiomerization'' required in
clavulanic acid formation(8) , we have postulated that the
branch point of these two pathways may lie beyond clavaminic acid (6)(19, 20) . Accordingly, we have proposed
that CS may be present in S. antibioticus, the organism
responsible for the production of 12 and 13 alone, and
that this multifunctional enzyme may play a role in the biosynthesis of
the clavams(20, 21) .
Figure 2: Structures of the clavam metabolites.
In this study we report the purification to homogeneity and thorough characterization of clavaminate synthase (CS3) from S. antibioticus. A comparison of the physical and kinetic properties of CS3 from S. antibioticus to the two isozymes from S. clavuligerus has been completed and has demonstrated that many common features exist among the three enzymes.
In and , V represents the
maximum velocity, A is the concentration of the variable
substrate, K is the Michaelis constant for A, and K
and K
are
saturation constants for a substrate which binds twice to the enzyme
with no intervening irreversible steps.
In radioactive assays
measuring carbon dioxide formation the assay components were identical
to those in the standard assay except for the substitution of
[1-C]
-KG diluted with radioinactive
material to a specific activity of 0.34 µCi/µmol. Incubations
were performed in 8-ml glass vials sealed with rubber septa and fitted
with plastic center wells (Kontes, Vineland, NJ). Each well contained a
piece of filter paper impregnated with 25 µl of 1 M benzethonium hydroxide in methanol. The assays were initiated by
syringe addition of enzyme and terminated with 50 µl of 50%
trifluoroacetic acid. After a 1-h incubation at 37 °C, the center
wells were transferred to scintillation vials for counting in 20 ml of
Opti-Fluor O mixture (Packard, Downers Grove, IL). Corrections were
applied for non-enzymatic decarboxylation of
-KG. The production
of succinate was measured with a coupled enzyme assay as described by
Beutler(28) .
The assay for the conversion of 2 to 3 was performed using reverse phase HPLC. The stationary phase
was Versapack, C-18 (10µm, 0.46 25 cm) and the mobile phase
was 100% H
O CS3 (21.6 µg) was incubated in 50 mM MOPS, pH 7. 0, 0.5 mM DTT, 0.1 mM ascorbate, 1.0
mM
-KG, 25 µM Fe
, and 1.2
mM2 at 30 °C. After 30 min of incubation the
reaction mixture was analyzed by HPLC. The product, 3, was
identified by coelution with an authentic sample.
The assay used to
monitor the conversion of 4 to 6 involved product
derivatization with phthalic o-dicarboxaldehyde (OPA) followed
by HPLC analysis(29, 30) . An OPA derivatization
mixture (45 mM OPA in 0.54 M sodium borate, pH 9.5,
containing 1% -mercaptoethanol) was prepared and placed on ice in
the dark. CS3 was incubated with 4 under standard conditions at
25 °C as above. Aliquots (15µl) were removed after 30 min,
mixed with 15µl of OPA derivatization mixture for 30 s, and
analyzed by HPLC using a Spherisorb octadecyl sulfate 2 C-18 column (5
µm, 0.46
25 cm). Derivatized products were monitored at 340
nm.
With homogeneous CS3 in hand, we set out to
determine whether CS3 performed each of the three transformations
catalyzed by the S. clavuligerus isozymes. Following
incubation of CS3 with deoxyguanidinoproclavaminic acid (2),
HPLC analysis of the reaction mixture confirmed the presence of
guanidinoproclavaminic acid (3) as evidenced by coelution with
an authentic sample. The conversion of proclavaminic acid (4) to
clavaminic acid (6) catalyzed by CS3 was monitored in two ways.
Following CS3 incubation, addition of imidazole to the reaction mixture
resulted in derivatization of 6 and production of an
,
-unsaturated acyl imidazole adduct whose chromophore was
monitored at 312 nm. The oxidative cyclization/desaturation reactions
were further observed by derivatizing the reaction mixture with OPA and
analyzing the derivatized products by reverse phase HPLC. By this
method we were also able to detect low levels of dihydroclavaminic acid (5).
N-terminal sequence analysis was carried out and
comparisons were made among all the CS enzymes (Table 2). This
limited comparison revealed that the higher degree of homology exists
between CS3 and CS1, the isozyme whose gene is not located in
the clavulanic acid biosynthetic operon. The amino acid composition of
CS3 was determined and the results are compiled with those translated
from the gene sequences of CS1 and 2 (Table 3)()(10) . The amino acid composition
of CS3 is similar to those of CS1 and CS2 and is typical of a globular
protein, with some notable exceptions. Amino acids utilizing codons
rich in A and T, for example phenylalanine and lysine, were present in
unusually low amounts. Conversely, amino acids such as arginine and
proline, whose codons are primarily G and C, appear more frequently in
CS3 than in most globular proteins(34) . This deviation from
the predicted amino acid distribution can be rationalized by the fact
that Streptomyces DNA has a high (74%) GC content (35) .
The UV/visible spectrum of CS3 was recorded from 240-600 nm and a single, broad absorbance maximum was observed at 280 nm. The lack of any longer wavelength absorbances in the spectrum of CS3 suggests that there are no tightly bound cofactors present. The molecular weight of CS3 has been estimated by several methods and these results were compared with those previously reported for CS1 and 2 (Table 4). The mass of CS3 was determined using matrix-assisted laser desorption (MALD) mass spectrometry, and a value of 35,839 ± 36 Da was obtained(33) . Analysis by SDS-PAGE revealed that CS3 migration is consistent with the molecular weight determined by MALD mass spectrometry (Fig. 3). This behavior distinguishes CS3 from its S. clavuligerus counterparts whose apparent molecular weights by SDS-PAGE differ significantly (3) from both the molecular weights translated from the gene sequence and those determined by MALD mass spectrometry (Table 4). CS3 behaves as a monomer when analyzed by Superose 12 fast protein liquid chromatography under non-denaturing conditions consistent with the results obtained for CS1 and CS2.
Figure 3: SDS-PAGE comparison of clavaminate synthases. Ten percent polyacrylamide gel was stained with Coomassie Brilliant Blue, and recombinant CS1 and CS2 were used as standards.
The transformation of 4 to 6 occurs
in a stepwise fashion in which each of the two oxidative half reactions
results in the decarboxylation of -KG to form succinate and
CO
. In parallel reactions utilizing
[1-
C]
-KG and radioinactive
-KG, the
evolution of
CO
and production of 6 were monitored. Two moles of CO
were evolved for each
mole of 6 produced (Fig. 4). Qualitatively, the formation
of succinate was confirmed using the succinic thiokinase coupled assay
of Beutler(28) .
Figure 4:
Comparison of CO evolution
with clavaminate formation. Parallel reactions under standard
conditions containing 1.0 mM
-KG were assayed for
clavaminate formation by spectrophotometric assay,
, or for
CO
formation by radiochemical assay,
.
We sought
to determine whether CS3 was susceptible to the same oxidative
self-inactivation observed for CS1 and CS2. CS3 is stable in a buffered
solution at 25 °C in the absence of 4 and all cofactors
except endogenous O (t
= 19
h) (Fig. 5). However, when CS3 was incubated under parallel
conditions with Fe
,
-KG, and O
,
rapid loss of catalytic activity resulted (t
= 52 s). Addition of catalase (0.1 mg/ml) provided partial
protection to CS3 from oxidative damage (t
= 46 min). The role of catalase appears to be specific as
substitution by BSA for catalase at the same concentration failed to
provide similar protection (t
= 72 s).
Figure 5:
Auto-inactivation of CS3 in the absence of
proclavaminic acid. CS3 was incubated in 50 mM MOPS buffer, pH
7.0, containing 0.5 mM DTT at 25 °C alone or with various
additives: , no additions;
, 10 µM Fe
, 100 µM ascorbate, 1.0 mM
-KG;
, same as
, plus 0.1 mg/ml
catalase.
The rapid inactivation of CS3 is consistent with data obtained for
CS1 and CS2 and further supports the suggestion that the mechanism of
oxygen activation is shared among all three enzymes. The observation
that catalase offers protection to the enzyme implies the formation of
hydrogen peroxide is occurring. However, the inability of catalase to
afford complete protection to CS3 indicates that diffusable hydrogen
peroxide cannot be the sole causative agent responsible for oxidative
damage leading to inactivation. The production of peroxide in the
incomplete reaction mixture implies that CS3 can catalyze a partial
reaction in which oxidative decarboxylation of -KG results in the
production of an activated iron-oxygen species. When 4 is
unavailable for oxidative reaction, this high energy oxygen species may
be redirected toward residues at or near the active site resulting in
self-inactivation.
A stepwise mechanism has been proposed
for CS1 and CS2 involving two discrete oxidative half reactions
occurring at the same active site. In the first, 4 is
oxidatively cyclized to 5, while consuming -KG and O
and producing CO
, succinate, and H
O. In
the second half reaction, 5 undergoes desaturation also
consuming
-KG and O
and releasing CO
,
succinate, and H
O. In the CS3 catalyzed conversion of 4 to 6 we sought to examine the dependence of velocity upon
the concentration of the substrate,
-KG. A linear
double-reciprocal plot was obtained when
-KG was supplied as the
variable substrate (Fig. 6). This result is consistent with the
stepwise mechanism proposed for CS1 and CS2 in which the binding of
-KG is separated by an irreversible step, in this case the release
of succinate and CO
. For a case in which the same substrate
binds twice and each binding event is not separated by an
irreversible step, theory predicts a parabolic double-reciprocal plot (37) . The velocity dependence upon substrate concentration in
this case is described by , however attempts to fit our
data to did not yield a satisfactory result.
Figure 6:
Double-reciprocal plot of the dependence
of initial velocity on -KG concentration. The concentration of
-KG was varied from 0.05 to 1.25 mM. The concentrations
of the nonvariable substrates were 1.5 mM for rac-proclavaminic acid and 50 µM for
Fe
. Velocity measurements were performed in duplicate
at 0.05, 0.113, and 1.25 mM
-KG. The line shown
represents the fit of the data to .
We have purified clavaminate synthase (CS3) from S.
antibioticus, an organism which does not produce clavulanic acid (1). This multifunctional enzyme performs the identical three
oxidative reactions catalyzed by the S. clavuligerus isozymes
in the biosynthesis of 1. All three synthases require the same
cofactors, namely Fe,
-KG, and O
and
have closely similar physical and kinetic properties ( Table 4and Table 5).
N-terminal sequence analysis demonstrated that CS3
shows greater homology to the CS1 isozyme than to CS2. While this comparison is made with only the first 20 amino acids
of each protein, it should be borne in mind that the genes for CS1 and
CS2 show very high levels of identity in their central regions with the
greatest divergence occurring at the N and C termini(10) .
Therefore, the similarities and differences seen in this sample may
well be more meaningful than its limited size might initially suggest.
Interestingly, the gene coding for CS1 is not present in the
clavulanic acid operon of S. clavuligerus. This organism
coproduces several clavam metabolites along with 1. It is
possible that the CS1 gene resides in a genomic region dedicated to the
production of clavams in S. clavuligerus. S. antibioticus,
however, produces CS yet fails to produce 1. We propose that CS3
is required for clavam biosynthesis and, therefore, might be expected
to be more homologous to the isozyme CS1 of S. clavuligerus.
Previous work has shown that proclavaminic acid (4) is an intermediate in clavam and clavulanic acid biosynthesis(19, 20) . We have demonstrated the ability of CS3 to hydroxylate deoxyguanidinoproclavaminic acid (2) to guanidinoproclavaminic acid (3), clearly implicating CS3 in the production of 4 in S. antibioticus. Further, proclavaminic amidino hydrolase activity has been detected in S. antibioticus, which provides a means of converting 3 to 4(21) . Additionally, CS3 possesses the ability to convert 4 to 6, a reaction known to occur in the biosynthesis of 1. If 6 is the in vivo product of the reaction of 4 with CS3, then the likelihood is increased that 6 plays a role in the biosynthesis of the clavams as well. Based on the stereochemical similarities that exist between 6 and the clavams, the intermediacy of 6 in their biosyntheses can be readily envisioned.
An extensive, shared biosynthetic pathway between the clavams and 1, including the intermediacy of 6, has been previously proposed (20) and the purification of CS3 from S. antibioticus strengthens this notion (Fig. 7). The partitioning of the two pathways is suggested to occur after the proposed intermediacy of clavaminic acid (6). An oxidative deamination occurs without decarboxylation but with stereochemical ring inversion to give the aldehyde, 7(8, 9) known to be the penultimate precursor of clavulanic acid (1). The same or similar intermediate, through decarboxylation but without inversion, has been proposed to afford the aldehyde 14. This intermediate is thought to be common to the individual members of the clavam family; for example, aldol condensation and further reactions can be envisioned to provide 12, while simple reduction gives 13. This proposal emphasizes the critical role CS3 plays in the biosynthesis of clavams 12 and 13 in S. antibioticus and relates to the open question of the location and organization of the clavam biosynthetic genes in S. clavuligerus.
Figure 7: Biosynthetic parallels among clavulanic acid and the clavams.