From The Oxford Centre for Molecular Sciences and the
Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, United
Kingdom, the § Department of Biochemistry, National Defence
Medical Centre, Taipei, Taiwan, Republic of China, the ** Structural
Biology Division, The Wellcome Trust Centre for Human Genetics and The
Oxford Centre for Molecular Sciences, Roosevelt Drive, Oxford OX3 7BN,
United Kingdom, and the
Université de
Liège, Laboratoire d'Enzymologie, CIP, Institut de Chimie B6,
B-4000, Liège, Belgium
Received for publication, January 4, 2001, and in revised form, February 14, 2001
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ABSTRACT |
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Deacetoxycephalosporin C synthase is an
iron(II) 2-oxoglutaratedependent oxygenase that catalyzes the
oxidative ring-expansion of penicillin N to deacetoxycephalosporin C. The wild-type enzyme is only able to efficiently utilize 2-oxoglutarate
and 2-oxoadipate as a 2-oxoacid co-substrate. Mutation of arginine 258, the side chain of which forms an electrostatic interaction with the
5-carboxylate of the 2-oxoglutarate co-substrate, to a glutamine
residue reduced activity to about 5% of the wild-type enzyme with
2-oxoglutarate. However, other aliphatic 2-oxoacids, which were not
co-substrates for the wild-type enzyme, were utilized by the R258Q
mutant. These 2-oxoacids "rescued" catalytic activity to the level
observed for the wild-type enzyme as judged by penicillin N and G
conversion. These co-substrates underwent oxidative decarboxylation as
observed for 2-oxoglutarate in the normal reaction with the wild-type
enzyme. Crystal structures of the iron(II)- 2-oxo-3-methylbutanoate
(1.5 Å), and iron(II)-2-oxo-4-methylpentanoate (1.6 Å) enzyme
complexes were obtained, which reveal the molecular basis for this
"chemical co-substrate rescue" and help to rationalize the
co-substrate selectivity of 2-oxoglutaratedependent oxygenases.
Deacetoxycephalosporin C synthase
(DAOCS)1 is an iron(II) and
2-oxoglutarate-dependent oxygenase that catalyzes the
conversion of penicillin N to deacetoxycephalosporin C in the
biosynthesis of cephem antibiotics in Streptomyces
clavuligerus (1). The subsequent hydroxylation of DAOC to
deacetylcephalosporin C (DAC) is catalyzed by a closely related enzyme,
deacetylcephalosporin C synthase (DACS). In Cephalosporium
acremonium a single, bifunctional protein,
deacetoxy/deacetylcephalosporin C synthase (DAOC/DAC synthase),
performs both reactions (2-4). S. clavuligerus also contains a 7 DAOCS is a member of the iron(II) and
2-oxoglutarate-dependent oxygenase family, which catalyze a
wide variety of oxidative reactions (6, 7). DAOCS, DACS, and DAOC/DAC
synthase belong to a subgroup of more closely related enzymes, which
have significant primary sequence homology to one another (6). Also
included in this group are two enzymes which do not use 2-oxoglutarate as a co-substrate, isopenicillin N synthase (IPNS), the
iron-dependent oxidase responsible for formation of the
penicillin nucleus, and 1-amino-1-carboxycyclopropane oxidase (ACCO)
which catalyzes the last step during ethylene biosynthesis in plants.
Mechanistic understanding of iron(II),
2-oxoglutarate-dependent oxygenases have been significantly
advanced by the recently determined crystal structures of DAOCS (1, 8,
9) and clavaminic acid synthase (10). The DAOCS crystal
structures revealed the presence of a number of arginine residues
within the active site, with arginine 258 (part of a conserved
RXS motif) being involved in co-substrate binding (1, 8).
The equivalent RXS motif residues in Aspergillus
nidulans IPNS, arginine 281, and serine 283, bind the
Here we report studies on the 2-oxoglutarate co-substrate-binding
function of arginine 258 in DAOCS by site-directed mutagenesis. Activity of the R258Q mutant was reduced in the presence of
2-oxoglutarate, but could be fully restored using aliphatic 2-oxoacids
as alternative co-substrates. The process is called "chemical
co-substrate rescue." The molecular basis for this phenomenon has
been investigated by crystallographic analyses of the R258Q mutant
complexed with iron(II) and alternative 2-oxoacids. The results have
implications for clinically observed mutations to phytanoyl-CoA
2-hydroxylase that result in Refsum's
disease.2
Materials--
All chemicals were obtained from the
Sigma-Aldrich Chemical Co. or E. Merck and were at least analytical
grade or higher. Oligonucleotides were synthesized by V. Cooper (Dyson
Perrins Laboratory, Oxford) using Applied Biosystem DNA synthesizers
(Models 380B and 394). Reagents were supplied by Roche Molecular
Biochemicals (ATP); MBI (1 kilobase and 100 base pair DNA gel markers);
Bio-Rad (mutagenesis reagents, competent cells, Bradford Reagent); New England Bio-Labs (enzymes for molecular biology); Promega (Wizard Plus
miniprep DNA purification system, Wizard Plus SV miniprep DNA
purification system); and Qiagen Ltd. (RNase A).
Site-directed Mutagenesis--
The primer
(5'-ACTGGAGGTCTGGCTGCTGC-3') was annealed to the DAOCS gene in the
pET24a vector (1) by heating to 70 °C and cooled over 30 min to
35 °C, and mutagenesis performed by the method of Kunkel et
al. (14, 15). The presence of the R258Q mutation was confirmed by
automated sequencing, using an ABI Prism sequencer.
Protein Expression, Purification, and Activity
Measurements--
Wild-type and R258Q mutant enzymes were purified as
described (1), except that a 80-320 mM NaCl gradient over
800 ml was used to elute the Q-Sepharose anion-exchange column. HPLC
assays were carried out as described in Ref. 1 with 5 mM of
the appropriate 2-oxoacid substituted for 2-oxoglutarate when assaying
the R258Q mutant. The products resulting from 2-oxoacid oxidation were
characterized by larger scale incubation (2 ml) with the required
2-oxoacid (10 µmol) in the presence and absence of penicillin G (20 µmol) (1). 2-Oxoglutarate conversion was assayed using
[14C1]2-oxoglutarate as reported (16).
Apparent kinetic parameters (± S.D.) were determined by HPLC assay (1)
and the spectrophotometric method of Dubus et
al.3 At least six
concentrations of the variable substrate was used (in triplicate) at a
single defined concentration of the second substrate (Table II). Data
from the HPLC (1) and spectrophotometric3 assays were
derived as described.
Crystallization, Data Collection, and Analysis of the R258Q
Mutant--
DAOCS was crystallized by vapor diffusion using the
hanging drop method at 14 °C, using protein (2 µl) and
precipitating solution (2 µl). The precipitating solution contained
100 mM HEPES-NaOH, pH 7.0, 1.5-1.7 M ammonium
sulfate, 3-6% (w/v) glycerol, and 5 mM 2-oxoglutarate,
2-oxo-4-methylpentanoate (
Images were processed with the program MOSFLM (17) and data reduced
with the CCP4 suite (18). Each data set was assessed for merohedral
twinning based on the H-statistic
(19).4 The approximate twin
fraction obtained was used in the program DETWIN (18), and a more
accurate value for the twin fraction was obtained by minimization of
the residual twinning.
The starting model was that of the 2-oxoglutarate complex (9) with all
water and ligands removed and confirmed with the iron-2-oxoglutarate
complex of DAOCS (8). Rigid body refinement, simulated annealing, and
12-19 rounds of energy minimization and B-factor refinement were
carried out using the CNS suite (20, 21). The iron and ligands were
built after 5-6 rounds of refinement. Iron-ligand distances were
determined using restrained refinement, with initial parameters based
on those previously observed (1). Water molecules were added using the
CNS procedure water_pick.inp (20). Visual inspection of maps was
accomplished using O (22).
Biochemical and Kinetic Analysis of the R258Q Mutant--
The
R258Q mutant was obtained via site-directed mutagenesis, and the enzyme
purified to >99% homogeneity (as judged by SDS-polyacrylamide gel
electrophoresis) (1). Analyses by ESI MS (observed 34,532 ± 6 Da;
calculated 34,525 Da) and DNA sequencing indicated that no undesired
mutations were present, while CD analyses suggested there were no gross
changes to the secondary structure of the enzyme.
Both the wild-type enzyme and the R258Q mutant were analyzed for their
ability to catalyze the conversion of penicillin N and penicillin G to
their respective products using discontinuous HPLC assays for cephem
production (Table I). The results
demonstrated that wild-type DAOCS has, at most, only traces of activity
with all 2-oxoacids tested other than 2-oxoglutarate and
2-oxoadipate.
The R258Q DAOCS mutant was able to convert 2-oxoglutarate to succinate
and carbon dioxide in the presence of either penicillin N and
penicillin G. Two experiments were carried out (in duplicate) to
investigate the coupling of penicillin and 2-oxoglutarate conversion by
the R258Q mutant. The level of conversion of penicillin N (28%) and
penicillin G (
The R258Q mutant was able to utilize several other 2-oxoacids as
co-substrates when penicillin N was a substrate, most notably 2-oxo-3-methylbutanoate, 2-oxo-4-methylpentanoate, and
2-oxo-5-thiahexanoic acid. The alternative 2-oxoacids were also
utilized when penicillin G was the prime substrate although the
activity was somewhat lower. As with the reaction with penicillin N,
the two best co-substrates were 2-oxo-3-methylbutanoate and
2-oxo-4-methylpentanoate. The lower activity for penicillin G compared
with penicillin N is probably due to poorer coupling of penicillin
oxidation to co-substrate conversion for the former. The increased
tolerance for alternative co-substrates by the single R258Q mutation to
the wild-type enzyme is noteworthy.
1H NMR and ESI MS analyses were used to characterize
products of the incubation of 2-oxo-4-methylpentanoate and
2-oxo-3-methylbutanoate with the R258Q mutant in the presence and
absence of penicillin G. No evidence for their modification, other than
oxidative decarboxylation to give 3-methylbutanoate and
2-methylpropanoate, respectively, was obtained. This implies that
2-oxo-4-methylpentanoate and 2-oxo-3-methylbutanoate fulfill the same
role as 2-oxoglutarate in the wild-type enzyme.
Apparent steady-state kinetic parameters for the R258Q mutant with the
"unnatural" 2-oxoacids were determined at a single concentration of
the second substrate. Analysis of the R258Q mutant showed that
2-oxo-4-methylpentanoate and 2-oxo-3-methylbutanoate are utilized with
a efficiency similar (as judged by
Kcat/Km) to that of
2-oxoglutarate with the wild-type enzyme (Tables II and III). Detailed
kinetic analyses have revealed that substrate inhibition of the
wild-type enzyme occurred at high concentrations of
2-oxoglutarate.3 A similar although less pronounced
phenomenon was observed for the R258Q mutant with
2-oxo-4-methylpentanoate and 2-oxo-3-methylbutanoate as co-substrates
with penicillin G as substrate. Due to the low levels of activity of
the R258Q mutant with 2-oxoglutarate in the presence of penicillin G it
was not possible to obtain accurate values for the kinetic parameters,
but the apparent Km for 2-oxoglutarate was increased
by >1000-fold compared with the wild-type enzyme.3
Crystallographic Analysis of the R258Q Mutant--
Crystal
structures of the R258Q mutant complexes with iron(II) and
2-oxo-4-methylpentanoate and iron(II) and 2-oxo-3-methylbutanoate were
determined to high resolution (1.5 and 1.6 Å, respectively) (Table
IV). The structures had similar overall
structures to the iron(II)-2-oxoglutarate complex of the wild-type
enzyme (root mean squared deviation C The R258Q mutant was chosen for analysis as it was hoped that the
glutamine side chain would allow for an active site with an intact
conformation, while allowing other 2-oxoacids with hydrophobic side
chains to bind. The utilization of alternative co-substrates has been
previously investigated for several oxygenases related to DAOCS.
2-Oxoadipate, which possesses an additional methylene group compared
with 2-oxoglutarate, has been shown to be a co-substrate for clavaminic
acid synthase (25), taurine dioxygenase (26), DAOC/DAC synthase (27),
and DAOCS from N. lactamdurans (28). The results show that the side chain of arginine 258 is a major
determinant of the 2-oxoacid co-substrate selectivity of DAOCS and, by
implication, related oxygenases. The decrease in activity of the R258Q
DAOCS mutant observed with 2-oxoglutarate is probably due to the loss
of a favorable ionic interaction between the 5-carboxyl group of
2-oxoglutarate and the guanidino group of arginine 258. Thus, it
appears that 2-oxo-4-methylpentanoate and 2-oxo-3-methylbutanoate are
utilized as efficient co-substrates by the R258Q mutant because they
possesses hydrophobic side chains similar to that of 2-oxoglutarate but
do not have the unfavorable polar interactions. As proposed for prolyl
hydroxylase (35), the structural and biochemical results imply that
DAOCS has three key subsites for 2-oxoglutarate binding,
viz. the iron-ligation site, the RXS motif
(Arg-258 and Ser-260) binding to the 5-carboxylate, and a hydrophobic
region binding to the C3- and C4-methylene groups of
2-oxoglutarate.
These results also demonstrate that the catalytic effectiveness of the
2-oxoacid co-substrate is dependent on which prime substrate is
utilized (i.e. penicillin N versus penicillin G). A factor in determining catalytic efficiency is the level of coupling of 2-oxoacid oxidation to penicillin oxidation. Several recent studies
on related enzymes have suggested that dioxygen binding to the
iron-2-oxoglutarate enzyme complex is promoted by the presence of the
prime substrate in the active site (10, 24, 36). Oxidative
decarboxylation of the 2-oxoacid co-substrate is thought to generate a
reactive ferryl species, which is responsible for oxidation of the
prime substrate (37). Crystallographic evidence for the presence of
this intermediate in the reaction of the iron(II)-dependent oxygenase, isopenicillin N synthase has been recently obtained (38).
The kinetic results further support the idea that cooperative binding
of the 2-oxoacid and penicillin sites is required for efficient
catalysis. The relatively large amount of uncoupled conversion of
2-oxoglutarate in the presence of penicillin G for the R258Q DAOCS
mutant may be a reflection of an "editing" mechanism used by the
enzyme to select for the correct or "correctly bound" prime
substrate (1).
Studies on human prolyl-4-hydroxylase (39) have shown that mutation of
His-501 lead to increases in the level of uncoupled conversion of
2-oxoglutarate by about 12-fold. This appears to be due to a relative
reduction in the rate of prolyl-residue hydroxylation compared with
that of 2-oxoglutarate conversion. The authors proposed that His-501
directed binding of the 2-oxoacid moiety of 2-oxoglutarate and
accelerated the breakdown of the ferryl intermediate. In the light of
the results presented in this paper it may be that His-501 is ligating
to the 5-carboxylate of the 2-oxoglutarate co-substrate in addition to
ligation by Lys-493, and that mutation led to a decrease in coupling of
2-oxoglutarate and prime substrate oxidation.
The metal-binding sites of non-heme oxygenases containing iron are
closely related to those of hydrolytic enzymes containing either zinc
or iron (40). Before the concentration of dioxygen reached present day
levels it is possible that iron-dependent enzymes were
involved in many non-redox reactions, e.g. hydrolytic reactions, reflecting the bioavailability of this metal (41). Upon
increases in dioxygen levels it is possible that some of these enzymes
evolved to use zinc, thereby avoiding deleterious oxidative damage,
while others evolved into oxygenases/oxidases. The increases in
dioxygen levels probably also caused proliferation of the Kreb's cycle
of which 2-oxoglutarate is an intermediate. The parallel increases in
dioxygen and 2-oxoglutarate concentrations may have also influenced the
evolution of selectivity for 2-oxoglutarate over other less abundant or
ubiquitous 2-oxoacids such as 2-oxoadipate.
2-Oxoacids are common metabolites and readily form bidentate ligands
with iron. The question therefore arises how and why the non-heme
iron(II) and 2-oxoglutarate-dependent oxygenases appear to
have evolved a (relatively strict) selectivity for 2-oxoglutarate. One
reason may be that 2-oxoglutarate is a common primary metabolite, but
other 2-oxoacids, e.g. pyruvate, are also ubiquitous. The proposed binding mode of 2-oxoglutarate at the active site of DAOCS
rationalizes the utilization of 2-oxoglutarate and its close analogue
2-oxoadipate, and the deselection of 2-oxoacids with hydrophobic side
chains. The results presented here and elsewhere (39) also demonstrate
that incorrect binding of 2-oxoacids can increase the extent of prime
substrate uncoupled oxidation. Furthermore, it seems that cooperativity
between the 2-oxoacid and the prime substrate-binding sites is required
for efficient catalysis. The exact mechanism for this cooperativity in
the catalytic process during and subsequent to dioxygen binding
requires further investigation. The evolution of the use of
2-oxoglutarate as a co-substrate may reflect a need for precision
(including ordered binding/release) during part of the catalytic
process. This may be best achieved using a relatively rigid
electrostatic interaction such as that formed between Arg-258 and the
5-carboxylate rather than a hydrophobic interaction or other
electrostatic/hydrogen bonding interactions. Thus, the selection of
2-oxoglutarate over pyruvate may also reflect mechanistic considerations.
We have termed the restoration of activity by the use of a modified
2-oxoacid "chemical co-substrate rescue." The chemical rescue of
mutant enzymes has been extensively investigated, although studies have
focused on restoring the role of a mutated amino acid residue,
e.g. by using organic amines (42, 43), ions (44, 45), or a
modified residue involved in metal binding (46). The rescue of activity
using modified (co)-substrates has been relatively underexplored,
although ATP derivatives have been elegantly used to investigate the
function of kinases (47). We have recently demonstrated chemical
co-substrate rescue of several clinically observed mutants of
phytanoyl-CoA 2-hydroxylase using alternative 2-oxoacids.2
The results in this paper give the first indications of the molecular and structural basis for this process, and suggest that many
non-heme-dependent oxygenases control 2-oxoacid
co-substrate selectivity in a similar manner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxylase, which is involved in the biosynthesis of
cephamycin C from DAC (5).
-carboxylate group of the
L-
-(
-aminoadipoyl)-L-cysteinyl-D-valine substrate (11). Both of these residues have been shown to be important
for substrate binding in the C. acremonium isoenzyme by
site-directed mutagenesis (12, 13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ketoisocaproate) or
2-oxo-3-methylbutanoate (
-ketoisovalerate). Crystals in the R3 space group resembling those of the wild-type enzyme (1) appeared after about 1 week. Prior to data collection crystals were
soaked under anaerobic conditions in mother liquor supplemented with 5 mM iron(II) sulfate, followed by cryo-protectant containing 25% (v/v) glycerol. Data was collected using a Rigaku rotating anode
generator fitted with 345 MAR Research image plate detector and at
Beamline 9.6 of the SRS, Daresbury, United Kingdom.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Relative penicillin N and penicillin G conversions (% of wild-type) by
the R258Q mutant (17 µg/assay) in the presence of alternative
2-oxoacids
5%) (relative to wild-type enzyme) was much lower
than the levels of 2-oxoglutarate conversion (67 and 29%, respectively), suggesting this mutation causes significant uncoupling of 2-oxoglutarate and penicillin oxidation. This contrasts with the
wild-type enzyme in which penicillin oxidation is tightly coupled (with
a ratio of 1.06:1 for the native enzyme using 2-oxoglutarate and
penicillin N (23), and 1.07:1 for the recombinant enzyme using
2-oxoglutarate and penicillin G (9)). This probably accounts (at least
in part) for the poor efficiency of penicillin oxidation by the R258Q
mutant. The possibility of penicillin oxidation being uncoupled from
2-oxoglutarate oxidation should be taken into consideration when
comparing the apparent kinetic parameters in Tables I,
II, and Table
III.
Kinetic parameters for wild-type DAOCS with various 2-oxoacids
co-substrates with penicillin N or penicillin G
Kinetic parameters for R258Q DAOCS with various 2-oxoacid co-substrates
with penicillin N or penicillin G
differences = 0.267 and
0.282 Å, respectively) (8). Both structures show that the 2-oxoacid
moiety is ligated to the iron in the active site in a bidentate manner
similar to that observed for 2-oxoglutarate in the wild-type enzyme (1, 8). The protein ligands are provided with the side chains of His-183,
Asp-185 and His-243, and, as before, the sixth ligand to the iron is
water. This water is proposed to be displaced by binding of the
"prime" substrate, allowing oxygen binding and initiation of the
catalytic cycle (1, 24). Most of the proteins side chains that bind to
2-oxoglutarate in the wild-type enzyme occupy similar positions to
these structures. However, the side chain of glutamine 258 is now
disordered. Binding of the side chains of 2-oxo-4-methylpentanoate and
2-oxo-3-methylbutanoate is apparently via hydrophobic interactions
involving those residues which normally "bind" to the C-3 and C-4
methylene groups of 2-oxoglutarate (i.e. Met-180, Ile-192,
Leu-204, Phe-225, Val-245, and Val-262) (Fig.
1). The result of this interaction is
that the co-substrate side chains are slightly displaced from the
positions occupied by the methylene groups of 2-oxoglutarate.
Crystallographic parameters for the structures of the R258Q mutant
complexed with iron(II) and either 2-oxo-3-methylbutanoate or
2-oxo-4-methylpentanoate
View larger version (58K):
[in a new window]
Fig. 1.
A, View of the crystal structure of the
iron(II)-2-oxo-3-methylbutanoate complex of the R258Q DAOCS mutant.
Hydrophobic residues, including the side chains of Met-180, Ile-192,
and Leu-204 apparently bind the side chain of the 2-oxoacid ligand. In
this view His-243, which would be directly behind the iron, has been
omitted for clarity. The electron density map is contoured at 1.59 .
B, Close up of the map of structure lacking
2-oxo-3-methylbutanoate ligand showing positive density
(blue). In this view His-183 would be directly behind the
iron, and has been omitted for clarity. The electron density map is
contoured at 2.46
and omit map at 1.5
. This figure was produced
using BOBSCRIPT (48, 49).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Butyrobetaine
hydroxylase is also able to utilize both 2-oxoadipate and oxaloacetate,
the latter of which is one methylene group shorter than 2-oxoglutarate,
albeit with greatly increased Km values (29). In
contrast, 2,4-dichlorophenoxyacetate dioxygenase is reported to be able
to utilize a number of alternative 2-oxoacids, albeit relatively
inefficiently (30). Other than IPNS and ACCO, the only known
non-heme-dependent oxygenase that does not utilize 2-oxoglutarate as its preferred "co-substrate" is
4-hydroxyphenylpyruvate dioxygenase. However, the structure of
4-hydroxyphenylpyruvate dioxygenase places it in the family of catechol
dioxygenases, (31) and it carries out a four-electron oxidation and
decarboxylation of the same 2-oxoacid, 4-hydroxyphenylpyruvate. It is
related to the 2-oxoglutarate-dependent oxygenases by
mechanism and can utilize 3-thienylpyruvate (32),
-ketoisocaproate
(2-oxo-4-methylpentanoate) (33), and 2-oxo-5-thiahexanoic acid (34) as
(co)substrates. In contrast,
-ketoisovalerate
(2-oxo-3-methylbutanoate) and
-keto-
-methyl-n-valerate (2-oxo-3-methylpentanoate), derived from valine and isoleucine, respectively, were not converted to their corresponding products (32).
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Leslie (University of Cambridge) for access to his program detwin, and Dr. R. C. Wilmouth (University of Oxford), Professors J. Hajdu (Uppsala University) and I. Andersson (Swedish University of Agricultural Sciences) for discussions. The Staff at Daresbury SRS are thanked for facilities and assistance.
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Sciences Research Council, the Engineering and Physical Sciences Research Council, the Medical Research Council, the Wellcome Trust, and the European Union Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rscb. org/) for the iron(II)-2-oxo-3-methylbutanoate complex (accession numbers 1hjg.pdb and r1hjgsf) and the iron(II)-2-oxo-4-methylpentanoate complex (accession numbers 1hjf.pdb and r1hjfsf).
¶ Contributed equally to the results of this work.
To whom correspondence should be addressed. Tel.: 1865-275677;
Fax: 1865-275674; E-mail: matthew.lloyd@chem.ox.ac.uk.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100085200
2 M. Mukherji, N. J. Kershaw, C. H. MacKinnon, I. J. Clifton, C. J. Schofield, A. S. Wierzbicki, and M. D. Lloyd, (2001) J. Chem. Soc. Chem. Commun., in press.
3 A. Dubus, M. D. Lloyd, H.-J. Lee, C. J. Schofield, J. E. Baldwin, and J.-M. Frere, Cell. Mol. Life Sci., in press.
4 I. J. Clifton, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: DAOCS, deacetoxycephalosporin C synthase; ACCO, 1-amino-1-carboxycyclopropane oxidase; DAC, deacetylcephalosporin C; DACS, deacetylcephalosporin C synthase; DAOC, deacetoxycephalosporin C; DAOC/DACS, deacetoxy/deacetylcephalosporin C synthase; ESI MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography.
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REFERENCES |
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