Contrasting fates for 6-α-methylpenicillin N upon oxidation by deacetoxycephalosporin C synthase (DAOCS) and deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS)

Article abstract of DOI:10.1016/S0960-894X(01)00470-X

6-α-Methylpenicillin N was synthesised via known routes from 6-aminopenicillanic acid, and tested as a substrate for recombinant DAOCS and DAOC/DACS. Incubation with DAOCS resulted in conversion of 2-oxoglutarate without oxidation of the penicillin substrate ('uncoupled turnover'). Incubation with DAOC/DACS resulted in oxidation to the cephem aldehyde. This is the first example of substrate-induced 'uncoupled turnover', which has been proposed to be an editing mechanism for these enzymes. Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(01)00470-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2511–2514
Contrasting Fates for 6-ꢀ-Methylpenicillin N upon Oxidation by
Deacetoxycephalosporin C Synthase (DAOCS) and
Deacetoxy/deacetylcephalosporin C Synthase (DAOC/DACS)
Christopher S. Hamilton, Akito Yasuhara, Jack E. Baldwin, Matthew D. Lloyd*
and Peter J. Rutledge*
The Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK
Received 7 June 2001; revised 4 July 2001; accepted 5 July 2001
Abstract—6-a-Methylpenicillin N was synthesised via known routes from 6-aminopenicillanic acid, and tested as a substrate for
recombinant DAOCS and DAOC/DACS. Incubation with DAOCS resulted in conversion of 2-oxoglutarate without oxidation of
the penicillin substrate (‘uncoupled turnover’). Incubation with DAOC/DACS resulted in oxidation to the cephem aldehyde. This is
the first example of substrate-induced ‘uncoupled turnover’, which has been proposed to be an editing mechanism for these
enzymes. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
recent crystallographic studies have suggested a radical
mechanism operates throughout the ring-expansion
reaction, with a ferric hydroxide complex abstracting a
hydrogen atom to give DAOC in the final step.9
Iron(II) and 2-oxoglutarate-dependent oxygenases play
a key role in the biosynthesis of cephem antibiotics. In
Streptomyces
clavuligerus,
two
sequence-related
enzymes, deacetoxycephalosporin C synthase (DAOCS)
and deacetylcephalosporin C synthase (DACS), are
responsible for the ring-expansion and hydroxylation of
penicillin N to give the key intermediate deacetylcepha-
losporin C (DAC). This is then converted to cephamy-
cin C via a series of reactions. In Cephalosporium
acremonium a single bifunctional enzyme, deacetoxy/
The ring-expansion and hydroxylation reactions cata-
lysed by DAOCS, DACS and DAOC/DACS all poten-
tially follow radical mechanisms. However, in order to
determine how 2-oxoglutarate conversion is coupled to
oxidation of the penam or cephem substrates, and
which of the two reactions is performed by DAOC/
DACS, crystallographic information about substrate
and product complexes of both these enzymes is
required in conjunction with functional and kinetic
studies. Obtaining crystals of the enzyme–penicillin N
complexes is difficult on account of the inherent instability
of these compounds under the required aqueous condi-
tions. In order to facilitate crystallisation of this complex,
synthesis of the stabilised substrate 6-a-methylpenicillin N
was undertaken, and this analogue has now been eval-
uated as a substrate for both DAOCS and DAOC/DACS.
The results reveal a fundamental difference in reactivity
between the two enzymes, in what appears to be the first
example of a substrate being ‘proof-read’8 during the
DAOCS-catalysed ring-expansion reaction.
deacetylcephalosporin
C synthase (DAOC/DACS),
which has primary sequence homology to the two mono-
functional S. clavuligerus enzymes, catalyses both the
ring-expansion and hydroxylation reactions. Cephalos-
porin C is formed by acetylation of DAC.1,2
Stereochemical studies on the ring-expansion reaction
catalysed by DAOC/DACS are consistent with a radical
mechanism.3,4 Radical mechanisms have also been pro-
posed for the hydroxylation reactions catalysed by the
related enzymes g-butyrobetaine hydroxylase5 and cla-
vaminate synthase.6 Even though the crystal structure
of DAOCS has been determined,7,8 many of the details
of its reaction mechanism remain obscure. However,
Synthesis of 6-ꢀ-Methylpenicillin N
*Corresponding authors. Tel.: +44-1865-275677/275693; fax: +44-
1865-275674; e-mail: matthew.lloyd@chem.ox.ac.uk; peter.rutledge@
chem.ox.ac.uk
6-a-Methylpenicillin N (1) was prepared from 6-amino-
penicillanic acid (6-APA, 2) and d-a-aminoadipic acid
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00470-X

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C. S. Hamilton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2511–2514
in six steps in 55.5% overall yield, using methods mod-
ified from those of Firestone10,11 (Scheme 1). 6-APA (2)
was protected as the benzyl ester (3), and converted into
the p-nitrobenzylidine imine (4). Methylation to 5 fol-
lowed by imine hydrolysis afforded the free amine (6),
for coupling with diprotected d-a-aminoadipic acid.
HBTU [O-(1H-benzotriazol-1-yl)-N,N0,N0-tetramethyl-
uronium hexa-fluorophosphate]-mediated coupling of
the two portions to 7,12 followed by hydrogenolytic
deprotection13 and HPLC purification gave 6-a-methyl-
penicillin N (1).14
lated was the ring-opened aldehyde (8) (Scheme 2).14
This product parallels that observed with penicillin N,16
and is probably produced via 9, 10, and 11. The latter
apparently undergoes facile non-enzymatic ring opening
in the presence of NH4HCO3 buffer. A small amount of
6-a-methylpenicillin N (1) was recovered from the crude
reaction mixture. No evidence for the presence of 9 or
10 was obtained, suggesting that the ring-expansion
reaction was rate limiting.
1
In contrast, H NMR analyses showed no evidence for
the conversion of 6-a-methylpenicillin N (1) to 6-a-
methyl-DAOC (9) by DAOCS (Scheme 2). 2-Oxogluta-
rate utilisation assays17 with both DAOCS and DAOC/
DACS showed that the presence of 6-a-methylpenicillin
N (1) stimulated co-substrate conversion markedly.
Parallel incubation studies suggest that the oxidation of
Incubation of 6-ꢀ-Methylpenicillin N with Enzymes
All incubations were carried out using highly purified
recombinant enzymes.15 When 6-a-methylpenicillin N
(1) was incubated with DAOC/DACS, the product iso-
Scheme 1. Synthesis of 6-a-methylpenicillin N (1). Reagents and conditions: (i) Et3N, PhCH2Br, acetone, 0 ꢀC, then rt, 2 days, 53%; (ii) p-nitro-
benzaldehyde, MgSO4, CH2Cl2, rt, 16 h, 94%; (iii) PhLi, THF, À78 ꢀC, then MeI, À78 ꢀC to rt, 0.5 h, 95%; (iv) p-TsOH, Girard’s Reagent P,
CH2Cl2/EtOH, 35 ꢀC, 18 h, 55%; (v) N-(p-nitrobenzyloxycarbonyl)-d-a-aminoadipic acid 1-p-nitrobenzyl ester, Et3N, HBTU, CH3CN, rt, 16 h,
52%; (vi) H2, Pd/C, NaHCO3, THF/H2O, rt, 24 h, then HPLC purification [ODS (250Â10 mm), gradient elution with 0–25% (v/v) acetonitrile in 10
mM aq NH4HCO3, 4 mL/min, l=254 nm, 5 AUFS], 41%.
Scheme 2. Conversion of 6-a-methylpenicillin N (1) with (a) DAOC/DACS and (b) DAOCS. 2-OG, 2-oxoglutarate; R, d-d-(a-aminoadipoyl).

C. S. Hamilton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2511–2514
2513
6-a-methylpenicillin N (1) by DAOCS is significantly
uncoupled from 2-oxoglutarate conversion, with a cou-
pling ratio of >1:29. In contrast, the oxidation of both
penicillin N and penicillin G by DAOCS is tightly cou-
pled to 2-oxoglutarate conversion.9,18 Thus, a large
change in catalytic efficiency has been brought about by
an apparently minor modification to the structure of the
natural substrate.
structures of enzyme–substrate complexes for both
enzymes are needed. However, in the absence of these
structures, the results reported here provide further
insight into the different selectivities of the two enzymes.
The differing reactivity of DAOCS and DAOC/DACS
with 6-a-methylpenicillin N (1) reflects a subtle differ-
ence in the active site geometries of the two proteins.
This difference in geometry is necessitated by the
requirement to accommodate a concave bicyclic peni-
cillin nucleus versus a ‘flattened’ unsaturated cephem
nucleus. DAOC/DACS accepts the analogue (1) as a
substrate in its active site and performs the ring expan-
sion and hydroxylation reactions on it, leading ulti-
mately to the aldehyde product (8). In contrast, the
uncoupled turnover observed with DAOCS indicates
that 6-a-methylpenicillin N (1) binds in the active site
well enough to stimulate 2-oxoglutarate conversion, but
not sufficiently tightly to be converted itself. It then
apparently dissociates without any other reaction
occurring. A similar difference in reactivity between the
two enzymes has been previously reported with a range
of substrate analogues19 (although it is difficult to be
certain whether this is truly a difference in reactivity or
more a reflection of the limitations of the various assays
used20). However, the structural difference between the
6-methyl analogue (1) and the natural substrate penicillin
N is comparatively small, and thus 1 could be expected to
react in an analogous manner with both enzymes.
Discussion and Conclusions
While DAOCS and DAOC/DACS have similar biolo-
gical functions, similar amino acid sequences and by
extrapolation similar structures, they apparently exhibit
significantly different substrate specificities. DAOC/
DACS reacts with a much broader range of substrates
than DAOCS, a fact most graphically illustrated by the
bifunctionality of the former enzyme and the contrast-
ing monofunctionality of the latter. Further structural
information is required before the precise molecular
basis for this difference in selectivity can be fully under-
stood. In particular, a structure for DAOC/DACS and
To date no X-ray crystallographic structure of DAOC/
DACS has been solved, but the enzyme shows high
levels of primary sequence homology with DAOCS. In
order to understand the molecular basis for the different
fates of 6-a-methylpenicillin N (1) with these two
enzymes, a model of the DAOC/DACS structure was
constructed21 and compared with the crystal structure
of DAOCS. This model suggests that the majority of
differences occur at the surface of the protein. Compar-
ison of the sequences for the regions responsible for
binding iron(II), 2-oxoglutarate (and substrate) suggests
that the active sites of the two enzymes are extremely
similar (Fig. 1).
Recent results22 support the hypothesis that binding of
the bicyclic penicillin nucleus in DAOCS is mediated by
Arg-162 and Arg-160, as had previously been pro-
posed.7,8 Assuming this hypothesis is correct, then the
only substitution within close proximity of the substrate
6-methyl group occurs at residue 73: a methionine in
DAOCS versus alanine in DAOC/DACS (Fig. 1). It is
plausible that the 6-a-methyl group results in unfavour-
able steric interactions between the side chain of Met-73
and the penicillin substrate in the active site of DAOCS.
In DAOC/DACS, however, the small alanine side chain
occupies the same position and thus unfavourable steric
interactions will be diminished.
However, DAOCS possesses four disordered loops
within the crystal structure7 (residues 83–90, 166–177,
196–200 and 249–257), and all but residues 196–200 are
within close proximity of the penicillin binding site. It is
probable that the three other disordered loops will
Figure 1. Binding of 6-a-methylpenicillin N (1) in the active site of: (a)
DAOCS; (b) DAOC/DACS. Arg-160 (DAOCS) or Arg-161 (DAOC/
DACS) have been removed for clarity, but are situated directly behind
Met/Ala-73. Figures represent models (ref 21) and were produced
using Bobscript.26,27

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C. S. Hamilton et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2511–2514
become ordered on penicillin binding, thus isolating the
active site during catalysis. Further experiments are
required in order to determine whether these residues
are important for efficient coupling of 2-oxoglutarate
conversion to penicillin oxidation.
12. Dourtoglou, V.; Gross, B. Synthesis 1984, 572.
13. Bodanszky, M.; Bodanszky, A. In The Practice of Peptide
Synthesis; Springer: Berlin, 1984; Vol. 22; p 153.
14. 1H NMR (200 MHz, D2O) d 1.35 (3H, s, Me), 1.43 (3H, s,
Me),
1.57
(3H,
s,
Me),
CHCH2CH2CH2CONH), 2.23 (2H, t,
1.46–1.88
(4H, m,
7.0 Hz,
J
CH2CH2CONH), 3.57 (1H, m, HO2CCHNH2), 4.11 (1H, s,
H-3), 5.27 (1H, s, H-5). IR (KBr disc) 1764 cmÀ1, strong (b-
lactam C¼O).
The factors that control the coupling of 2-oxoglutarate
conversion to penicillin oxidation by DAOCS are subtle
and hitherto poorly defined. However, it is known that
modifications to either the C-terminus9 or Arg-25823
(the residue that binds the 5-carboxylate group of 2-
oxoglutarate) both lead to substantial uncoupling of
these reactions. Low levels of uncoupled 2-oxoglutarate
conversion have been proposed to be a mechanism of
‘proof-reading’ in order to deselect incorrect substrates,
or correct substrates incorrectly bound.8
15. DAOCS and DAOC/DACS were produced using Escher-
ichia coli BL21 (DE3) with the pET24a-derived plasmids
pHL1 and pKC200 (constructed by Dr. K. S. Hewitson, Uni-
versity of Oxford), respectively. Purification to ca. 95% purity
was achieved using anion-exchange and gel filtration chroma-
tographies.
6-a-Methylpenicillin N (1) (1.5 mg) was incubated with
DAOCS (6.47 mg, 116 IU/mg using penicillin G as sub-
strate20) as previously reported8 for 90 min. 1H NMR analysis
(500 MHz) of the quenched reaction suggested <2% conver-
sion to cephem products.
More information, and in particular structural infor-
mation, is required before the factors behind the cou-
pling and uncoupling of these processes can be fully
elucidated. However, the differing fates of 6-a-methyl-
penicillin N (1) upon oxidation with DAOCS and
DAOC/DACS brings some further insight to this
interesting puzzle.
6-a-Methylpenicillin N (1) (1.5 mg) was incubated8 with
DAOC/DACS (2.86 mg, 135 IU/mg with 6-a-methylpenicillin
1
N as substrate20). H NMR analyses (500 MHz) of the quen-
ched reaction mixture suggested >95% conversion to (8).
HPLC purification [ODS (250Â4.6 mm), isocratic elution with
25 mM NH4HCO3 and 1% (v/v) acetonitrile, 1 mL/min,
l=254 nm, 2 AUFS] resulted in isolation of (8) (107 mg) with
a retention volume of 4.4 mL: 1H NMR (500 MHz, D2O) d
1.55–1.65 (5H, m, Me and CH2CH2CONH), 1.70–1.85 (2H,
m, CH2CH2CH2CONH), 2.35 (2H, m, CH2CONH), 3.28
(0.6H, s, residual MeOH), 3.40 (1H, d, J 17 Hz, one of SCH2),
3.50 (1H, m, H2NCH), 3.55 (1H, d, J 17 Hz, one of SCH2),
4.97 (1H, s, NHCHSCH2), 9.05 (1H, s, CHO); m/z (Àve ESI
MS) 401.33 ([MÀH]À, 100%; calcd 401.4). A small amount of
1 was recovered as a broad peak with a retention volume of
6.6–7.0 mL: m/z (Àve ESI-MS) 372.3 [MÀH]À, (100%).
16. Baldwin, J. E.; Goh, K.-C.; Schofield, C. J. J. Antibiot.
1992, 45, 1378.
Acknowledgements
We thank Dr. K. S. Hewitson for plasmid pKC200, Dr.
B. Odell and Dr. R. T. Aplin for NMR and mass spec-
trometric analyses, respectively, and Dr. R. M. Adling-
ton and Professor C. J. Schofield for helpful discussions.
The BBSRC, EPSRC, MRC, Wellcome Trust and E.U.
Biotechnology programme funded this work.
17. Lee, H.-J.; Lloyd, M. D.; Harlos, K.; Schofield, C. J. Bio-
chem. Biophys. Res. Commun. 2000, 267, 445.
18. Baldwin, J. E.; Crabbe, M. J. C. FEBS Lett. 1987, 214,
357.
References and Notes
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DAOCS: iron(II):2-oxoglutarate complex (1rxg.pdb) with the
programme O,24 followed by model refinement within CNS.25
Residues 310–332 of DAOC/DACS could not be adequately
modelled, but are predicted to form at least one further helix.
Models for 6-a-methylpenicillin N and dioxygen were pro-
duced using Quanta (Molecular Simulations), docked into the
protein structures and refined.
22. Lipscomb, S. J.; Lee, H.-J.; Baldwin, J. E.; Schofield, C. J.;
Lloyd, M. D. Manuscript in preparation.
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