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 (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) Eur. J. Biochem. 269, 2735–2739 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02945.x EXPERIMENTAL PROCEDURES 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 Kb and 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 andextendedinthe5¢ 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 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 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 m M NaCl gradient over 80 mL. Fractions (5 mL) containing DAOCS were pooled and (NH 4 ) 2 SO 4 solution added to a final concentration of 1.2 M .Thesamplewas filtered, loaded onto a RESOURCE-PHE column equili- brated with the above buffer and 1.2 M (NH 4 ) 2 SO 4 at 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 M penicillin 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 m M NH 4 HCO 3 in 15% (v/v) methanol at 1 mLÆmin )1 , followed by a gradient to 25 m M NH 4 HCO 3 in 80% (v/v) methanol over 20 mL. Table 1. Primers used to 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]. Mutation Primer sequence Method R74I 5¢-CCCGTCCCCACCATGATCCGCGGCTTCACCGGG-3¢ USE R74Q 5¢-CCCGTCCCCACCATGCAGCGCGGCTTCACCGGG-3¢ USE R75I 5¢-CCCGTCCCCACCATGCGCATCGGCTTCACCGGG-3¢ USE 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 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 k cat and K 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 (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 and penicillin 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 )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 substrate 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 k cat for penicillin N was increased for both the R306L and R307Q mutants with smaller changes in the K m value. The R306L mutation had little effect on kinetic values using penicillin G as substrate (Table 3), whereas the R307Q mutation increased the K m value by 10-fold, but had little effect on the k 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 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 Ôuncoupled turnoverÕ. This is clear when penicillin G is used as substrate, and may be a consequence of the higher K m 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 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 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. REFERENCES 1. Lloyd, M.D., Lee, H J., Harlos, K., Zhang, Z.H., Baldwin, J.E., Schofield, C.J., Charnock, J.M., Garner, C.D., Hara, T., Terwisscha van Scheltinga, A.C. et al. (1999) Studies on the active site of deacetoxycephalosporin C synthase. J. Mol. Biol. 287, 943–960. 2. 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Biochem. 269) 2739 . 5¢-CCCGTCCCCACCATGATCCGCGGCTTCACCGGG-3¢ USE R74Q 5¢-CCCGTCCCCACCATGCAGCGCGGCTTCACCGGG-3¢ USE R75I 5¢-CCCGTCCCCACCATGCGCATCGGCTTCACCGGG-3¢ USE R75Q 5¢-CCCGTCCCCACCATGCGCCAGGGCTTCACCGGG-3¢. 5¢-CCCGTCCCCACCATGCGCCAGGGCTTCACCGGG-3¢ USE R160Q 5¢-TAGCGGAACTGCAGCAGCG-3¢ Kunkel R162A 5¢-CGCTGCTGCGGTTCGCATACTTCCCGCAGGTC-3¢ PCR R162Q 5¢-CGCTGCTGCGGTTCCAATACTTCCCGCAGGTC-3¢