The role of arginine residues in substrate binding and catalysis
by deacetoxycephalosporin C synthase
Sarah J. Lipscomb
1,
*, Hwei-Jen Lee
1,2,
*, Mridul Mukherji
1
, Jack E. Baldwin
1
, Christopher J. Schofield
1
and Matthew D. Lloyd
1,3
1
Oxford Centre for Molecular Sciences and the Dyson Perrins Laboratory, Oxford, UK;
2
Department of Biochemistry,
National Defence Medical Centre, Taipei, Taiwan;
3
Department of Pharmacy and Pharmacology, The University of Bath, UK
Deacetoxycephalosporin C synthase (DAOCS) catalyses the
oxidative ring expansion of penicillin N, the committed step
in the biosynthesis of cephamycin C by Streptomyces clavu-
ligerus. Site-directed mutagenesis was used to investigate the
seven Arg residues for activity (74, 75, 160, 162, 266, 306 and
307), selected on the basis of the DAOCS crystal structure.
Greater than 95% of activity was lost upon mutation of Arg-
160 and Arg266 to glutamine or other residues. These results
are consistent with the proposed roles for these residues in
binding the carboxylate linked to the nucleus of penicillin N

DAOCS contains eight arginine residues within, or close
to, its active site that may be involved in catalysis. Arg258
has already been shown by structural and mutagenesis
studies to bind the 5-carboxylate of the 2-oxoglutarate [13].
Mutagenesis of this residue to glutamine reduced
2-oxoglutarate conversion. However, other aliphatic 2-oxo-
acids, which are not cosubstrates for wild-type DAOCS,
had higher levels of activity as they interact more
favourably with the mutated cosubstrate binding site. Here
we report site-directed mutagenesis studies on the other
arginine residues located within the active site (74, 75, 160,
162, 266, 306 and 307) that, together with the crystallo-
graphic analyses, support the proposed roles for arginines
160, 162 and 266, and suggest roles for the other arginine
residues.
Scheme 1. Conversion of penicillin N (1) to cephamycin C (4). 2-OG,
2-oxoglutarate; R,
D
-d-(a-aminoadipoyl)-; *, methyl group incorpor-
ated into cephem ring by DAOCS. The putative high-energy iron
intermediate [Fe(IV) ¼ O/Fe(III)-O.] is shown boxed. Note that the
precise arrangement of the ligands around the iron is uncertain [12].
Correspondence to M. D. Lloyd, The Department of Pharmacy and Pharmacology, The University of Bath, Claverton Down,
Bath BA2 7AY, UK. Fax: + 44 1225 386114, E-mail:
Abbreviations: DAC, deacetylcephalosporin C; DACS, deacetylcephalosporin C synthase; DAOC, deacetoxycephalosporin C; DAOCS,
deacetoxycephalosporin C synthase; Dnase 1, deoxyribonuclease 1; EDTA, ethylenediaminetetraacetic acid; ESI MS, electrospray ionization
mass spectrometry; USE, unique site elimination.
*Note: these authors contributed equally to this work.
(Received 4 January 2002, revised 12 April 2002, accepted 19 April 2002)
Eur. J. Biochem. 269, 2735–2739 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02945.x

reported [1].
Purification and assay of DAOCS mutants
Wild-type DAOCS and the Arg160 and 162 mutants were
purified to  85% homogeneity (by SDS/PAGE analysis)
from  25 g of frozen recombinant E. coli cells by anion-
exchange and gel filtration chromatographies as previously
described [1]. The Q-sepharose column was eluted with an
80–320 m
M
NaCl gradient over 800 mL. The required
fractions were pooled, concentrated to  8 mL and further
purified using a Superdex-200 column (86 · 3.2 cm,
692 mL) equilibrated in gel filtration buffer [1].
The remaining mutants were purified on a smaller scale to
 85% homogeneity (by SDS/PAGE analysis) using the
following protocol. Frozen recombinant E. coli cells (1.5–
3.5 g) were resuspended in 50 m
M
Tris/HCl, 1 m
M
EDTA,
pH 7.5 and 2 m
M
dithiothreitol (20 mL) and treated with
lysozyme (6 mg). The sample was stirred for 10 min before
addition of MgCl
2
(5 m
M
)andDnase1(20lg). After a

3mLÆmin
)1
, and protein eluted with a 0.96–0.36
M
(NH
4
)
2
SO
4
gradient over 120 mL. Fractions (5 mL) were
analysed by SDS/PAGE following trichloroacetic acid
precipitation. Purified protein was exchanged into 50 m
M
Tris/HCl pH 7.5 using an Econo-Pak column (Bio-Rad)
and concentrated to  15 mgÆmL
)1
. Purified mutants were
analysed by circular dichroism analysis as previously
described [1]. Highly purified wild-type enzyme and mutants
were also analysed by ESI MS [wild-type: 34 551 Da
(predicted), 34 550 ± 9 Da (observed); R160Q: 34 525 Da
(predicted), 34 524 ± 7 Da (observed); R162Q: 34 525 Da
(predicted), 34 525 ± 9 Da (observed)].
Activity assays
Radioactive 2-oxoglutarate conversion assays were conduc-
ted as previously described [17], using 0.1 m
M
penicillin N,
10 m

R75Q 5¢-CCCGTCCCCACCATGCGCCAGGGCTTCACCGGG-3¢ USE
R160Q 5¢-TAGCGGAACTGCAGCAGCG-3¢ Kunkel
R162A 5¢-CGCTGCTGCGGTTCGCATACTTCCCGCAGGTC-3¢ PCR
R162Q 5¢-CGCTGCTGCGGTTCCAATACTTCCCGCAGGTC-3¢ PCR
R266I 5¢-AGTGTGTTCTTCCTCATCCCCAACGCGGACTTC-3¢ USE
R266Q 5¢-AGTGTGTTCTTCCTCCAGCCCAACGCGGACTTC-3¢ USE
R306L 5¢-GATGTGCGCAGGATGTTCA-3¢ Kunkel
R307Q 5¢-CTTGGATGTCTGGCGGATGTT-3¢ Kunkel
2736 S. J. Lipscomb et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Retention volumes for product and substrate were 18.3 and
19.5 mL, respectively. Approximately 0.15 mg of enzyme
wasusedineach100lL assay.
1
H NMR (500 MHz)
spectroscopy was used to confirm the presence of the
expected cephem products [1]. Note the radioactive and
HPLC assays are carried out under different conditions.
RESULTS
Construction and purification of mutants
The following single mutants were constructed by site-
directed mutagenesis: R74I, R74Q, R75Q, R160Q, R162Q,
R162A, R266I, R266Q, R306L and R307Q. The following
double mutants were constructed using a second round of
mutagenesis: R74I/R266I, R74Q/R266I, R74I/R266Q and
R74Q/R266Q. Sequencing of the clones revealed that only
the desired mutations were present except in the R75I
mutant, which also contained an additional mutation,
D270G. Analysis of the DAOCS structure [1,11,12],
indicated that this mutation would be on the surface of
the protein. All mutants and wild-type enzyme were

m
values were similar to the values for the wild-type enzyme.
2-Oxoglutarate conversion by the R74I and R74Q
mutants was also significantly stimulated by the presence
of penicillin G (Table 2, entries 2 and 3). However, penicillin
G oxidation was considerably reduced relative to the wild-
type enzyme, i.e. it was less than stoichiometric for
2-oxoglutarate conversion. The results for the R75Q mutant
in the presence of penicillin G showed little or no evidence
for 2-oxoglutarate stimulation or penicillin G oxidation,
suggesting that this mutation had abolished or severely
affected ÔprimeÕ substrate binding. The low levels of
penicillin G oxidation by the R74I, R74Q and R75Q
mutants prevented further kinetic analysis of these mutants
with this substrate.
When Arg266 was mutated (R266L, R266Q) (Table 2,
entries 13–14) 2-oxoglutarate conversion was very low and
the same whether penicillin N, penicillin G or no penicillin
Table 3. Kinetic parameters. Top, kinetic parameters for penicillin N
conversion (10–50 lm) by wild-type DAOCS and arginine mutants by
HPLC assay. Parameters were determined [1] at 2 mm 2-oxoglutarate
using at least five different concentrations of penicillin substrate, at
least in duplicate. Values are reported ± SD. Bottom, kinetic
parameters for penicillin G conversion (0.5–3.0 mm) of wild-type
DAOCS and arginine mutants by HPLC assay. Parameters were
determined [1] at 2 mm 2-oxoglutarate using at least five different
concentrations of penicillin substrate, at least in duplicate. Values are
reported ±SD.
Mutant K
m

are corrected for conversion in the absence of any substrate. HPLC
assays [1] were used to assess penicillin conversion. Results are nor-
malized to penicillin N conversion by wild-type enzyme (26 nmolÆ
min
)1
Æmg
)1
) and are based on at least duplicate readings. Standard
deviations are  15% and 10% for 2-oxoglutarate and penicillin
conversion, respectively. ND, not determined.
DAOCS enzyme
Penicillin N Penicillin G
2-OG HPLC 2-OG HPLC
1. Wild-type 100 100 79 65
2. R74I 100 76 53 6
3. R74Q 209 45 93 5
4. R75I/D270G 83 62 31 42
5. R75Q 45 25 <5 8
6. R74I/R266I <5 <2 <5 <2
7. R74Q/R266I <5 <2 <5 <2
8. R74I/R266Q <5 <2 40 4
9. R74Q/R266Q <5 <2 <5 <2
10. R160Q ND ND <5 4
11. R162Q ND ND <5 3
12. R162A ND ND <5 <2
13. R266I <5 <2 <5 3
14. R266Q <5 <2 <5 3
15. R306L 200 200 41 64
16. R307Q 38 144 26 39
Ó FEBS 2002 Role of arginine residues in DAOCS (Eur. J. Biochem. 269) 2737

cat
value. Note
the increased specific activity for the R306L mutant may
reflect a decreased rate of inactivation for this mutant.
DISCUSSION
The reaction catalysed by the iron(II) and 2-oxoglutarate-
dependent oxygenases involves conversion of 2-oxogluta-
rate and dioxygen to give succinate, carbon dioxide and an
enzyme-bound reactive oxidizing intermediate, believed to
be a high-energy ferryl [Fe(IV) ¼ O/Fe(III)-O.] species
[1,4]. The latter is used to effect the oxidative conversion of
the prime substrate (in the case of DAOCS, the penicillin).
For many 2-oxoglutarate-dependent oxygenases, 2-oxo-
glutarate conversion occurs at a low rate in the absence of
prime substrates but is stimulated by their presence [4,19].
Studies with some oxygenases (e.g. clavaminic acid syn-
thase [20,21]) have suggested that prime substrate binding
activates the enzyme–iron(II)-2-oxoglutarate ternary com-
plex to oxygen binding, thereby initiating catalysis and
ensuring coupling of 2-oxoglutarate conversion to prime
substrate oxidation.
In the case of DAOCS it has been previously shown that
mutations to residues involved in 2-oxoglutarate or penicil-
lin binding [12,13] can result in substantial uncoupling of
penicillin oxidation from 2-oxoglutarate (i.e. there is a less
than stoichiometric oxidation of the penicillin substrate).
Uncoupling of 2-oxoglutarate conversion can also appar-
ently result from mutations to residues not directly involved
in substrate or cosubstrate binding [17]. Stimulation of
2-oxoglutarate conversion is therefore evidence for ÔprimeÕ

together, the results of this and previous studies [12,13]
suggest that correct orientation of the penicillin substrate
with respect to the putative high-energy ferryl intermediate
is important for optimum coupling between 2-oxoglutarate
and penicillin conversion.
Recent studies have suggested that the C-terminus of
DAOCS (including arginines 306 and 307) is involved in
conformational changes during catalysis. It is possible that
DAOCS and other 2-oxoglutarate dependent oxygenases
have evolved to maximize coupling between 2-oxoglutarate
and ÔprimeÕ substrate oxidation. This is probably required in
order to control the reactive oxidizing species produced
during catalysis and to avoid inactivation of the enzyme.
Evidence for the oxidative inhibition of TfdA under
uncoupled conditions has been reported [23].
Re-engineering of DAOCS to accept hydrophobic peni-
cillin substrates is a desirable objective, as this may allow
fermentation of starting materials for production of semi-
synthetic cephem antibiotics. The results in this paper
suggest that point mutations to C-terminal residues (per-
Fig. 1. Proposed binding mode for penicillin N to the iron(II)-2-oxo-
glutarate complex of DAOCS, showing possible involvement of selected
residues.
2738 S. J. Lipscomb et al. (Eur. J. Biochem. 269) Ó FEBS 2002
haps in conjunction with C-terminal truncation [12] or other
active site mutations [24]) may allow improved conversion
of unnatural substrates (e.g. penicillin G) whilst maintaining
tight coupling between substrate oxidation and 2-oxoglut-
arate conversion.
ACKNOWLEDGEMENTS

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10. Baldwin,J.E.,Adlington,R.M.,Kang,T.W.,Lee,E.&Schofield,
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Baldwin, J.E., Schofield, C.J., Hajdu, J. & Andersson, I. (1998)
Structure of a cephalosporin synthase. Nature 394, 805–809.
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Ó FEBS 2002 Role of arginine residues in DAOCS (Eur. J. Biochem. 269) 2739