A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans

Appl Microbiol Biotechnol (2010) 86:163–175 DOI 10.1007/s00253-009-2234-y

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans Stephen G. Bell & Alison Dale & Nicholas H. Rees & Luet-Lok Wong

Received: 8 July 2009 / Revised: 14 August 2009 / Accepted: 28 August 2009 / Published online: 25 September 2009 # Springer-Verlag 2009

Abstract Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized β-ionone to predominantly 3-R-hydroxy-β-ionone and 4-hydroxyβ-ionone, respectively. CYP111A2 oxidized linalool to 8hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo wholecell substrate oxidation systems that could produce up to 6.0 gL−1 of 5-exo-hydroxycamphor at rates of up to 64 μM (gram of cell dry weight)−1 min−1. These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.

Electronic supplementary material The online version of this article (doi:10.1007/s00253-009-2234-y) contains supplementary material, which is available to authorized users. S. G. Bell (*) : A. Dale : N. H. Rees : L.-L. Wong Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK e-mail: [email protected]

Keywords Cytochrome P450 . Novosphingobium aromaticivorans . Electron transfer . Ferredoxin reductase . Whole-cell biotransformations

Introduction Cytochrome P450 (CYP) enzymes are heme-dependent monooxygenases that catalyze the insertion of an oxygen atom from atmospheric dioxygen into carbon-hydrogen bonds in a diverse range of organic compounds (Ortiz de Montellano 2005; Sigel et al. 2007). Dioxygen activation requires two electrons (Eq. 1) that are commonly derived from reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and transferred to the P450 enzyme by electron transfer proteins (Hannemann et al. 2007; Munro et al. 2007a, b). R
H þ O2 þ Hþ þ NADðPÞH ! R
OH þ H2 O þ NADðPÞþ

ð1Þ

The C–H bond oxidation activity of P450 enzymes has many physiological roles including hormone and secondary metabolite biosynthesis as well as xenobiotic metabolism and degradation (Guengerich 2001a, b; Cryle et al. 2003; Isin and Guengerich 2007). The identification and characterization of efficient electron transfer chains for P450 enzyme systems will enhance their potential for use in, for example, environmental biotechnology and biohydroxylation for synthesis. Microorganisms with broad degradative properties are rich sources of new enzyme systems and metabolic pathways. Novosphingobium aromaticivorans is a gramnegative, aerobic, and oligotrophic bacterium that can degrade a wide variety of polyaromatic hydrocarbons (Fredrickson et al. 1991; Romine et al. 1999a, b; Shi et

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al. 2001; Janikowski et al. 2002). It has also been implicated in initiating primary biliary cirrhosis (Selmi and Gershwin 2004; Bogdanos and Vergani 2009). The N. aromaticivorans genome consists of the chromosomal DNA (3.56 Mbp) and two circular plasmids (pNL1, 180 kbp and pNL2, 480 kbp; Fredrickson et al. 1999). The sequence of pNL1 has been published and the sequencing of the chromosomal DNA and pNL2 is complete (Romine et al. 1999a, b; Copeland et al. 2006). The pNL1 plasmid contains a high proportion of genes encoding proteins involved in the catabolism and transport of aromatic compounds, including many oxygenase enzymes (Fredrickson et al. 1999; Romine et al. 1999a, b; Pinyakong et al. 2003; Basta et al. 2005). Eleven potential CYP genes are located on the chromosomal DNA with a further five on the pNL2 plasmid. We recently reported the cloning, expression, and purification of twelve of the sixteen potential CYP enzymes from N. aromaticivorans (Bell and Wong 2007). A [2Fe-2S] ferredoxin Arx (Saro_1477) that was genetically associated with CYP101D2 (Saro_1478) was also produced heterologously in Escherichia coli. Putidaredoxin reductase (PdR) of the CYP101A1 (P450cam) system from Pseudomonas putida was used successfully to mediate Arx reduction by NADH. This hybrid PdR/Arx class I electron transfer chain reconstituted monooxygenase activity of the P450 enzymes CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2, albeit at relatively low catalytic product formation rates of 90–170 nmol (nmol-P450)−1 min−1. CYP101D1 and CYP101D2 were found to catalyze the oxidation of camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized β-ionone while CYP111A2 was a linalool hydroxylase. The majority of characterized bacterial P450 systems are three-component class I electron transfer chains (reductase/redoxin/P450) which are also found in mitochondrial P450 systems (Hannemann et al. 2007; Munro et al. 2007a, b). However, the orphaned nature of the majority of the CYP genes in N. aromaticivorans is commonly encountered. Close clustering of the CYP gene with the genes encoding the electron transfer proteins, such as those found for CYP101A1, CYP176A1, and CYP108D1, is the exception rather than the rule (Koga et al. 1989; Peterson et al. 1992, Zotchev and Hutchinson 1995; Hawkes et al. 2002; Chun et al. 2007). For instance, of the 174 CYP genes from 45 different Streptomyces species, only 18 are clustered with a ferredoxin (Parajuli et al. 2004). In these organisms, genes encoding all possible electron transfer proteins could be expressed and the proteins assessed for activity (Chun et al. 2007). Alternatively known reductase/redoxin systems can be recruited to reconstitute the activity in vitro and in vivo but such

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cross-reactions between different P450 electron transfer chains are often slow (Agematu et al. 2006; Momoi et al. 2006; van Beilen et al. 2006). In an effort to discover and characterize new class I electron transfer chains, we searched the genome of N. aromaticivorans for genes that encode proteins with high amino acid sequence homology to PdR. Here, we report the cloning and expression of one such gene (ArR) in E. coli and the production and purification of the recombinant flavin-dependent ferredoxin reductase ArR. The catalytic activities of the ArR/Arx/CYP systems were found to be up to five times higher than the hybrid PdR/ Arx/CYP systems. Whole-cell substrate oxidation systems expressing ArR, Arx and a N. aromaticivorans P450 enzyme have been constructed, enabling in vivo preparative scale reactions for product isolation and characterization and paving the way to potential biotransformation applications.

Materials and methods General The genomic DNA of N. aromaticivorans (ATCC 700278D5) was obtained from ATCC-LGC Promochem, UK. The pCWori+vector was from Richard Dalquist (University of Oregon, USA) and Roland Wolf (University of Dundee, UK). The pET28a vector was from Merck Biosciences. Enzymes for molecular biology were from New England Biolabs, UK. KOD polymerase (Merck Biosciences, UK) was used for the polymerase chain reaction (PCR) steps. General DNA and microbiological experiments were carried out by standard methods. General reagents were from Sigma/Aldrich or Merck, UK. NADH and yeast alcohol dehydrogenase were from Roche Diagnostics, UK. Isopropyl-β-D-thiogalactopyranoside (IPTG), dithiothreitol (DTT) and buffer components were from Melford Laboratories, UK. K3[Fe(CN)6], 2,6dichrolophenolindophenol (DCIP) and organic substrates were from Sigma-Aldrich. Proteins were stored at −20°C in 50 mM Tris, pH7.4, containing 50% v/v glycerol. Glycerol was removed immediately before use by gel filtration on a 5-mL PD-10 column (GE Healthcare, UK) by eluting with 50 mM Tris, pH 7.4. UV/Vis spectra and spectroscopic activity assays were recorded at 30±0.5°C on a Varian CARY-50 or CARY-1E spectrophotometer. Gas chromatography (GC) analyses were performed on a ThermoFinnegan Trace GC instrument equipped with an auto-sampler and a DB-1 fused silica column (7 m×0.25 mm) or a CP-SIL 8CBfused silica column (15 m×0.32 mm; Varian) using helium as the carrier gas and flame ionization detection. The

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injector and flame ionization detector were held at 200°C and 250°C, respectively. Nuclear magnetic resonance (NMR) spectra were acquired on a Varian Unity plus spectrometer operating at 500 MHz (1H) and 125 MHZ (13C). Electrospray protein mass spectrometry was carried out on a Micromass Platform II instrument. Enzymes and molecular biology The ArR gene (Saro_0216) was amplified by 25 cycles of strand separation at 95°C for 1 min followed by annealing at 42°C and extension at 68°C for 2 min. The oligonucleotides used were as follows (the Nde I and Hind III restriction sites introduced for cloning into the multiple cloning site region of the pCWOri+vector are underlined): 5′- ttaattcatatggccagcgaagttcaggcag-3′ and 5′-ttaattaagct tattggatccctaggccagcatctccttgagc-3′. The amplified gene was incorporated into the pCWOri+vector and fully sequenced by automated DNA sequencing on an ABI 3730 DNA Analyser by the Geneservice DNA sequencing facility at the Department of Biochemistry, University of Oxford. The PCR and cloning of the CYP and Arx genes have been reported elsewhere (Bell and Wong 2007). Protein expression and purification Recombinant ArR was produced in E. coli DH5α harboring a pCWori+plasmid containing the ArR gene and purified using a method similar to those used for PdR from P. putida (Peterson et al. 1990). A single colony was inoculated into 200 mL Luria-Bertani broth (LB) containing 100 μg mL−1 carbenicillin (LBcarb) and grown at 37°C overnight. This culture was then inoculated at a ratio of 25 mL L−1 into 6× 1-L of LBcarb medium containing 0.4% v/v glycerol, and grown at 37°C for 4–6 h to OD600 ∼1. Recombinant protein production was induced by the addition of 0.4 mM IPTG (from a 1 M stock in water). The incubator temperature was lowered to 30°C, and the culture was then grown for at least another 6 h. The cell pellet harvested from the culture by centrifugation was resuspended in 200 mL buffer T (50 mM Tris, pH 7.4, 1 mM DTT) and the cells were lysed by sonication. Cell debris was removed by centrifugation at 37,000g for 30 min at 4°C. The supernatant was loaded on to a DEAE Fastflow Sepharose column (200 mm×50 mm; GE Healthcare) and the protein eluted using a linear salt gradient of KCl (50–250 mM) in buffer T developed over eight column bed-volumes at a flow rate of 8 mL min–1. The yellow protein-containing fractions were collected and concentrated by ultrafiltration. The protein was desalted using multiple cycles of ultrafiltration. The final purification step was anion-exchange chromatography using a Source-Q column (120 mm×26 mm; GE Healthcare) and eluting with a linear gradient of 50–200 mM KCl

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in buffer T developed over 20 column bed-volumes at a flow rate of 8 mL min−1. Purified fractions (A280/A458 95% and 20% high spin, respectively) while CYP111A2, like CYP111A1 (P450lin), showed >95% high spin heme upon linalool binding (Bell and Wong 2007). The substrate dissociation constant (Kd) for the various substrate/CYP enzyme combinations was determined by titrating the enzymes with substrate (Fig. S5). The substrate binding was found to be tighter for those substrates which induced the greater spin-state shift (e.g., β-ionone/ CYP101B1 and linalool/CYP111A2; Table 1). When the value for Kd becomes comparable to that of the enzyme concentration (e.g., linalool/CYP111A2 and β-ionone/ CYP101B1) the hyperbolic function becomes an inaccurate model with which to estimate Kd and the data were analyzed using the tight-binding quadratic equation (Eq. 3). When ArR was used as the ferredoxin reductase with Arx to reconstitute monooxygenase activity with these P450 enzymes the steady-state turnover rates increased by 50% to 400% over those observed previously with PdR (Table 1). The efficiency of NADH utilization for product formation in all the reactions was high, ranging from 60% to 95%. There was no obvious relationship between the spin-state shift or substrate dissociation constant and the rates and efficiency of product formation (Table 1). The ArR/Arx system supported very slow NADH turnover and gave little or no product formation with the other CYP enzymes from N. aromaticivorans for which

Table 1 Substrate binding and catalytic activity of various CYP enzymes from Novosphingobium aromaticivorans using the ferredoxin reductases ArR and PdR P450 enzyme/ substrate

Spin-state shift

Binding constant

ArR/Arx NADH consumption rate

CYP101D2/ camphor CYP101D1/ camphor CYP101D1/2adamantanone CYP101B1/βionone CYP101C1/βionone CYP111A2/linalool CYP101A1/ camphora

PdR/Arx Product formation rate

Coupling NADH consumption rate

Product formation rate

Coupling

40%

3.09±0.13 745±46

741±28

97%

156±4.6

153±4.5

96%

40%

6.0±0.29 431±2.0

408±5.0

90%

157±9.0

131±8.6

84%

60%

5.39±0.37 427±19

386±13

90%

159±9.7

128 ±7.0

81%

144±2

69%

154±6.8

92.2±4.7

60%

429±10

282±26

66%

239±2.9

174±4.8

73%

0.264±0.04 676±35 0.45±0.05 754±16

463±31 753±33

68% 100%

195±22 24.6±3.1

136±11.7 0.7±0.1

69% 3%

≥95% 20% ≥95% 100%

0.086±0.03 209±8 44.8±4.9

Rates are reported as mean±S.D. (n≥3) and given in nmol (nmol-CYP)−1 min−1 . Coupling is the percentage efficiency of NADH utilization for the formation of products. The background consumption rates in the absence of substrate were 25.7±2.7 for ArR/Arx and 11.2±0.9 for PdR/Arx a

From Westlake et al. Eur. J. Biochem. 265, 929–935 (1999). The activity data shown is for PdR/Pdx and ArR/Pdx, respectively

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substrate binding has been observed. For example, CYP108D1 (α-terpineol, 40%; phenanthrene, 90%), CYP153C1 (octane, 80%), and CYP219A1 (isolongifolen9-one, >95%) gave no product formation with the ArR/Arx or the PdR/Pdx electron transfer chains (data not shown; Bell and Wong 2007). Product formation The products of camphor and 2-adamantanone oxidation by CYP101D1 and CYP101D2 had been identified as 5-exohydroxycamphor and 5-hydroxy-2-adamantanone (Fig. 2; Bell and Wong 2007). CYP101B1 and CYP101C1 showed lower activity and selectivity with camphor. CYP101C1 oxidized β-ionone to 4-hydroxy-β-ionone (75%) with one other, unidentified product. This latter compound was the major product of β-ionone oxidation by CYP101B1 (90%) where 4-hydroxy-β-ionone was the minor product (10%; Fig. 2; Bell and Wong 2007). The stereochemistry at the four positions of the 4-hydroxy-β-ionone product from both these CYP101 enzymes was shown to be single

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isomer and the same as the previously characterized product of β-ionone oxidation by the A74G/F87V/L188Q mutant of CYP102A1 (P450BM-3) from Bacillus megaterium (Urlacher et al. 2006) by GC coelution on a chiral phase column. The unidentified product from linalool oxidation by CYP111A2 and β-ionone oxidation by CYP101B1 and CYP101C1, were purified from preparative scale reactions and characterized by GC-MS (Bell and Wong 2007) and NMR spectroscopy. The ArR/Arx electron transfer chain was used to reconstitute an in vitro substrate oxidation system. Yeast alcohol dehydrogenase and ethanol were used to regenerate the NADH cofactor. The products were extracted from the supernatant using dichloromethane and purified by silica column chromatography. The efficacy of the systems were high, typically a total of >50 mg of purified products were obtained from a 200-mL reaction mixture containing 100 nM CYP enzyme, which corresponded to total turnover numbers (TTN) of >10,000. One reason for the high TTNs is the benign nature of the substrates and products. Higher TTNs may be possible with the use of a second organic phase that allows product removal (Maurer et al. 2005; Schewe et al. 2009). The product distributions in these preparative scale turnovers were identical to those observed in the activity assays, with no evidence of further oxidation of the initial products, indicating that the enzymes were highly selective as well as active catalysts. The second β-ionone oxidation product was identified as 3R-hydroxy-β-ionone by a combination of the TOCSY and nuclear Overhauser enhancement spectroscopy (NOESY) spectra. The regiochemistry was readily assigned by tracing the coupling pattern and the stereochemistry was established from the NOESY spectrum (Scheme 1). The product of linalool oxidation by CYP111A2 was readily identified from the 1H NMR spectrum as 8-hydroxy-linalool by the lower intensity of the methyl resonance indicating the loss of one of the methyl groups and the appearance of a twoproton resonance at 4.12 ppm assigned to the CH2 group of the allylic alcohol (Fig. 2). The 1H and 13C spectra were fully assigned and consistent with 8-hydroxy-linalool (Cuvigny 1988). Hence, CYP111A2 shows the same selectivity as CYP111A1 (P450lin; Ullah et al. 1990). In vivo substrate oxidation

Fig. 2 Substrates utilized and products formed in this work by the various CYP enzymes of N. aromaticivorans

In order to investigate the feasibility of using these P450 enzymes for synthesis via biotransformation, polycistronic ArR/Arx/CYP101 expression systems were constructed using commercially available vectors (Fig. 3). Two vectors (pRSFDuet-1 and pETDuet-1) were used, each expressing two genes (ArR and Arx in pETDuet-1, and Arx and the CYP in pRSFDuet-1). One copy of the Arx gene was

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Scheme 1 The pattern of nOes observed in the NOESY spectrum of the 3-hydroxy-β-ionone product

included on each plasmid to increase the production of the ferredoxin protein relative to the CYP enzyme in the cells and maximize the turnover activity of the in vivo systems. In this two-vector approach, each gene has its own promoter and this should overcome the lower expression and activity of the tricistronic system we reported for CYP101A1 (Bell et al. 2001) and for other similar systems described recently (Kim and Ortiz de Montellano 2009). The pETDuet-Arx-ArR and one of the relevant pRSFDuet-Arx-CYP plasmids were transformed into E. coli hosts, and the cells were grown and proteins overproduced as described in the experimental section. All four of the P450 enzyme systems tested (CYP101B1, CYP101C1, CYP101D1, and CYP101D2) produced 5 to 6 gL−1 of a dark red-brown cell pellet in 2YT media indicating successful expression of the ferredoxin and P450 components of the system (there was some blue color in cells expressing CYP101B1, presumably due to indigo formation (Gillam et al. 1999; Bell et al. 2001)). The P450 concentration was quantitated by measuring the ferrous-CO spectra of the cell lysate. All four CYP enzymes were expressed to good levels (per liter culture: CYP101B1, 29 mg; CYP101C1, 16 mg; CYP101D1, 44 mg; and CYP101D2, 30 mg). The expression levels of the ArR and Arx enzymes are more difficult to measure but using the pETDuet-Arx-ArR and pRSFDuet-Arx plasmids, an estimate of the Arx concentration of ∼3 μM was obtained which is more than double the concentration of the P450 component in the cell (0.4–1µM). The harvested cell pellets were resuspended in buffered EMM and substrate (4 mM camphor for CYP101D1 and CYP101D2 or 2 mM β-ionone for CYP101B1 and CYP101C1) was added. Samples were removed periodically and monitored by GC for substrate consumption and product formation. When all the substrate had been consumed, further aliquots of substrate were added. Both the CYP101D1/Arx/ArR and CYP101D2/Arx/ArR systems were capable of rapidly oxidizing camphor to 5-

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exo-hydroxycamphor. A 500-mL suspension of 2.4–2.9 g of cell wet weight (equivalent to 0.50–0.61 g of cell dry weight (cdw)) containing either of these systems was found to oxidize 4 mM of camphor completely within 50 min. When all of the camphor had been converted, the 5-exohydroxycamphor product was further oxidized to 5oxocamphor. Time-course analysis by GC showed that the rates of 5-exo-hydroxycamphor formation were approximately 64 and 56 μM (gram of cdw)−1 min−1 for the CYP101D1 and CYP101D2 systems, respectively (Fig. 4). The addition of further aliquots of 4 mM camphor after 2 and 4 h, and 2 g of solid camphor (to each 500 ml) after 6 h led to a total product formation (5-exo-hydroxycamphor and 5-oxocamphor) of up to 21 and 38 mM after 20 h for the CYP101D1 and CYP101D2 systems, respectively (Fig. 5). This corresponds to approximately 3.5 and 6.3 g L−1 of total product, respectively. The CYP101D2 system had converted all of the camphor substrate added (∼38 mM) into product in the 20-h time period of the whole-cell reaction. The CYP101D2 system produced 6.0 gL−1 of 5exo-hydroxycamphor with the remaining product being 5oxocamphor. These systems are more active than other polycistronic and fusion protein systems that have been reported in the literature using CYP101A1 (Sibbesen et al. 1996; Bell et al. 2001; Mouri et al. 2006; Robin et al. 2009). The CYP101B1/Arx/ArR and CYP101C1/Arx/ArR systems oxidized β-ionone to predominantly 3-hydroxy-βionone and 4-hydroxy-β-ionone, respectively. However, further oxidation of the products was prevalent, particularly for the CYP101C1 system. The rate of product formation for the CYP101B1 system was ∼3.2 μM (g of cdw)−1 min−1 while the rate with the CYP101C1 was ∼1.1 μM (g of cdw)−1 min−1. After 20 h, ∼750 μM (0.16 gL−1) of product was detected in the CYP101C1 system and ∼4 mM (0.83 g L−1) in the CYP101B1 system. The slower rates of βionone turnovers cannot be rationalized in terms of the amount of proteins in the cells or the in vitro activity of the enzymes. However, the solubility of β-ionone is much lower in water than that of camphor (48 mg L−1 vs. 1.2 g L−1) which may affect the amount of substrate that can be transferred into E. coli through the cell membrane. The higher solubility of the 3-hydroxy-β-ionone and 4hydroxy-β-ionone products than the β-ionone substrate may account for the greater degree of further oxidation in the β-ionone turnovers compared with the camphor turnovers.

Discussion The P450 enzymes of the CYP101 and CYP111 families of N. aromaticivorans are able to oxidize terpenoid

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Fig. 3 Construction of the in vivo expression system

compounds. A broad range of substrates including other terpenoid compounds (CYP219A1), alkanes (CYP153C1), polycyclic aromatic hydrocarbons, and substituted aromatics (CYP108D1, CYP203A2) are substrates for other P450 enzymes from this bacterium. These enzymes, along with the many other oxygenase and catabolic enzymes, may enable N. aromaticivorans to thrive in the oligotrophic environments in which it is found. We have shown that ArR (Saro_0216) is a FADdependent ferredoxin reductase that functioned as an efficient class I electron transfer chain with the [2Fe-2S]

ferredoxin Arx (Saro_1477) to support monooxygenase activity of all four CYP101 enzymes as well as CYP111A2 from N. aromaticivorans. This contrasts with the low cross-reactivity observed between the CYP101A1 and CYP111A1 (P450lin) electron transfer chains (Ullah et al. 1990; Bernhardt and Gunsalus 1992; Ropp et al. 1993). The five N. aromaticivorans enzymes, therefore, may be expected to have similar thermodynamic properties and ferredoxin recognition sites on the heme proximal face (Sequence alignments of the proteins are shown in Figs. S1, S2, and S3). Structural and thermodynamic

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173

CYP101D1 camphor

5000

CYP101D2 camphor 4000

CYP101B1 ionone CYP101C1 ionone

3000 2000 1000 0 0

50

100

150

200

250

Time (min)

Fig. 4 Product formation (μM (g of cdw)−1) of the ArR/Arx/CYP whole-cell systems: CYP101B1/β-ionone (filled triangle), CYP101C1/β-ionone (filled circle), CYP101D1/camphor (filled diamond), and CYP101D2/camphor (filled square)

characterization of these enzymes should provide insights into the ferredoxin/CYP interaction in class I P450 enzymes. It is not yet known if the physiological functions of these five CYP enzymes are linked (for example in a degradation pathway); the use of a single electron transfer system for a number of enzymes suggests that this is a possibility. The significance of the location of the CYP101B1 gene on pNL2 and of the ArR, Arx, CYP111A2 and the other CYP101 genes on the chromosome is as yet unknown. When ArR was used to support camphor oxidation by the Pdx/CYP101A1 system, the activity was almost two orders of magnitude lower than the reaction supported with PdR. Similarly, Pdx and Arx only supported slow camphor oxidation by CYP101D2 and CYP101A1, respectively (Supplementary data, Table S2; Bell and Wong 2007). Hence, despite the sequence similarities between the electron transfer proteins of the two systems, the low cross-reactivity showed that the ferredoxin reductase/ferredoxin and ferredoxin/CYP recognition interactions must be significantly different. The low cross-reactivity observed between the ArR/Pdx and PdR/Arx electron transfer proteins from N. aromaticivorans and P. putida contrasts with previous studies which showed a high retention of activity between PuR/Pdx and PdR/Pux enzymes from P. putida and Rhodopseudomonas palustris CGA009 (Bell et al. 2008). This highlights the importance of fully characterizing the electron transfer proteins of CYP enzymes from different bacteria in order to maximize the hydroxylation activity. The feasibility of using P450 enzymes for biotransformations depends on maximizing the catalyst lifetime whilst removing the need to use expensive biological cofactors. In vitro cofactor regeneration or replacement of the natural cofactors using chemical or electrochemical methods have all

been attempted with a variety of P450 enzymes, with different degrees of success (Bernhardt 2006; Chefson and Auclair 2006). Alternatively, all the proteins in a P450 enzyme system can be expressed in a single heterologous host to form a catalytically competent in vivo substrate oxidation system (Bell et al. 2001; Julsing et al. 2008). The rapid and selective conversion of camphor to 5-exo-hydroxycamphor by the CYP101D1 and CYP101D2 systems on a 3.2– 6.0 gL−1 scale in 20 h under nonoptimized conditions suggests that there may be potential for these class I electron transfer systems in synthetic biological hydroxylation. The lower oxidation rates with less soluble substrates such as β-ionone will prove more challenging and may require the addition of second organic phases and reagents which promote the solubility and the transfer of the substrate across the cell membrane (Meyer et al. 2006; Morrish et al. 2008). Nevertheless the formation of up to 0.83 gL−1 of β-ionone oxidation products is an encouraging first step. In summary, the ferredoxin reductase ArR from N. aromaticivorans mediates fast electron transfer to the [2Fe-2S] ferredoxin Arx to support monooxygenase activity of CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2, in all cases with high coupling efficiency for product formation. Structural, thermodynamic, and kinetic studies of ArR, Arx, and the P450 enzymes will provide new information on protein recognition and the electron transfer mechanism in class I P450 systems. In vivo systems expressing all three components of these systems have been constructed and shown to function efficiently and with high yields in E. coli whole-cell biotransformation reactions.

450000

5-exo-hydroxy camphor

400000

GC detector response

Product µM (g of cdw) −1

6000

time = 0 time = 20 min time = 60 min

350000

time =180 min

300000

time = 20 hr

250000 200000 150000 100000

camphor

5-oxocamphor

internal standard

50000 0 4.5

5.5

6.5

7.5

8.5

9.5

10.5

Time (min)

Fig. 5 Time course of the hydroxylation of camphor by the ArR/Arx/ CYP101D2 whole-cell system in EMM media; camphor RT 4.78 min, 5-exo-hydroxycamphor RT 7.16 min, 5-oxocamphor RT 6.11 min and 9-fluorenol 9.61 min

174 Acknowledgements This work was supported by the Higher Education Funding Council for England.

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