Enzyme assays Wild-type DAOCS and all mutants were assayed for their ability to convert 2-oxoglutarate to succinate and carbon dioxide [17], and penicillin N and penicillin G to their co
Trang 1The role of arginine residues in substrate binding and catalysis
by deacetoxycephalosporin C synthase
Sarah J Lipscomb1,*, Hwei-Jen Lee1,2,*, Mridul Mukherji1, Jack E Baldwin1, Christopher J Schofield1 and Matthew D Lloyd1,3
1
Oxford Centre for Molecular Sciences and the Dyson Perrins Laboratory, Oxford, UK;2Department of Biochemistry,
National Defence Medical Centre, Taipei, Taiwan;3Department 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
(Arg160 and Arg162) and the carboxylate of the
a-amino-adipoyl side-chain (Arg266) The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS
Keywords: cephalosporin; penicillin; b-lactam; 2-oxogluta-rate; nonhaem iron(II) oxygenase
Deacetoxycephalosporin C synthase (DAOCS; Swissprot
P18548) is an iron(II) and 2-oxoglutarate-dependent
oxygenase that catalyses the conversion of penicillin N
(Scheme 1, 1) to deacetoxycephalosporin C (DAOC 2) in
Streptomyces clavuligerus[1–7] The subsequent
hydroxyla-tion of DAOC to give deacetylcephalosporin C (DAC 3) is
catalysed by a closely related oxygenase,
deacetylcephalo-sporin C synthase (DACS; Swissprot P42220) The DAC 3
product is then converted into cephamycin C 4 in a process
involving oxidation at the C-7 position (Scheme 1)
Our understanding of the catalytic mechanism of DAOCS [8–10] has been significantly advanced by the determination of the crystal structure of wild-type and mutant DAOCS with various ligands [1,11–13] However, a detailed understanding of the ring-expansion reaction requires structural information for DAOCS complexed with penicillin substrates and deacetoxycephem products Attempts to cocrystallise DAOCS in the presence of various substrates and products have been hampered by their instability under the crystallization conditions
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: M.D.Lloyd@bath.ac.uk
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)
Trang 2E X P E R I M E N T A L P R O C E D U R E S
Materials
All chemicals were obtained from the Sigma–Aldrich
Chemical Co or Merck and were of analytical grade or
higher Reagents were also supplied by: Amersham
Biosciences (protein chromatography systems and columns,
U.S.E Mutagenesis Kit); Bohringer–Mannheim (ATP);
MBI (1 Kband 100 bp DNA gel markers); Bio-Rad
(Kunkel mutagenesis reagents); New England Bio-Labs
(enzymes for molecular biology); Novagen (pET vectors);
Sigma-Genosys (mutagenesis primers); Phenomenex
(HPLC columns); Promega (Wizard Plus miniprep DNA
purification system, Wizard Plus SV miniprep DNA
purification system); Stratagene (competent cells, PCR
Script vector, QuikChange mutagenesis kit)
Site-directed mutagenesis
The R162Q and R162A mutants were constructed using
the Stratagene QuikChange system, with the DAOCS
gene subcloned into the PCR-Script vector Primers
(0.1 nmol) (Table 1) were made in complementary pairs
and extended in the 5¢ to 3¢ direction The R160Q,
R306L and R307Q mutants were constructed by the
Kunkel method [14,15] The remaining mutants were
constructed using the unique site elimination (USE)
system [16] Automated DNA sequencing (Department of
Biochemistry, University of Oxford, UK) before
sub-cloning into the pET11a or pET24a vectors confirmed
the sequences of all mutants The required plasmids were
transformed into Escherichia coli XL1 Blue and E coli
BL21 (DE3) and expressed and grown as previously
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 mM 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 mMTris/HCl, 1 mMEDTA,
pH 7.5 and 2 mMdithiothreitol (20 mL) and treated with lysozyme (6 mg) The sample was stirred for 10 min before addition of MgCl2 (5 mM) and Dnase 1 (20 lg) After a further 10 min stirring, the sample was sonicated (4· 20 s) and centrifuged at 24 000 g for 20 min The supernatant was filtered through a 0.22-lm filter and loaded onto a HiPrep Q-Sepharose (16/10) XL column pre-equilibrated with the above buffer at 5 mLÆmin)1 Protein was eluted using a 120–400 mMNaCl gradient over 80 mL Fractions (5 mL) containing DAOCS were pooled and (NH4)2SO4solution added to a final concentration of 1.2M The sample was filtered, loaded onto a RESOURCE-PHE column equili-brated with the above buffer and 1.2M (NH4)2SO4 at
3 mLÆmin)1, and protein eluted with a 0.96–0.36M (NH4)2SO4gradient over 120 mL Fractions (5 mL) were analysed by SDS/PAGE following trichloroacetic acid precipitation Purified protein was exchanged into 50 mM 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 mMpenicillin N,
10 mMpenicillin G or water as substrates Assays based on the detection of deacetoxycephem products used the repor-ted HPLC system [1] Assay mixtures containing penicil-lin G were purified by HPLC with a Hypersil C4 column (250· 4.6 mm) using 5 mL of 25 mMNH4HCO3in 15% (v/v) methanol at 1 mLÆmin)1, followed by a gradient to
25 mM NHHCO in 80% (v/v) methanol over 20 mL
Table 1 Primers usedto construct DAOCS mutants Bold residues denote the mutation site USE, unique site elimination Selection primers: (pstI–ncoI) 5¢-CGTGACACCACGATGC*CATGGGCAATGGCAACAACG-3¢; (ncoI–pstI) 5¢-CGTGACACCACGATGCCTGCA*GCAA TGGCAACAACG-3¢ *denotes cleavage site for selection primers PCR, Stratagene QuikChange Mutagenesis Kit Kunkel, Method developed by Kunkel [14,15].
R74I 5¢-CCCGTCCCCACCATGATCCGCGGCTTCACCGGG-3¢ USE R74Q 5¢-CCCGTCCCCACCATGCAGCGCGGCTTCACCGGG-3¢ USE R75I 5¢-CCCGTCCCCACCATGCGCATCGGCTTCACCGGG-3¢ USE R75Q 5¢-CCCGTCCCCACCATGCGCCAGGGCTTCACCGGG-3¢ USE
R162A 5¢-CGCTGCTGCGGTTCGCATACTTCCCGCAGGTC-3¢ PCR R162Q 5¢-CGCTGCTGCGGTTCCAATACTTCCCGCAGGTC-3¢ PCR R266I 5¢-AGTGTGTTCTTCCTCATCCCCAACGCGGACTTC-3¢ USE R266Q 5¢-AGTGTGTTCTTCCTCCAGCCCAACGCGGACTTC-3¢ USE
Trang 3Retention volumes for product and substrate were 18.3 and
19.5 mL, respectively Approximately 0.15 mg of enzyme
was used in each 100 lL assay 1H 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
R E S U L T S
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
purified to 85% purity (by SDS/PAGE analyses) using
anion-exchange and gel filtration or anion–exchange and
hydrophobic interaction chromatographies Circular
di-chroism analyses of the mutants suggested that no gross
changes in structure had occurred compared to the
wild-type enzyme
Enzyme assays
Wild-type DAOCS and all mutants were assayed for their
ability to convert 2-oxoglutarate to succinate and carbon
dioxide [17], and penicillin N and penicillin G to their corresponding deacetoxycephem products [1,18] (Table 2) The levels of 2-oxoglutarate conversion were corrected for the rate observed in the absence of a penicillin substrate, i.e these results represent stimulation of 2-oxoglutarate con-version Steady-state kinetic analyses were carried out on wild-type enzyme and those mutants catalysing significant penicillin oxidation
Arginines 74, 75 and 266
In the case of the R74I, R74Q and R75Q mutants (Table 2, entries 2, 3 and 5), 2-oxoglutarate conversion was signifi-cantly stimulated by the presence of penicillin N (> 25%, the level of wild-type DAOCS), indicating that substrate binding occurs The slightly lower levels of penicillin N oxidation compared to 2-oxoglutarate conversion may be indicative of partial uncoupling of the two reactions More detailed kinetic analyses of penicillin N conversion by the arginine 74 and 75 mutants (Table 3) showed that kcatand
Kmvalues 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 prime 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 (m M ) k cat (s)1) k cat /K m ( M )1 Æs)1) Penicillin N
Wild-type 0.033 ± 0.016 0.020 ± 0.007 606 R74I 0.033 ± 0.007 0.010 ± 0.002 303 R74Q 0.025 ± 0.010 0.006 ± 0.001 240 R75I/D270G 0.086 ± 0.020 0.060 ± 0.010 697 R75Q 0.065 ± 0.040 0.030 ± 0.010 461 R306L 0.056 ± 0.008 0.160 ± 0.027 2857 R307Q 0.027 ± 0.003 0.070 ± 0.004 2592 Penicillin G
Wild-type 0.7 ± 0.1 0.050 ± 0.006 71 R306L 0.6 ± 0.1 0.070 ± 0.003 116 R307Q 7.0 ± 0.7 0.060 ± 0.010 8
Table 2 Activity assay results for wild-type and mutant DAOCS with
penicillin N andpenicillin G Assays for 2-oxoglutarate conversion [17]
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)1Æmg)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
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
Trang 4substrate was present Similarly, 2-oxoglutarate conversion
was not stimulated by penicillin substrates in the R74I/
R266I, R74Q/266I or R74Q/R266Q mutants (Table 2,
entries 6–9) These results suggest penicillin binding has
been abolished or severely affected in the arginine-266
mutants
Arginines 160 and 162
No stimulation of 2-oxoglutarate conversion was observed
in the presence of penicillin G for the R160Q, R162Q and
R162A mutants (Table 2, entries 10–12), consistent with
loss of penicillin binding
Arginines 306 and 307
The specific activity analyses suggested that mutation of
Arg306 to leucine, and to a lesser extent Arg307 to
glutamine, enhanced penicillin N conversion compared to
the levels of wild-type DAOCS (Table 2, entries 15–16)
However, no enhancement in activity compared to
wild-type levels was observed when penicillin G was used as a
substrate (Table 2, entries 15–16) Steady-state kinetic
analyses (Table 3) showed that kcat for penicillin N was
increased for both the R306L and R307Q mutants with
smaller changes in the Kmvalue The R306L mutation had
little effect on kinetic values using penicillin G as substrate
(Table 3), whereas the R307Q mutation increased the Km
value by 10-fold, but had little effect on the kcatvalue Note
the increased specific activity for the R306L mutant may
reflect a decreased rate of inactivation for this mutant
D I S C U S S I O N
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 uncouppenicil-ling 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 prime
substrate binding, but not necessarily of its oxidation
The effects of the DAOCS arginine mutations reported in
this paper fall into three types Some mutations prevent or
seriously hinder productive penicillin (prime) substrate
binding to the enzyme (arginines 160, 162 and 266), as demonstrated by the lack of stimulation of 2-oxoglutarate conversion and the almost complete absence of any deacetoxycephem product Other mutants, e.g R74Q, apparently bind the penicillin substrate sufficiently well to stimulate 2-oxoglutarate conversion, but probably not in the optimal manner for penicillin oxidation leading to some
uncoupled turnover This is clear when penicillin G is used
as substrate, and may be a consequence of the higher Km value for this substrate [1,22] The third type of mutant, represented by arginines 306 and 307, are located in the C-terminus They appear to modulate the activity of the C-terminus, which encloses the DAOCS active site during catalysis [12] and prevents premature loss of the penicillin substrate before oxidation
Penicillin substrate binding to DAOCS The results in this paper give support to the proposed mode of penicillin binding by DAOCS [1,12] a key feature
of which is that arginines 160 and 162 bind to the carbonyl and/or carboxylate of the bicyclic b-lactam nucleus, with the pro-S methyl group projecting towards the iron (Fig 1) We also propose that Arg266 forms a salt bridge with the carboxyl group of the a-aminoadipoyl- side-chain
of penicillin N (1), with arginines 74 and 75 apparently playing a less important role in side-chain binding Taken 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 prime 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.
Trang 5haps 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
A C K N O W L E D G E M E N T S
We thank Dr R T Aplin for mass spectrometric analyses and Mrs E.
McGuinness and Dr B Odell for NMR analyses Mrs W J Sobey is
thanked for technical assistance The Biotechnology and Biological
Sciences Research Council, Engineering and Physical Sciences Research
Council, Medical Research Council, the Wellcome Trust and the
European Union are thanked for financial assistance.
R E F E R E N C E S
1 Lloyd, M.D., Lee, H.-J., Harlos, K., Zhang, Z.H., Baldwin,
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active site of deacetoxycephalosporin C synthase J Mol Biol.
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2 Schofield, C.J., Baldwin, J.E., Byford, M.F., Clifton, I.J., Hajdu, J.
& Roach, P.L (1997) Proteins of the penicillin biosynthesis
pathway Curr Opin Struct Biol 7, 857–864.
3 Baldwin, J.E & Schofield, C.J (1992) The Chemistry of b-lactams.
Blackie Academic and Professional, London.
4 Prescott, A.G & Lloyd, M.D (2000) The iron (II) and
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8 Townsend, C.A., Theis, A.B., Neese, A.S., Barrabee, E.B &
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the ring expansion of penicillin N to deacetoxycephalosporin C.
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9 Pang, C.P., White, R.L., Abraham, E.P., Crout, D.H.G., Lutstorf,
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11 Valega˚rd, K., Terwisscha van Scheltinga, A.C., Lloyd, M.D.,
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12 Lee, H.-J., Lloyd, M.D., Harlos, K., Clifton, I.J., Baldwin, J.E & Schofield, C.J (2001) Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS) J Mol Biol 308, 937–948.
13 Lee, H.-J., Lloyd, M.D., Clifton, I.J., Harlos, K., Dubus, A., Baldwin, J.E., Frere, J.-M & Schofield, C.J (2001) Probing the cosubstrate selectivity of deacetoxycephalosporin C synthase: The role of arginine-258 J Biol Chem 276, 18290–18295.
14 Kunkel, T.A (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection Proc N atl Acad Sci USA 82, 488– 492.
15 Kunkel, T.A., Roberts, J.D & Zakour, R.A (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection Methods Enzymol 154, 367–382.
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18 Baldwin, J.E., Adlington, R.M., Coates, J.B., Crabbe, M.J.C., Crouch, N.P., Keeping, J.W., Knight, G.C., Schofield, C.J., Ting, H.H., Vallejo, C.A., Thorniley, M & Abraham, E.P (1987) Purification and initial characterization of an enzyme with dea-cetoxycephalosporin C synthetase and hydroxylase activities Biochem J 245, 831–841.
19 Myllyharju, J & Kivirikko, K.I (1997) Characterization of the iron and 2-oxoglutarate-binding sites of prolyl 4-hydroxylase EMBO J 16, 1173–1180.
20 Zhou, J., Gunsior, M., Bachmann, B., Townsend, C.A & Solo-man, E.I (1998) Substrate binding to the a-ketoglutarate-dependent non-haem iron enzyme clavaminate synthase 2: Coupling mechanism of oxidative decarboxylation and hydroxy-lation J Am Chem Soc 120, 13539–13540.
21 Zhang, Z.-H., Ren, J., Stammers, D.K., Baldwin, J.E., Harlos, K.
& Schofield, C.J (2000) Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase Nat Struct Biol 7, 127–133.
22 Dub us, A., Lloyd, M.D., Lee, H.-J., Schofield, C.J., Baldwin, J.E.
& Frere, J.-M (2001) Substrate selectivity studies on deacetox-ycephalosporin C synthase using a direct spectrophotometric assay Cell Mol Life Sci 58, 835–843.
23 Liu, A., Ho, R.Y.N., Que, L., Ryle, M.J., Phinney, B.S & Hausinger, R.P (2001) Alternative reactivity of an alpha-ketoglutarate-dependent iron (II) oxygenase: enzyme self hydro-xylation J Am Chem Soc 123, 5126–5127.
24 Lee, H.-J., Schofield, C.J & Lloyd, M.D (2002) Active site mutations of recombinant deacetoxycephalosporin C synthase Biochem Biophys Res Commun 292, 66–70.