Rebecca R. Chaoa,
James J. De Vossb and
Stephen G. Bell*a
aDepartment of Chemistry, University of Adelaide, SA 5005, Australia. E-mail: stephen.bell@adelaide.edu.au; Fax: +61 8 8303 4380
bSchool of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Qld 4072, Australia
First published on 3rd June 2016
The cytochrome P450 enzyme, CYP199A4, demethylated 4-methoxybenzoic acid, but not 4-methoxyphenylacetic acid, with high product formation activity. The oxidative demethylation of 3-(4-methoxyphenyl)propionic acid was 8-fold more active than 4-methoxyphenylacetic acid and 4-methoxycinnamic acid was efficiently oxidised at a product formation rate of 180 nmol nmol-P450−1 min−1. Accordingly the oxidation of cinnamic acid derivatives was investigated in order to determine the potential of CYP199A4 to act as a biocatalyst for this important class of biological molecules. 4-Methoxy- and 4-methyl-cinnamic acids bound tightly to CYP199A4 and were better substrates for CYP199A4 than cinnamic acid itself. The oxidations of both 4-methoxy- and 4-methyl-cinnamic acids was 100% selective for attack at the para substituent. Certain dimethoxy substituted cinnamic acids were demethylated more efficiently than 4-methoxycinnamic acid and retained the selectivity for the para-methoxy substituent. Only very low product turnover was observed with 3,5-dimethoxycinnamic acid. 4-Isopropylcinnamic acid was hydroxylated and desaturated by CYP199A4 at the isopropyl group. Cinnamic acids with a para-substituted alkyl- and alkyloxy–cinnamic acid framework were a good fit for the active site of the CYP199A4 enzyme and as a consequence were efficiently and selectively oxidised. Whole-cell oxidations resulted in high yields of product and CYP199A4 could be developed for applications in the biocatalytic oxidation of cinnamic acid derivatives and related phenylpropanoids.
Mechanistically, C–H bond hydroxylation occurs via hydrogen abstraction by compound I, followed by oxygen rebound to give the hydroxylated organic.2,9 O-Demethylation of 4-methoxybenzoic acid by CYP199A4 occurs via the same pathway, initially yielding a hemiacetal which spontaneously decomposes to yield 4-hydroxybenzoic acid and formaldehyde.2,6,10,11 The alkene desaturation product is hypothesised to arise from initial hydrogen atom abstraction followed by either a second hydrogen atom abstraction from an adjacent carbon or one-electron oxidation by compound II. The latter pathway generates a carbocation which then loses a proton to form the alkene.2,6
A detailed knowledge of the types of substrates that can bind in the enzyme active site and the interactions responsible for binding is of paramount importance in achieving the activity and turnover numbers required for synthetic applications.12–15 The stable, soluble nature and high activity of the CYP199A4 enzyme means it is well suited for larger scale processes involving C–H bond oxidation such as hydroxylation and oxidative demethylation.4,6,16–19 Altering the benzene ring, the carboxylate group or the removal of the methoxy substituent of 4-methoxybenzoic acid all reduced the substrate binding and monooxygenase activity of CYP199A4.4,6,20,21 Replacement of the methoxy group with alternate substituents is tolerated as are small additional functional groups on the benzene ring.6 We have shown that CYP199A4 can bind and O-demethylate 3,4-dimethoxybenzoic acid with total regioselectivity at the 4-methoxy group and demethenylate 3,4-methylenedioxybenzoic acid.4,20 Others have reported that the CYP199A subfamily member, CYP199A2 from R. palustris CGA009, which shares >85% sequence identity with CYP199A4, can catalyse the oxidation of larger substrates such as 2-naphthoic acid, indole-6-carboxylic acid and 3-hydroxy- and 4-hydroxy-cinnamic acids (m- and p-coumaric acids, respectively).4,16–18,22–24 Mutant forms of CYP199A2 have been shown to enhance the biocatalytic properties the enzyme, allowing the whole-cell oxidation of cinnamic acid, benzyl alcohols and phenols using an E. coli system.19 However, detailed in vitro analysis of the substrate binding interactions and enzyme activity with these substrates has not been reported.
Several crystal structures of substrate-free and substrate-bound forms of CYP199A4 and CYP199A2 have been determined.5,6,10 In the substrate-bound forms (PDB codes; 4DO1, 4EGM and 4EGN), the substituent para to the carboxylate is oriented towards the heme-iron which is consistent with exclusive attack at this position. The carboxylate group of the substrate interacts with the polar side chains of Arg92, Ser95, Ser244 and, via a bridging water molecule, Arg243 (Fig. 1). In addition, hydrophobic interactions with the benzene ring help to position the substrate. The crystal structures of CYP199A4 in complex with the larger indole-6-carboxylic acid and 2-naphthoic acid substrates have also been solved (PDB codes; 4EGO and 4EGP). In the indole-6-carboxylic acid-bound structure, the benzene rings, the carboxylate groups and the bridging water molecules are at virtually identical locations to those observed with 4-methoxybenzoic acid.5 Although 2-naphthoic acid is similar in size to indole-6-carboxylic acid, its crystallographically observed binding mode is significantly different. The carboxylate group is located ca. 1.9 Å closer to the N-terminal end of the I helix. The water molecule found in the 4-methoxybenzoic acid-bound form, which interacts with the substrate and Arg243, is displaced and the carboxylate group of 2-naphthoic acid interacts directly with Arg243.5 The hydrogen bonds between the carboxylate oxygen atom and the side chains of Ser95 and Ser244 are maintained. Together, these structures show that CYP199A4 is able to accommodate larger planar and acidic substrates.5 By way of contrast, it has been shown that small divergences from 4-methoxybenzoic acid structure can result in a dramatic reduction in the enzyme activity and in the affinity for the substrate. For example, the activity of CYP199A4 with 4-methoxyphenylacetic acid, which is non planar, is 240-fold lower than that with 4-methoxybenzoic acid and there is a three orders of magnitude reduction in the substrate binding affinity.21 This highlights the specificity of the hydrophilic interactions between the CYP199A4 enzyme and the aromatic acid group of the substrate.
Fig. 1 The active site of 4-methoxybenzoic acid-bound CYP199A4 (PDB code 4DO1). The amino acid residues which confer the substrate-specificity of the enzyme have been shown. 4-Methoxybenzoic acid is shown in yellow, the heme in grey and the amino acids in green. |
To further understand these interactions, we compare the binding and kinetic properties of CYP199A4 with selected cinnamic acid derivatives (Fig. 2). These cinnamic acids belong to the phenylpropanoid family of natural products and have a variety of biological activities and functions.25,26 Cinnamic acids have an unsaturated planar carboxylate containing side chain and will be compared with related non-planar substrates. The requirement of a para-substituent in these larger substrate and the effect of other substituents at the other locations of the benzene ring will also be investigated. The aim is to provide a better understanding of the structural features required for tight binding to this family of CYP enzymes. In addition the size and shape of substrates that can be efficiently oxidised by CYP199A4 will be determined which will facilitate its use as a biocatalyst on a synthetically useful scale.
Substrate | % HS | Kd/μM | NADH | PFRa | Cb % |
---|---|---|---|---|---|
a PFR: product formation rate.b C: the coupling efficiency; the percentage of NADH consumed in the reaction that led to the formation of products.c Not determined due to absence of spin state shift.d No significant NADH turnover activity was observed.e Minimal levels of product formation were observed (Fig. S3).f Determined at A370 due to interfering absorption of the substrate.g No significant product observed. | |||||
4-Methoxycinnamic acid | 70% | 3.6 ± 0.1 | 560 ± 10 | 180 ± 9 | 32 ± 1 |
Cinnamic acid | <5% | —c | 24 ± 1 | —e | —e |
3-(4′-Methoxyphenyl)propionic acid | 20% | 31 ± 1 | 98 ± 5 | 43 ± 3 | 43 ± 2 |
4-Methylcinnamic acid | 70% | 21 ± 0.4 | 202 ± 7 | 85 ± 1 | 42 ± 1 |
3-Hydroxycinnamic acid | <5% | —c | 9 ± 0.1 | —e | —e |
4-Methoxyphenylacetic acid21 | 5% | 690 ± 70 | 21 ± 1.5 | 5.0 ± 0.6 | 23 ± 2 |
4-Hydroxycinnamic acid | 0% | —c | 17 ± 0.5 | 0.6 ± 0.04 | 3.4 ± 0.2 |
3,4-(Methylenedioxy)cinnamic acid | 30% | 120 ± 2 | 51 ± 0.4f | 6.6 ± 0.2 | 13 ± 0.5 |
4-Isopropylcinnamic acid | >95% | 3.4 ± 0.1 | 190 ± 13 | 131 ± 10 | 69 ± 3 |
3,5-Dimethoxy-4-hydroxycinnamic acid | <5% | —c | 50 ± 2 | —g | —g |
3,5-Dimethoxycinnamic acid | <5% | —c | 22 ± 1 | 0.7 ± 0.02 | 3.3 ± 0.2 |
3-Methoxy-4-hydroxycinnamic acid | <5% | —c | —d | —g | —g |
3-Hydroxy-4-methoxycinnamic acid | 25% | 224 ± 13 | 408 ± 25 | 239 ± 5 | 59 ± 3 |
3,4-Dimethoxycinnamic acid | 20% | 840 ± 30 | 400 ± 20 | 302 ± 17 | 75 ± 4 |
2,4-Dimethoxycinnamic acid | 10% | 86 ± 4.5 | 441 ± 30 | 282 ± 25 | 64 ± 2 |
2,3,4-Trimethoxycinnamic acid | <5% | —c | 39 ± 2 | 24 ± 1 | 61 ± 3 |
3,4,5-Trimethoxycinnamic acid | <5% | —c | —d | —g | —g |
We have previously shown that a substituent para to the carboxylate moiety of benzoic acid was important for binding and efficient catalytic activity with both CYP199A2 and CYP199A4 (ref. 6, 20 and 22) and 4-methoxybenzoic acid was the optimal substrate for both with CYP199A4 (≥95%, spin state shift and a Kd of 0.28 μM).4,5,10 Accordingly, we tested the binding of 4-methoxycinnamic acid with CYP199A4. This substrate induced a spin state shift of 70% on binding to CYP199A4 and the dissociation constant was determined to be 3.6 μM (Table 1 and Fig. 3 and 4). By way of contrast, 4-methoxyphenylacetic acid binds poorly: spin state shift 5%; Kd 690 μM.21 3-(4-Methoxyphenyl)propionic acid induced a lower spin shift to the high-spin state (20%) and weaker binding (Kd, 31 μM) with CYP199A4 than both 4-methoxybenzoic and 4-methoxycinnamic acids. However the magnitude of this shift and the binding affinity is higher than is observed with 4-methoxyphenylacetic acid and cinnamic acid (Table 1, Fig. 3 and 4).
Due to the favourable results obtained with 4-methoxycinnamic acid, we tested a range of substituted cinnamic acids with CYP199A4 to better determine the optimal size and shape of the molecules which complement the substrate binding pocket (Fig. 2). Additional substitutions at the 2- and 3-positions of 4-methoxycinnamic acid tended to reduce the affinity for binding to CYP199A4. 3-Hydroxy-4-methoxy-, 3,4-dimethoxy, 2,4-dimethoxy- and 3,4-methylenedioxy-cinnamic acids all induced significantly lower spin state shifts upon addition to CYP199A4 and resulted in weaker binding when compared to 4-methoxycinnamic acid (Table 1 and Fig. S1†). 2,4-Dimethoxycinnamic acid bound the most tightly to CYP199A4 of any of the disubstituted substrates investigated despite possessing one of the lowest spin state shifts observed (Table 1). Substrates containing methoxy groups at the 3- or 5-positions with a hydrogen atom or a hydroxyl group at the 4-position such as 3,5-dimethoxycinnamic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid) resulted in little observable shift in the spin state upon binding to CYP199A4 (Table 1 and Fig. S1†). Two trimethoxy substituted cinnamic acids were tested with CYP199A4. Addition of neither 2,3,4-trimethoxycinnamic nor 3,4,5-trimethoxycinnamic acids induced a significant spin state shift (Table 1).
The effect of the size and type of the para substituent was also investigated with alkyl substituted cinnamic acids. Upon binding to CYP199A4, 4-methylcinnamic acid induced a similar spin state shift to 4-methoxycinnamic acid but the binding was 6-fold weaker (Table 1 and Fig. 3 and 4). The increased size of the alkyl para-substituent in 4-isopropylcinnamic acid resulted in the largest spin state shift observed, ≥95% high-spin and a comparable binding affinity to 4-methoxycinnamic acid (Table 1 and Fig. S2†). The trend in the CYP199A4 substrate binding affinity for the alkyl and methoxy para-substituted cinnamic acids mirrors that of equivalent para-substituted benzoic acids (methoxy > isopropyl > methyl) despite the different trend in the spin state shifts.5 On the whole, appropriately substituted cinnamic acids appear to be good target substrates for oxidation by CYP199A4.
Fig. 5 NADH oxidation assays of 4-methoxycinnamic acid (red), 4-methylcinnamic acid (black), 3-(4′-methoxyphenyl)propionic acid (green), 4-methoxyphenylacetic acid (blue), cinnamic acid (cyan), 4-hydroxycinnamic acid (purple) and 4-methoxybenzoic acid (magenta). See Experimental section for details and Table 1. |
Scheme 1 The products formed from CYP199A4 enzyme turnovers with different cinnamic acid derivatives (abbreviations; CA, cinnamic acid; MCA, methoxycinnamic acid). |
In accord with the reduced spin state shift and weaker binding of 3-(4′-methoxyphenyl)propionic acid to CYP199A4, the rate of NADH oxidation was lower than that for 4-methoxycinnamic acid (98 min−1, Table 1 and Fig. 5). The coupling efficiency was higher than that observed with 4-methoxycinnamic acid and 4-methoxyphenylacetic acid, resulting in a product formation rate of 43 min−1 (Table 1). A single product was observed in the HPLC analysis of the turnover which co-eluted with 3-(4′-hydroxyphenyl)propionic acid (Scheme 1 and Fig. S3†).
The addition of cinnamic acid and 3-hydroxycinnamic acid to CYP199A4 resulted in no appreciable increase in the rate of NADH oxidation above that observed in absence of substrate. 4-Hydroxycinnamic acid induced a very small increase in NADH consumption (Table 1 and Fig. 5). Chromatographic analysis of the CYP199A4 catalysed oxidation of these two substrates revealed little product formation (Fig. S3†) but 3-hydroxycinnamic acid did yield very low levels of 3,4-dihydroxycinnamic acid (Fig. S3†). HPLC analysis of the cinnamic acid turnover showed trace amounts of 3- and 4-hydroxycinnamic acids, though most if not all of the 3-hydroxycinnamic acid could potentially be accounted for from impurities in the substrate (Fig. S3†).
Minimal increases in the rate of NADH oxidation above that of the background NADH oxidase activity of the HaPux/HaPuR/CYP199A4 system, in the absence of substrate, were observed with 3-methoxy-4-hydroxy-, 3,5-dimethoxy- and 3,5-dimethoxy-4-hydroxy-cinnamic acids (Table 1). No product arising from substrate oxidation could be identified from CYP199A4 mediated oxidation of 3-methoxy-4-hydroxycinnamic acid or 3,5-dimethoxy-4-hydroxycinnamic acid, but very low levels of a single product were detected from 3,5-dimethoxycinnamic acid (Fig. S3†). GC-MS analysis revealed a product with a mass consistent with 3-hydroxy-5-methoxycinnamic acid (expected mass: 338.6, observed mass: 338.3; Fig. S4 and S5†), the product from a single oxidative demethylation.
Product formation from CYP199A4 catalysed oxidation of 3,4-(methylenedioxy)cinnamic was 30-and 50-fold slower than that observed with 4-methoxy- and 3,4-dimethoxy-cinnamic acids (Table 1). The lower amount of product formed is mainly due to low coupling efficiency, 13% (Table 1). The sole product was identified as 3,4-dihydroxycinnamic acid arising from demethenylation (Fig. 6 and Scheme 1). The oxidation of 3,4-dimethoxy-, 2,4-dimethoxy- and 3-hydroxy-4-methoxy-cinnamic acids by CYP199A4 resulted solely in the demethylation of the methoxy group at the 4-position and formation of a single product (Fig. S3 and S6†). Despite the lower spin state shifts and weaker binding to the enzyme compared to 4-methoxycinnamic acid, the rate of NADH oxidation was high in all cases (Table 1). In fact, the levels of metabolite generated exceeded that of 4-methoxycinnamic acid due to higher coupling efficiencies (59–75%) and resulted in product formation rates in excess of 200 min−1 (Table 1).
The turnover of 3,4,5-trimethoxycinnamic acid by CYP199A4 did not result in any NADH oxidation activity above that of background NADH oxidase activity and no product formation was observed. 2,3,4-Trimethoxycinnamic was oxidised by CYP199A4 to generate a single product (Fig. 6 and Scheme 1). Although the NADH oxidation activity was low the coupling efficiency of this turnover was high resulting in a product formation rate of 24 min−1 (Table 1). GC-MS analysis indicated that the product arose from a single oxidative demethylation (substrate expected mass: 310.4, observed mass: 310.0; oxidative demethylation product; expected mass: 368.6, observed mass: 368.0; Fig. S4 and S5†). The product was assigned as 4-hydroxy-2,3-dimethoxycinnamic acid based on the exclusive attack on the para-substituent reported so far for CYP199A4.
Substitution of the methoxy group with a methyl group resulted in a reduction in the NADH oxidation activity and product formation rate of the enzyme, 85 min−1 (Table 1). However, the coupling of the CYP199A4 turnover of 4-methylcinnamic acid was higher than that of 4-methoxycinnamic acid (Table 1). As a result, the relative reduction in activity reported is not as dramatic as that observed for the CYP199A4 catalysed turnover of 4-methylbenzoic acid compared to 4-methoxybenzoic acid.6 A single product was observed in the CYP199A4 mediated oxidation of 4-methylcinnamic acid (Fig. 6). This was generated in higher yield using a whole-cell oxidation system (vide infra), purified via semi-prep HPLC and characterised via NMR as 4-hydroxymethylcinnamic acid (Scheme 1 and Fig. S6†).
Despite the higher spin state shift of CYP199A4 with 4-isopropylcinnamic acid, the enzyme-catalysed oxidation of this substrate was slower than that of 4-methylcinnamic acid (Table 1). Four products were observed in the analysis of the in vitro turnovers by HPLC and GC-MS (Scheme 2 and Fig. S6†). These products were also formed when using the whole-cell oxidation system, although in different proportions. The masses of the derivatised products indicated one resulted from dehydrogenation, two others from hydroxylation and another from further oxidation of the alkene or an alcohol product (Fig. S5 and S6 and Table S2†).6,10,28 The products were generated using a whole-cell oxidation system and three were isolated and purified by HPLC. They were identified by NMR as 4-(1′-hydroxyisopropyl)cinnamic acid, 4-(2′-hydroxyisopropyl)cinnamic acid and 4-(1′,2′-epoxyisopropyl)cinnamic acid (Scheme 1 and ESI†). The epoxide product presumably arises from further oxidation of 4-(prop-1′-en-2′-yl)cinnamic acid, the desaturation product of 4-isopropylcinnamic acid (Scheme 2).
Fig. 7 HPLC analysis of the whole-cell oxidation of cinnamic acid derivatives; turnover at 4 hours (black), turnover overnight (red). 200 μM 9-hydroxyfluorene standard RT 23.8 min. (a) 4-Methylcinnamic acid – substrate RT 22.8 min, product RT 9.3 min. (b) 3-(4′-Methoxyphenyl)propionic acid – substrate RT, 18.0 min, product, RT 9.6 min. (c) 4-Methoxycinnamic acid – substrate RT 17.9 (cis) and 18.1 (trans) mins, product RT 11.0 min. The additional peak at 7.5 min is consistent with further oxidation of the 4-hydroxycinnamic acid product to 3,4-dihydroxycinnamic acid (Table S1†). |
In the CYP199A4 whole-cell turnovers of 4-methylcinnamic acid and 4-methoxycinnamic acid, all of the substrate (>95%) was converted to product at both 4 and 24 hours (Fig. 7 and S7†). 3-Hydroxy-4-methoxycinnamic and 3-(4′-methoxyphenyl)propionic acids were the only other substrates turnovers where all of the material added (2 mM) was consumed within 4 hours (Fig. 7 and S7†). However, only in the turnover of 3-hydroxy-4-methoxycinnamic acid was all the additional substrate consumed after 24 hours. Significant levels of the substrate (∼20%) remained in the 3-(4-methoxyphenyl)propionic acid turnover after 24 hours (Fig. S7†). In the turnover of 2,4-dimethoxycinnamic acid, most of the substrate (∼75%) was depleted and the product formation was high after 4 hours but a higher ratio of substrate to product was observed after 24 hours (Fig. S7†). The turnover of 3,4-dimethoxycinnamic acid resulted in conversion of approximately half the substrate in the first 4 hours (∼960 μM 3-methoxy-4-hydroxycinnamic acid), and a similar proportion of starting material and product was observed at 24 hours (Fig. S7†). Oxidation of 4-methoxyphenylacetic acid by CYP199A4 generated the next highest level of product with 320 and 510 μM of 4-hydroxyphenylacetic acid being obtained after 4 and 24 hours, respectively (Fig. S7†).
The levels of product in the turnovers in which the substrate lacks an alkyl or methoxy para-substituent, were very low (≤120 μM after 24 hours). No monooxygenase product was observed at either 4 or 24 hours with cinnamic, 3-hydroxycinnamic and 4-hydroxy-3,5-dimethoxycinnamic acids (Fig. S7†). There was no demethylated metabolite in the turnover of 3,5-dimethoxycinnamic acid after 4 hours but low levels of product (65 μM) were observed after 24 hours (Fig. S7†). Low levels of 3,4-dihydroxycinnamic acid were found in the whole-cell oxidation of 4-hydroxycinnamic acid at both 4 and 24 hours (∼30 and 120 μM, respectively, Fig. S7†). Control experiments showed that no product was formed in the whole-cell system when the P450 was excluded (Fig. S8†).
The turnover of 4-isopropylcinnamic acid generated products arising from hydroxylation at both the α- and β-carbons. Metabolites arising from desaturation pathways were also observed, in agreement with the products reported from the oxidation of 4-isopropylbenzoic acid by CYP199A4. Where the cinnamic acid substrate lacked a substituent or contained a hydroxy group at the para-position the turnovers generated very low levels of product highlighting the selectivity of the enzyme for this position. It may be the case that substrates containing a para-hydroxy substitution are close enough coordinate to the heme-iron which may interfere with the observation of a type I spin state shift and the commencement of the P450 catalytic cycle. In the turnover of 3,5-dimethoxybenzoic acid by CYP199A4, a small amount of 3-hydroxy-5-methoxybenzoic acid was observed. It has been hypothesised that this arises from a substrate methoxy group being oriented toward the heme-iron and therefore close enough to be oxidised.20 If only one substituent is present at the 3-position, it is likely to point away from the heme in a similar orientation to that observed in the crystal structure of 3,4-dimethoxybenzoic acid-bound CYP199A4 (PDB code; 4EGN).5
The impact of additional substitutions in the cinnamic acid structure on substrate binding to and the activity of CYP199A4 is generally representative of what was previously observed with benzoic acids.4,5,20,22 However, the punitive effect of the additional substitutions at the 3-position of the cinnamic acids was greater than the equivalent modification of benzoic acids and this may be related to their larger size. The larger trimethoxycinnamic acids seem to be too bulky for the substrate binding pocket of CYP199A4 or their shape many force the substrate to bind in such a fashion that the water bound to the heme iron is not displaced.
Overall, the trend in the levels of product formed in the in vitro CYP199A4 turnovers were mirrored in the whole-cell assays. Complete conversion of suitable substrates at concentrations up to 4 mM could be achieved using low cell density whole-cell oxidation in shake flasks. There was higher than expected levels of product observed in the whole-cell turnovers of 4-hydroxycinnamic acid when compared to the in vitro turnover studies. Recent work has shown that the E. coli hydroxylase complex, HpaBC, is capable of oxidising this substrate to 3,4-dihydroxycinnamic acid which may account for some of this.29 However control whole-cell experiments in the absence of the P450 showed that no detectable caffeic acid was being produced (Fig. S8†).
The selectivity of CYP199A4 oxidation of the cinnamic acids for the para-substituent is interesting as chemical oxidants might be expected to target the more reactive alkene group. Indeed chloroperoxidase oxidises cinnamic acid and its derivatives at the alkene group.30 Cinnamic acids and their derivatives are phenylpropanoids which are naturally found in plants. They are constituent components of the biosynthetic pathways of many natural products encompassing coumarins and flavonoids.25,26 They have a range of medicinal properties including anticancer, antimicrobial and antioxidant activities.25 Therefore new biocatalytic methods to facilitate the synthesis of cinnamic derivatives could enhance their use and effectiveness. For example ozagrel, an antiplatelet agent inhibiting thromboxane A2, is related to 4-methylcinnamic acid.31 CYP199A4 could have a role as a biocatalyst for the modification of substituted cinnamic acid derivatives and the oxidation of these substrates could be improved using protein engineering techniques to optimise the activity. For example the F185L mutant of CYP199A2 is reported to give increased levels of product with 4-hydroxycinnamic acid and is capable of oxidising cinnamic acid to 3,4-dihydroxycinnamic acid.23
The expression and purification of CYP199A4, HaPux and HaPuR have been described elsewhere.4–6 The CYP199A4 protein concentration was calculated using ε419 = 119 mM−1 cm−1 as previously described.5
For dissociation constant determination CYP199A4 was diluted to 1.3–5.5 μM using 50 mM Tris, pH 7.4, in 2.5 mL and 0.5–2 μL aliquots of the substrate were added using a Hamilton syringe from 1, 10 or 100 mM stock solutions in ethanol or DMSO (Fig. 3 and S2†). The maximum Soret band peak-to-trough difference (ΔA) in absorbance was recorded between 700 nm and 250 nm. Further aliquots of substrate were added until the peak-to-trough difference of the Soret band did not change. The dissociation constants, Kd, were obtained by fitting ΔA against total substrate concentration [S] to a hyperbolic function:
For gas chromatography analysis, 990 μL of the reaction mixture was mixed with 10 μL of an internal standard solution (20 mM 9-hydroxyfluorene) and 3 μL of 3 M HCl. The mixture was extracted three times with 400 μL of ethyl acetate and the organic extracts were combined and dried over MgSO4. Solvent was evaporated under a stream of nitrogen and the sample dissolved in 150 μL acetonitrile. Excess (35 μL) BSTFA + TMCS (99:1) was added and the mixture left for at least 120 min to produce the trimethylsilyl ester of the carboxylic acid group and trimethylsilyl ether of the alcohol, if formed. These reaction mixtures were used directly for GC-MS analysis. The retention times for the trimethylsilyl (TMS) derivatives are given in the ESI (Table S2†).
In order to isolate and identify products for which no standards were available, and to assess the activity of the CYP199A4 system in E. coli, we utilised a whole-cell oxidation system comprising of the plasmids pETDuetHaPux/HaPuR and pRSFDuetHaPux/CYP199A4, the construction and use of has been described previously.4 Protein production was induced at late log phase by adding IPTG (to 0.1 mM final concentration) and cooling the growth to room temperature. The cells from a 250 mL growth (∼6 g cell wet weight L−1) were harvested via centrifugation (5000g, 10 min) and used immediately. They were resuspended in double the volume of E. coli minimal media (EMM; K2HPO4 7 g, KH2PO4 3 g, Na3citrate 0.5 g, (NH4)2SO4 1 g, MgSO4 0.1 g, 20% glucose (20 mL) and glycerol (1% v/v) in one litre).33 For small scale turnovers this cell suspension was split into 30 mL aliquots in 250 mL Erlenmeyer flasks. The substrates were added (from a 100 mM stock) to the resuspended cells to a concentration of 2 mM and the reactions were then shaken at 30 °C and 200 rpm. Samples for HPLC analysis were taken after 4 hours at which point a further 2 mM aliquot of substrate was added and a second sample was taken after 24 hours. The supernatant was separated from the cells by centrifugation before analysis. Control experiments in the absence of the plasmid containing the CYP199A4 gene were performed with cell containing just the pETDuetHaPux/HaPuR using the same method as above.
For large scale growths 200 mL of the cell suspension was added to a 2 L baffled flask and 2 mM aliquots of substrate were added at 1, 3 and 6 hours. After 20 hours, the supernatant (200 mL) was acidified, extracted in ethyl acetate (3 × 100 mL), washed with brine (100 mL) and dried with MgSO4. The organic extracts were pooled and the solvent was removed by vacuum distillation and then under a stream of nitrogen. The products were purified using an Agilent 1100 HPLC equipped with Supelcosil LC-18 semi-prep column (5 μm particle size, 25 cm × 10 mm) and a fraction collector. A gradient, 20–50% of acetonitrile (with trifluoroacetic acid, 0.1%) in water (TFA, 0.1%) was used with UV detection at 240, 254 and 280 nm. Those fractions containing a single product (≥95%) were combined for characterisation. The solvent was removed under reduced pressure and the purified product was dissolved in deuterated DMSO. NMR spectra were acquired on an Agilent DD2 spectrometer operating at 500 MHz for 1H and 126 MHz for 13C. A combination of 1H and 13C experiments were used to determine the structures of the products (see ESI†).
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11025h |
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