CYP199A4 catalyses the efficient demethylation and demethenylation of para-substituted benzoic acid derivatives

Article abstract of DOI:10.1039/c5ra08730a

The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris strain HaA2, can efficiently demethylate 4-methoxybenzoic acid via hemiacetal formation and subsequent elimination of formaldehyde. Oxidative demethylation of a methoxy group para to the carboxyl moiety is strongly favoured over reaction at one in the ortho or meta positions. Dimethoxybenzoic acids containing a para-methoxy group were also efficiently demethylated exclusively at the para position. The presence of additional methoxy substituents reduces the substrate binding affinity and the activity compared to 4-methoxybenzoic acid. The addition of the smaller hydroxy group to the ortho or meta positions or of a nitrogen heteroatom in the aromatic ring of the 4-methoxybenzoate skeleton was better tolerated by the enzyme and these analogues were also readily demethylated. There was no evidence of methylenedioxy ring formation with 3-hydroxy-4-methoxybenzoic acid, an activity which is observed with certain plant CYP enzymes with analogous substrates. CYP199A4 is also able to deprotect the methylenedioxy group of 3,4-(methylenedioxy)benzoic acid to yield 3,4-dihydroxybenzoic acid and formic acid. This study defines the substrate range of CYP199A4 and reveals that substrates without a para substituent are not oxidised with any significant activity. Therefore para-substituted benzoic acids are ideal substrate scaffolds for the CYP199A4 enzyme and will aid in the design of optimised probes to investigate the mechanism of this class of enzymes. They also allow an assessment of the potential of CYP199A4 for synthetic biocatalytic processes involving selective oxidative demethylation or demethenylation.

Full text of DOI:10.1039/c5ra08730a

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CYP199A4 catalyses the efficient demethylation and demethenylation of
para-substituted benzoic acid derivatives
Tom Coleman1, Rebecca R. Chao1, John B. Bruning2, James J. De Voss3 and Stephen G.
Bell1,*
1 Department of Chemistry, University of Adelaide, SA, 5005, Australia; email:
stephen.bell@adelaide.edu.au
2 Department of Molecular and Cellular Biology, University of Adelaide, Adelaide, SA,
5005, Australia.
3 School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Qld,
4072, Australia
TOC
CYP199A4, a cytochrome P450 enzyme from Rhodopseudomonas palustris HaA2, is able to
efficiently demethylate a range of benzoic acids at the paraꢀposition. It can also catalyse
demethenylation reactions.

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Abstract
The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris strain HaA2,
can efficiently demethylate 4ꢀmethoxybenzoic acid via hemiacetal formation and subsequent
elimination of formaldehyde. Oxidative demethylation of a methoxy group para to the
carboxyl moiety is strongly favoured over reaction at one in the ortho or meta positions.
Dimethoxybenzoic acids containing a paraꢀmethoxy group were also efficiently
demethylated exclusively at the para position. The presence of additional methoxy
substituents reduces the substrate binding affinity and the activity compared to 4ꢀ
methoxybenzoic acid. The addition of the smaller hydroxy group to the ortho or meta
positions or of a nitrogen heteroatom in the aromatic ring of the 4ꢀmethoxybenzoate skeleton
was better tolerated by the enzyme and these analogues were also readily demethylated.
There was no evidence of methylenedioxy ring formation with 3ꢀhydroxyꢀ4ꢀmethoxybenzoic
acid, an activity which is observed with certain plant CYP enzymes with analogous
substrates. CYP199A4 is also able to deprotect the methylenedioxy group of 3,4ꢀ
(methylenedioxy)benzoic acid to yield 3,4ꢀdihydroxybenzoic acid and formic acid. This
study defines the substrate range of CYP199A4 and reveals that substrates without a para
substituent are not oxidised with any significant activity. Therefore paraꢀsubstituted benzoic
acids are ideal substrate scaffolds for the CYP199A4 enzyme and will aid in the design of
optimised probes to investigate the mechanism of this class of enzymes. It also allows an
assessment of the potential of CYP199A4 for synthetic biocatalytic processes involving
selective oxidative demethylation or demethenylation.
2

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Introduction
Cytochrome P450 (CYP) enzymes are hemeꢀdependent monooxygenases with physiological
roles that include hormone and secondary metabolite biosynthesis, as well as xenobiotic
metabolism.1ꢀ3 CYP enzymes primarily catalyse the insertion of an oxygen atom from
dioxygen into carbonꢀhydrogen bonds of organic molecules. Mechanistically, hydrogen
abstraction by Compound I is followed by oxygen rebound to give the hydroxylated
compound.4, 5 Many other types of activity are also known and include C–C bond cleavage
and formation, oxidative dealkylation and various ring alterations.2, 3, 6, 7 The stable, soluble
nature of bacterial CYP enzymes and the wide range of compounds they oxidise are of
interest for applications in synthesis involving C–H bond oxidation.8, 9 The ability to engineer
these bacterial CYP enzymes via rational or random methods can further enhance their
activities and broaden their substrate range.8, 10 A detailed knowledge of the enzymeꢀsubstrate
interactions is critical in these efforts in order to maximise the catalytic efficiency of the CYP
systems and to achieve the turnover numbers required for synthetic applications.
Rhodopseudomonas palustris is a family of purple photosynthetic bacteria that are
isolated in temperate, aquatic sediments and possess extraordinary metabolic versatility.
Bacteria classified as Rhodopseudomonas spp. can grow with or without light or oxygen, fix
nitrogen and have a wide range of biodegradative abilities.11 Studies have shown that
different Rhodopseudomonas species share many characteristics but different strains have
unique sets of functional genes to take advantage of the microenvironment in which they are
found. We recently reported the properties of the complete CYP199A4 class I P450 system,
from the R. palustris strain HaA2, which uses an electron transfer chain consisting of a [2Feꢀ
2S] ferredoxin (HaPux) and a flavinꢀdependent ferredoxin reductase (HaPuR) to mediate
heme reduction by NADH.12ꢀ17 CYP199A4 and the closely related CYP199A2 enzyme, from
R. palustris strain CGA009, have high affinities for 4ꢀmethoxybenzoic acid.12, 13, 17 The
3

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benzoic acid skeleton is important for binding and replacing it with a benzyl alcohol or a
18
benzaldehyde reduces the strength of substrate binding dramatically.12,
Altering the
benzene ring to a saturated cyclohexyl system or removal of the substituent at the paraꢀ
position both have a deleterious effect on substrate binding and activity.12, 18
In addition to 4ꢀmethoxybenzoic acid, CYP199A4 can bind and Oꢀdemethylate 3,4ꢀ
dimethoxybenzoic acid (veratric acid) solely at the 4ꢀmethoxy group to yield 3ꢀmethoxyꢀ4ꢀ
hydroxybenzoic acid (vanillic acid). However the spin state shift observed upon substrate
binding is reduced, the binding is not as tight and the activity is lower.17 CYP199 enzymes
have also been reported to be biocatalysts for the oxyfunctionalisation of various aromatic
carboxylic acids including 2ꢀnaphthoic acid, indoleꢀ6ꢀcarboxylic acid and cinnamic acids.19ꢀ21
Recently mutant forms of the CYP199A2 enzyme have been shown to catalyse the oxidation
of benzyl alcohols and phenols using a wholeꢀcell oxidation system. However detailed
analysis of the substrateꢀenzyme interactions were not presented.22
The crystal structure of substrateꢀfree and 4ꢀmethoxybenzoic acidꢀbound forms of
CYP199A4 have been determined (PDB: 4DNZ and 4DO1).13, 18, 23 In the substrateꢀbound
structures the substrate carboxylate group interacts with polar and basic amino acid side
chains while hydrophobic interactions with the benzene ring help hold the substrate in
position. These interactions account for the efficiency and substrate specificity of the
enzyme. In the crystal structure of 4ꢀmethoxybenzoic acidꢀbound CYP199A4, the substrate
carboxylate group forms hydrogen bonds directly to the side chains of Arg92, Ser95 and
Ser244 and, via a bridging water molecule, Arg243. The addition of the substrate triggers
movement in these residues and is accompanied by the binding of a chloride ion.23 These
structural changes cap the entrance to the active site and protect the heme from the external
solvent. The methyl group of the paraꢀmethoxy substituent is oriented towards the heme iron
(3.9 Å), which is consistent with exclusive attack at this position, resulting in demethylation
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to form 4ꢀhydroxybenzoic acid. The crystal structure of CYP199A4 with 3,4ꢀ
dimethoxybenzoic acid (PDB: 4EGN) reveals that the introduction of a methoxy group at the
meta position causes steric clashes with active site residues, Phe182 and Phe185, which
presumably leads to the reduced efficiency of binding and therefore activity.18
The chemical removal of methyl protecting groups often requires harsh conditions
and unselective reagents and very few catalysts are available for this reaction.24, 25 Alkyl
methyl ethers usually require, a Lewis acid and a nucleophile for efficient demethylation
while aryl methyl ethers, such as 4ꢀmethoxybenzoic acid, can be demethylated in the absence
of the Lewis acid but in both instances elevated temperatures are required. CYP enzymeꢀ
catalysed Oꢀdemethylation reaction proceeds under ambient conditions and are therefore
attractive targets for carrying out selective catalytic oxidative demethylation and the
deprotection of methoxy groups under mild conditions.
Here we compare the binding and kinetic properties of CYP199A4 with selected
substituted methoxybenzoic acid substrates. These studies will provide a basis for
understanding how benzoic acids with differently sized substituents at the orthoꢀ and metaꢀ
positions or a heteroatom in the benzene ring interact with and are oxidised by the enzyme.
We also investigate if CYP199A4 is able to catalyse the demethenylation of 3,4ꢀ
(methylenedioxy)benzoic acid. This will provide a basis for the future synthesis of small
molecule probes designed to investigate the subtleties of the mechanism of action of CYP
enzymes as well as the design of better biocatalysts using this system.
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Results and discussion
Substrate binding studies on the CYP199A4 system
We had shown previously that binding of 4ꢀmethoxybenzoic acid to CYP199A4 induces a
≥ 95% Type I spin state shift (Table 1) while 3,4ꢀdimethoxybenzoic acid (veratric acid) only
shifts the spin state to 70% highꢀspin.17 Binding of 3,4ꢀdimethoxybenzoic acid to CYP199A4
was also two orders of magnitude (29.5 ꢀM) weaker compared to 4ꢀmethoxybenzoic acid
(0.28 ꢀM). A comparison of the crystal structures of substrateꢀbound forms of CYP199A4
with these two substrates reveals that the phenyl rings, the carboxylate group and 4ꢀmethoxy
substituent are, to all practical purposes, superimposable. The 3ꢀmethoxy substituent of 3,4ꢀ
dimethoxybenzoic acid points away from the heme and it makes contact with the benzene
ring of Phe182 and Phe185. This causes Phe182 to move, resulting in changes in the location
of the side chains of Leu396 and Asp251. These steric clashes presumably are the main
contributing factor to the weaker binding of this substrate and the relative location of the two
methoxy groups to the heme iron provides a compelling rationale for demethylation only at
the para substituent. In this work a range of substituted benzoic acid derivatives was tested to
further probe the limits of substrate shape and electronic structure on CYP199A4 substrate
binding and activity (Fig. 1a).
2ꢀMethoxyꢀ and 3ꢀmethoxybenzoic acid both induced a significantly lower spin state
shift to the highꢀspin form than 4ꢀmethoxybenzoic acid, with the 3ꢀmethoxy variant giving a
greater shift than the 2ꢀmethoxy isomer (Table 1, Fig. 1b and Fig. S1). Benzoic acid bound to
CYP199A4 and induced a spin state shift of 30% which is higher than that induced by 2ꢀ
methoxybenzoic acid but lower than those of 3ꢀ and 4ꢀmethooxybenzoic acids.
Dimethoxybenzoic acids which lack the substituent at the paraꢀposition also induced
minimal spin state shifts in CYP199A4, though that of 3,5ꢀdimethoxybenzoic acid was higher
than that of 2,5ꢀdimethoxybenzoic acid (Table 1 and Fig. S1). This highlights that the paraꢀ
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methoxy substituent, or equivalent, is a requirement for removal of the water bound to the
heme iron when substrate binds. This agrees with previous data obtained for CYP199A2
where the addition of benzoic acid induces a lower shift than 4ꢀmethoxybenzoic acid and
both 2ꢀchlorobenzoic acid and 3ꢀchlorobenzoic acid bind poorly compared to 4ꢀ
chlorobenzoic acid.12 The paraꢀmethoxy group of 4ꢀmethoxybenzoic acid interacts with
Phe182 and Phe298.23 These interactions could stabilise the binding of this substrate over
benzoic acid alone. The lack of the paraꢀmethoxy group and the presence of groups at the
orthoꢀ or metaꢀ positions, which could result in steric clashes with active site amino acids or
the heme, reduce the binding affinity of these benzoic acids. As the presence of a substituent
para to the carboxylic acid was found to be important for substrate binding we decided to
explore the effect of additional substituents at the orthoꢀ and metaꢀpositions. Therefore we
studied the binding of differently substituted 4ꢀmethoxybenzoic acids (Fig. 1a).
2,4ꢀDimethoxyꢀ, 2ꢀhydroxyꢀ4ꢀmethoxy and 3ꢀhydroxyꢀ4ꢀmethoxyꢀbenzoic acids
induced a spin state shift of ≥ 90%, all of which are greater than that observed with 3,4ꢀ
dimethoxybenzoic acid (Table 1, Fig. 1 and Fig. S1). 3ꢀHydroxyꢀ4ꢀmethoxy benzoic acid
with the smaller hydroxy group at the metaꢀposition appears to be better accommodated in
the substrate binding pocket than 3,4ꢀdimethoxybenzoic acid which has a larger methoxy
moiety at the analogous position. In addition, having the methoxy group ortho to the
carboxylate group, as in 2,4ꢀdimethoxybenzoic acid, appears not to disrupt binding as much
as the 3,4ꢀdimethoxybenzoic acid. We also investigated the effect of inserting a heteroatom at
the metaꢀposition of the benzene ring. 6ꢀMethoxynicotinic acid induced a ≥ 95% type I spin
state shift (Fig. S1), indicating that a nitrogenꢀcontaining aromatic ring does not dramatically
affect substrate binding.
The size of the substrate and the nature of the substitutions which can bind in the
active site of CYP199A4 were further tested by investigating 3,4ꢀ(methylenedioxy)benzoic
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acid, which is structurally related to both 3,4ꢀdimethoxybenzoic acid and 3ꢀhydroxyꢀ4ꢀ
methoxybenzoic acid. This substrate induces a shift to 70% highꢀspin which is comparable to
that observed with the larger 3,4ꢀdimethoxybenzoic acid but lower than that observed with 3ꢀ
hydroxyꢀ4ꢀmethoxybenzoic acid (Table 1 and Fig. 1). The methylenedioxy five membered
ring may be orientated towards the heme in a similar position to that of the pyrole ring of
indoleꢀ6ꢀcarboxylic acidꢀbound CYP199A4 (PDB: EGO, binding of this substrate induces a
20% shift to the highꢀspin form)18 or in a similar location to that of the methoxy groups of
3,4ꢀdimethoxybenzoic acid (PDB: 4EGN).23
While the spin state shift gives an indication of substrate binding neatly in the
substrate binding pocket it does not provide detailed information on how tightly the substrate
may be bound. To further analyse substrate binding, the dissociation constant (Kd) was
determined for each substrate using UVꢀVis titration experiments (Table 1, Fig. 2 and S2). 6ꢀ
Methoxynicotinic acid binds tightly to CYP199A4 (Kd = 0.46 ꢁM), highlighting that the
heterocyclic ring does not significantly impair binding. 3ꢀHydroxyꢀ4ꢀmethoxybenzoic acid
bound less tightly (Kd = 1.7 ꢁM) than 4ꢀmethoxybenzoic acid and 6ꢀmethoxynicotinic acid
but more tightly than 3,4ꢀdimethoxybenzoic acid, confirming that the smaller hydroxyl group
at this position is better accommodated in the active site of CYP199A4. 2ꢀHydroxyꢀ4ꢀ
methoxybenzoic acid bound as tightly as 4ꢀmethoxybenzoic acid. Despite the higher spin
state shift observed on substrate binding, 2,4ꢀdimethoxybenzoic acid bound more weakly
than 3,4ꢀdimethoxybenzoic acid and the larger methoxy substituent at the orthoꢀposition must
also impair substrate binding though not dissociation of the water ligand from the heme iron
(Table 1 and Fig. 2). The most likely cause of the reduced binding affinity is steric clashes
with amino acids in the active site of CYP199A4.
3,4ꢀ(Methylenedioxy)benzoic acid bound more tightly than both 3,4ꢀ
dimethoxybenzoic acid and 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid suggesting improved
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interactions with the substrate binding pocket despite the lower spin state shift observed
(Table 1 and Fig. S2). This may be due to combination of the more hydrophobic
methylenedioxy ring, compared to the hydroxyl group in 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid,
and the smaller size of the substrate compared to 3,4ꢀdimethoxybenzoic acid. 3ꢀ
Methoxybenzoic acid bound less tightly to CYP199A4 than any of the substrates which
contain a methoxy group at the paraꢀposition (Table 1). 3ꢀMethoxybenzoic acid bound to
CYP199A4 two orders of magnitude more weakly than 4ꢀmethoxybenzoic acid and, despite
its higher spin state shift, less tightly than benzoic acid suggesting the 3ꢀmethoxy group
interacts less favourably with the substrate binding pocket. The binding of 3,5ꢀ
dimethoxybenzoic acid was 20ꢀfold weaker than 3ꢀmethoxybenzoic acid in line with the low
spin state shift observed. The negligible spin state shifts induced by the 2ꢀmethoxybenzoic
and 2,5ꢀdimethoxybenzoic acids prevented the accurate determination of the dissociation
constant (Kd > 1.5 mM).
Overall the data indicates that a substituent para to the carboxylate moiety is
beneficial for substrate binding and that additional, preferably smaller, substituents can be
accommodated at the orthoꢀ and metaꢀpositions.
Activity and product formation assays
The rates of NADH oxidation and product formation varied considerably across the range of
benzoic acid substrates though all were lower in activity when compared to 4ꢀ
methoxybenzoic acid (Table 1 and Fig. 3).17, 18 CYP199A4 had no observable NADH
oxidation or product formation activity with 2ꢀmethoxybenzoic and 2,5ꢀdimethoxybenzoic
acids (Fig. S3a and b). CYP199A4 in the presence of 3ꢀmethoxybenzoic acid did oxidise
NADH but no product from substrate oxidation could be detected via HPLC or GCꢀMS
analysis (Fig. S3c) and similar results were obtained for benzoic acid (Fig. S3). Furthermore
very little hydrogen peroxide was generated in these turnovers. The percentage of reducing
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NADH equivalents channelled into hydrogen peroxide formation was 2.2 ± 0.7% for 3ꢀ
methoxybenzoic acid and 4.7 ± 0.3% for benzoic acid (Table 1) suggesting the elevated
NADH oxidation rate is most likely due to the oxidase uncoupling pathway. This presumably
arises as the 3ꢀmethoxy substituent or any other C−H bond is not close enough to the reactive
ironꢀoxo intermediate to be oxidised.26, 27 Of the substrates which lack a 4ꢀmethoxybenzoic
acid moiety only 3,5ꢀdimethoxybenzoic acid yielded any observable product. The rate of
NADH oxidation is very slow and the levels of product formation are extremely low with a
single product being observed by HPLC (Table 1 and Fig. 4a). In the absence of an authentic
product standard the turnover was derivatised with BSTFA/TMSCl and analysed by GCꢀMS
(Fig. 5a). The product has a mass of 312.1 AMU which corresponds to a doubly TMSꢀ
derivatised species arising from a single oxidative demethylation of the substrate to yield 3ꢀ
hydroxyꢀ5ꢀmethoxybenzoic acid (Scheme 1 and Fig. S4a). The observation of even a small
amount of product with 3,5ꢀdimethoxybenzoic acid is interesting as the related 3ꢀ
methoxybenzoic acid is not oxidatively demethylated by CYP199A4 and this information
when combined with the significantly different spin state shift on substrate binding suggests a
different binding mode for these two molecules in the active site.
In the CYP199A4 substrateꢀbound crystal structures with 4ꢀmethoxybenzoic, 4ꢀ
ethylbenzoic and 3,4ꢀdimethoxybenzoic acids, (PDB codes: 4DO1, 4EGM and 4EGN,
respectively) the benzoic acid components of the substrates are superimposed. The
substituent in the para position is held over the heme iron for efficient oxidation while the 3ꢀ
methoxy substituent of 3,4ꢀdimethoxybenzoic acid points away from the heme towards
Phe182. Given that substrates which do not have a 4ꢀmethoxy substituent give rise to low
levels of product formation it is likely that the other substituents, such as a 3ꢀmethoxy group,
point away from the heme. In the case of 3,5ꢀdimethoxybenzoic acid if one methoxy group
points at Phe182 the other must be orientated towards the heme. Despite not sterically
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displacing the ironꢀbound water molecule and modifying the spin state, it is close enough to
result in low levels of a single demethylated product. 3,5ꢀDimethoxybenzoic acid was docked
into the substrate binding pocket of 4ꢀmethoxybenzoic acidꢀbound CYP199A4 (PDB:
4DO1), minus the substrate, and energy minimised. Compared to the structure of 3,4ꢀ
dimethoxybenzoic acid and other analogues the benzoic acid moiety was tilted by 15 ° (Fig.
6a). In this position the 3ꢀmethoxy methyl carbon is only 5.0 Å from the heme iron (the
oxygen of the same methoxy group is 0.2 Å closer). The 3ꢀmethoxy carbon is also 2.8 Å from
both Leu98, and the closest heme carbon atom and 3.0 Å from a heme nitrogen. The 5ꢀ
methoxy group points towards Phe182 with both the oxygen and methyl carbon being 2.9 Å
from the closest atom of this residue (Fig. 6a). This altered binding orientation, compared to
monosubstituted benzoic acids, may explain the observation of an oxidative demethylation
product, while the long FeꢀC (OꢀMe) distance gives rise to the reduced product formation
rate, which is two orders of magnitude lower, compared to 4ꢀmethoxybenzoic acid (Fig. 6a).
The binding orientation of 3,5ꢀdimethoxybenzoic acid is much less favourable, resulting in a
20ꢀfold decrease in binding and a 5000ꢀfold decrease in activity relative to 4ꢀmethoxybenzoic
acid.
The oxidation of all the remaining substrates by CYP199A4 resulted in the formation
of a single product arising through demethylation of a methoxy group at the paraꢀposition, or
in the case of 3,4ꢀ(methylenedioxy)benzoic acid, demethenylation of the methylenedioxy
group (Scheme 1). The products were identified via coꢀelution of authentic standards via
HPLC or GCꢀMS analysis of the trimethylsilyl derivatives (Fig. 4, and S3). Levels of product
formation were determined using HPLC or GCꢀMS after calibrating with the product
standards.
The CYP199A4 catalysed turnover of 6ꢀmethoxynicotinic acid displayed a NADH
oxidation rate of 353 nmol (nmolꢀCYP)−1 min−1 (Table 1 and Fig. 3), which demonstrates the
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pyridine heterocyclic ring has a detrimental effect on the enzyme’s activity despite the
substrate binding data indicating that 6ꢀmethoxynicotonic acid can bind effectively to the
CYP199A4 enzyme. The product was confirmed as arising from oxidative demethylation via
HPLC coꢀelution of the turnover with an authentic sample of 6ꢀhydroxynicotinic acid (Fig.
4b). GCꢀMS analysis of the BSTFA/TMSClꢀderivatised turnover mixture yielded product of
the predicted mass (m/z of 283.2 AMU vs. the expected m/z of 283.1 AMU). There was no
evidence of Nꢀoxide formation via GCꢀMS or HPLC. The product formation rate was 286
nmol (nmolꢀCYP)−1 min−1 indicating that the coupling of NADH reducing equivalents to
product formation was high (81%). Therefore the fourꢀfold lower activity of the 6ꢀ
methoxynicotinic acid compared to 4ꢀmethoxybenzoic acid is due to slower turnover rate of
the catalytic cycle. The incorporation of a nitrogen atom meta to the carboxylate group in the
aromatic ring (ortho to the methoxy group) results in a lower activity. This nitrogen contains
a nonꢀbonded lone pair which is not part of the aromatic ring and the nitrogen is considered
to be electron withdrawing. The substrate may be orientated with the nitrogen either directed
towards the heme ring or pointing at the Phe182 and Phe185 residues. Docking of the 6ꢀ
methoxynicotinic acid substrate in the CYP199A4 active site with the nitrogen in this
position, followed by energy minimisation, resulted in a structure where this nitrogen was
only 5.2 Å from the heme iron (Fig. 5b). This compares to the 6ꢀmethoxy carbon which is
only 3.9 Å away. We note the lack of any Nꢀoxide formation which would be evidence of the
substrate being orientated with the nitrogen closer the heme. The Nꢀcontaining heterocycle is
less sterically hindered than a meta substituted benzene ring and therefore 6ꢀalkyl substituted
nicotinic acid derivatives could be useful probes to further investigate the partition between
desaturation and hydroxylation of alkyl side chains by CYP199A4 using the electron
withdrawing pyridine ring.
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2,4ꢀDimethoxybenzoic acid is efficiently turned over to yield one product which was
confirmed by GCꢀMS to correspond to a single demethylation (Fig. 5b and S3d). Based on all
the previous data obtained for substituted 4ꢀmethoxybenzoic acids with CYP199A4 it is
likely that the product is a result of oxidative demethylation para to the carboxyl group to
yield 2ꢀmethoxyꢀ4ꢀhydroxybenzoic acid (Fig. S5). The turnover activity and coupling of 2,4ꢀ
dimethoxybenzoic acid with CYP199A4 were lower than 3,4ꢀdimethoxybenzoic acid. Both
2ꢀ and 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acids were turned over by CYP199A4 generating a
single product (2,4ꢀ and 3,4ꢀdihydroxybenzoic acids, respectively, Fig. 4c and Fig. S3e). The
product formation rate of the 3ꢀhydroxy analogue was higher than that of 2ꢀhydroxyꢀ4ꢀ
methoxybenzoic acid (Table 1). This is in line with the NADH oxidation activities but not the
tighter binding of the 2ꢀhydroxy analogue. The activity and coupling of the substrates with a
hydroxyl or methoxy substituent at the 3ꢀposition were higher than those with the equivalent
substitutions at the 2ꢀposition (Table 1).
Certain plant P450 enzymes, such as CYP719A can catalyse the oxidative cyclisation
of orthoꢀhydroxymethoxyꢀcontaining substrates resulting in the formation of
a
methylenedioxy ring. This reaction is important, for example, in isoquinoline alkaloid
biosynthesis.28, 29 There was no evidence for the formation of a methylenedioxy bridge in the
CYP199A4 catalysed turnover of 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid. Methylenedioxyꢀbridge
formation is proposed to occur in the plant enzymes by P450ꢀdependent hydroxylation of the
methoxy group yielding a hemiacetal which cyclises to form the methylenedioxy bridge by
an ionic mechanism, involving a methylene oxonium ion intermediate (Scheme 2). The
evidence for this mechanism is that no oxygen is incorporated into the product. The
hemiacetal precursor to ring formation is the same as that which would be generated in the
oxidative demethylation reaction of 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid. In CYP199A4 3ꢀ
hydroxyꢀ4ꢀmethoxybenzoic acid may be bound in a similar fashion to 3,4ꢀdimethoxybenzoic
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acid with the substituent in the 3ꢀposition pointing away from the heme iron (Fig. S5). In this
orientation the methyl of the 4ꢀmethoxy group points towards the heme iron and away from
the 3ꢀhydroxy group allowing rapid hemiacetal formation and demethylation rather than
methylenedioxy ring formation. If the hydroxy group points towards the heme it would be
located ∼4.3 Å from the iron. The plant P450 enzymes, which carry out this transformation,
must bind their substrates in such a way relative to the heme ironꢀoxo intermediate which
favours this reaction over oxidative demethylation or convert the intermediate radical directly
to the cationic methylene oxonium ion.
As a substrate 3,4ꢀ(methylenedioxy)benzoic acid generates a single monooxygenase
product, 3,4ꢀdihydroxybenzoic acid (Fig. 4d). The demethenylation reaction proceeds with
lower activity than the demethylation of 4ꢀmethoxybenzoic acid, 3,4ꢀdimethoxybenzoic acid
and 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid. This would be unexpected if hydrogen abstraction
was the rate determining step due to the favourable electronic effect of two oxygen atoms.
The substrate must be held in the substrate binding pocket with the methylene CꢀH bonds
located in a less favourable position for attack by compound I compared to the methyl
moieties of the methoxy groups of the other substrates. This may be due to the constrained
nature of the five membered ring resulting in the methylene group being bound further away
from the heme iron. Docking of 3,4ꢀ(methylenedioxy)benzoic acid into the substrate binding
pocket followed by energy minimisation indicated it could bind in a similar orientation to
indoleꢀ6ꢀcarboxylic acid (PDB: EGO) which places the methylenedioxy bridge only 4.0 Å
from the heme iron (Fig. 6c), compared to 3.8 Å for the methoxy carbon of 4ꢀ
methoxybenzoic acid (PDB: 4DO1). However the substrate could also bind in a similar
fashion to 3,4ꢀdimethoxybenzoic acid (PDB: 4EGN) which would place the methylene
carbon 5.7 Å away from the heme iron (Fig. 6c and Fig. S5).
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Demethenylation is thought to occur by a threeꢀstep process involving initial
oxidation of the methylene carbon, followed by a rearrangement and hydrolysis generating
formic acid rather than formaldehyde, which is produced in oxidative demethylation (Scheme
3). To confirm this we analysed the turnovers of 3,4ꢀmethylenedioxy benzoic acid and 4ꢀ
methoxybenzoic acid derivatives for the presence of formaldehyde and formic acid. The
turnovers of 4ꢀmethoxybenzoic, 3,4ꢀdimethoxybenzoic and 3ꢀhydroxyꢀ4ꢀmethoxybenzoic
acids generated formaldehyde, as measured by the Purpald assay, in line with the levels of
product observed (Fig. 7 and Fig. S6). Nugatory levels of formic acid were observed in these
assays (formate assay, Megazyme). The turnover of 3,4ꢀ(methylenedioxy)benzoic acid
showed the opposite results with low levels of formaldehyde but significant quantities of
formic acid being produced in agreement with the proposed mechanism. To confirm the
absence of 3,4ꢀ(methylenedioxy)benzoic acid in the turnover of 3ꢀhydroxyꢀ4ꢀ
methoxybenzoic acid, we also analysed this turnover reaction for the generation of formic
acid as well as formaldehyde. Only formaldehyde was found to be present and the lack of any
formic acid rules out further processing of any generated methylenedioxy compounds during
CYP199A4 mediated turnover of 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid.
Kinetic isotope studies on the reaction of mammalian P450s on methylenedioxy
containing drug molecules have led to the proposal that an alternative pathway driven by
peroxide based chemistry may compete with the mechanism outlined (Scheme 3).30 The
levels of uncoupling in the CYP199A4 catalysed reaction are relatively low therefore
CYP199A4 could be used to study the mechanism of demethenylation in more detail. In
addition there was no evidence of inhibition of turnover rates after initial oxidation of the
methylenedioxy group (Fig. 4) as has been observed in the CYP2A6 metabolism of
paroxetine which is proposed to be due to binding of a carbene species to the heme iron
(Scheme 3 and Fig. 3).31
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Overall we have shown that CYP199A4 catalyses the efficient oxidative
demethylation of a range of paraꢀsubstituted benzoic acid derivatives with concomitant
formation of formaldehyde. Substituents of moderate size at the orthoꢀ and metaꢀpositions
can be tolerated and oxidative demethenylation, which generates a diol and formic acid, is
also catalysed efficiently by this enzyme. The binding of the benzoic acid substrates in
CYP199A4 must be very tightly controlled as oxidation only occurs on the para substituent.
With other P450s blocking the position of the closest C−H bonds, where hydroxylation is
preferred, often leads to oxidation at another position often at an adjacent carbon.32, 33
Therefore CYP199A4 and para substituted benzoic acids are ideal enzymeꢀsubstrate
combinations which ensure that only the desired functional groups is oxidised. This could be
utilised for selective demethylation or hydroxylation reactions on multiply substituted
substrates. Importantly these reactions would be catalytic and performed under ambient
conditions which is not the case for the currently available chemical methods. In addition
differently para substituted benzoic acid substrates, which are readily available, are ideal
substrate scaffolds with which to study aspects of cytochrome P450 mechanism including
heteroatom dealkylation, hydroxylation and desaturation activities.
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Experimental Section
General
General reagents, organic substrates and N,Oꢀbis(trimethylsilyl)trifluoroacetamide with
trimethylchlorosilane (BSTFA/TMSCl, 99:1) were from SigmaꢀAldrich, TCI or Merck,
Australia. Buffer components, NADH and isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG) were
from Astral Scientific, Australia. UV/Vis spectra and spectroscopic activity assays were
recorded at 30 ± 0.5 °C on an Agilent CARYꢀ60 or Varian CARYꢀ5000 spectrophotometer.
Enzymes and molecular biology
General DNA and microbiological experiments were carried out by standard methods.34 The
expression and purification of CYP199A4, HaPux, and HaPuR have been described
elsewhere.14, 17, 35 Proteins were stored at –20 °C in 50 mM Tris, pH 7.4 containing 50% v/v
glycerol. Glycerol was removed immediately before use by gel filtration on a 5ꢀmL PDꢀ10
column (GE Healthcare, UK) by eluting with 50 mM Tris, pH 7.4. The CYP199A4 protein
concentration was calculated using ε419 = 119 mM−1cm−1.17, 23
Activity assays
In vitro NADH turnover rate assays were performed with mixtures (1.2 mL) containing 50
mM Tris, pH 7.4, 0.5 ꢁM CYP199A4, 5 ꢁM HaPux, 0.5 ꢁM HaPuR and 100 ꢁg mL−1 bovine
liver catalase. The buffer solution was oxygenated before use and the mixture equilibrated at
30 °C for 2 min. Substrates were added from a 100 mM stock solution in ethanol to a final
concentration of 1 mM. NADH was added to ca. 320 ꢁM (final A340 = 2.00) and the
absorbance at 340 nm was monitored. The rate of NADH oxidation was calculated using ε340
= 6.22 mM–1cm–1.
Substrate binding: spin state determination and binding titrations
The highꢀspin heme content was estimated (to approximately ±5%) by comparison with a set
of spectra generated from the sum of the appropriate percentages of the spectra of the
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substrateꢀfree (>95% lowꢀspin, Soret maximum at 418 nm) and camphorꢀbound (>95% highꢀ
spin, Soret maximum at 392 nm) forms of WT CYP101A1.
For dissociation constant determination CYP199A4 was diluted to 0.5 – 2.0 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. The maximum peakꢀtoꢀ
trough difference (ꢂA) in absorbance between 700 nm and 250 nm was recorded. Further
aliquots of substrate were added until the peakꢀtoꢀtrough difference did not change. The
dissociation constants, Kd, were obtained by fitting ꢂA against total substrate concentration
[S] to a hyperbolic function:
ꢁAmax ×[S]
ꢁA =
Kd +[S]
where ꢂAmax is the maximum absorbance difference. Several substrates exhibited tight
binding, with Kd < 2 µM. In these instances the data were fitted to the tight binding quadratic
equation:36
([E]+[S]+ Kd ) − {([E]+[S]+ Kd )2 − 4[E][S]}
ꢁA
=
ꢁAmax
2[E]
where ꢂAmax is the maximum absorbance difference and [E] is the enzyme concentration.
Analysis of metabolites
After the NADH had been consumed in substrate oxidation incubations, 132 ꢁL of the
reaction mixture was mixed with 2 ꢁL of an internal standard solution (10 mM 9ꢀ
hydroxyfluorene in ethanol). This was mixed with 66 ꢁL of acetonitrile before analysis by
HPLC. Reverse phase HPLC was performed using an Agilent 1260 Infinity pump equipped
with an Agilent Eclipse Plus C18 column (250 mm x 4.6 mm, 5 µm) and an autoinjector and
UV detector. A gradient, 20 ꢀ 95%, of acetonitrile in water (both containing trifluoroacetic
acid, 0.1%) was used and the absorbance was monitored at 254 nm (unless otherwise stated).
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The retention times for the substrates and products were as follows; 2ꢀmethoxybenzoic acid,
11.9 min; 3ꢀmethoxybenzoic acid, 13.0 min; 4ꢀmethoxybenzoic acid, 12.5 min; 4ꢀ
hydroxybenzoic acid, 6.8 min; 3ꢀhydroxybenzoic acid, 8.2 min; 6ꢀmethoxynicotinic acid, 7.0
min; 6ꢀhydroxynicotinic acid, 5.1 min; 2ꢀhydroxyꢀ4ꢀmethoxybenzoic acid, 13.9 min; 2,4ꢀ
dihydroxybenzoic acid, 8.5 min; 3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid, 7.9 min; 3,4ꢀ
dihydroxybenzoic acid, 5.3 min; 3,4ꢀ(methylenedioxy)benzoic acid, 12.2 min; 3,4ꢀ
dimethoxybenzoic acid, 12.0 min; 3ꢀmethoxyꢀ4ꢀhydroxybenzoic acid, 7.5 min; 2ꢀmethoxyꢀ4ꢀ
hydroxybenzoic acid, 7.4 min; 2,4ꢀdimethoxybenzoic acid 11.9 min; 3,5ꢀdimethoxybenzoic
acid 13.8 min; 3ꢀhydroxyꢀ5ꢀmethoxybenzoic acid 9.9 min; 2,5ꢀdimethoxybenzoic acid, 13.7
min.
For gas chromatography analysis 990 ꢁL of the reaction mixture was mixed with 10
ꢁL of an internal standard solution (10 mM 9ꢀhydroxyfluorene in ethanol) and 2 ꢁL of
concentrated HCl. The mixture was extracted three times with 400 ꢁL of ethyl acetate and the
organic extracts were combined and dried over MgSO4. Solvent was evaporated under a
stream of dinitrogen and the sample dissolved in 200 ꢁL acetonitrile. Excess (25 ꢁL) BSTFA
+ TMSCl (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. The resultant mixtures were used directly for GC analysis. The oven temperature
was held at 120 ºC for 3 min and then increased at 10 ºC min−1 up to 220 ºC before being
held for a further 7 min. The retention times for the trimethylsilyl (TMS) derivatised
substrates and products were as follows; 4ꢀmethoxybenzoic acid, 7.8 min; 4ꢀhydroxybenzoic
acid, 11.2 min; 6ꢀmethoxynicotinic acid, 7.8 min; 6ꢀhydroxynicotinic acid, 10.2 min; 2,4ꢀ
dimethoxybenzoic acid, 14.2 min; 2ꢀhydroxyꢀ4ꢀmethoxybenzoic acid, 16.1 min; 3,5ꢀ
dimethoxybenzoic acid, 13.7 min; 3ꢀhydroxyꢀ5ꢀmethoxybenzoic acid, 15.2 min.
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Products were calibrated against authentic product standards where available. When
authentic samples were not available the coupling was estimated based on the closest
available compound from those above. For example for 2ꢀmethoxyꢀ4ꢀhydroxybenzoic acid,
for which no authentic standard was available, the product peaks were calibrated against both
the substrate (2,4ꢀdimethoxybenzoic acid) and 2,4ꢀdihydroxybenzoic acid to determine the
HPLC detector response (the responses of both were very similar). These were then used to
estimate the concentration of the product peak.
Formaldehyde, formic acid and hydrogen peroxide assays
The formaldehyde content of turnovers was determined using mixtures (600 µL) containing
Tris buffer, pH 7.4, 50 mM (312 µL), the turnover mixture (240 µL) and Purpald (48 µL
from 168 mM stock in 2M NaOH). The reaction of formaldehyde and Purpald was allowed to
develop with shaking for 15 min before quantifying the formaldehyde Purpald complex
(Scheme S1) using the absorbance at 550 nm and subsequent comparison to a calibration
curve (Fig. S6).37
The formic acid content was determined using a formic acid assay kit from
Megazyme. The turnover (1.1 mL), H2O (1.0 mL), phosphate buffer (pH 7.6, 0.2 mL) and βꢀ
NAD (0.2 mL) were combined and equilibrated for 5 min. The absorbance at 340 nm was
measured and formate dehydrogenase (50 µL) was added to consume formic acid and form
NADH; this was allowed to react for 15 min. The increase in absorbance at 340 nm was used
to quantify the formic acid. The concentration of hydrogen peroxide formed by uncoupling
during NADH oxidation was determined by the horseradish peroxidase/phenol/4ꢀ
aminoantipyrine assay as described previously.38
Docking Studies
Ligand coordinates and restraints were created using PHENIX eLBOW (PMID: 19770504).
Ligands were initially docked manually into the active site of CYP199A4 (PDB: 4DO1), with
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the 4ꢀmethoxybenzoic acid removed, using Coot (PMID: 20383002). Each model was
individually subjected to regularization and model refinement with ICMꢀPro (to carry out
energy minimization, optimise model geometry, and alleviate clashing side chains). The
proteinꢀligand complexes were subjected to 300 rounds of Cartesian MMFF energy
minimization using ICM Pro (Molsoft, LLC). Side chains were globally optimised and the
backbone was annealed to improve the model using the ICM Pro Monte Carlo algorithm.39
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Conclusions
The substrate range of CYP199A4 has been assessed using a range of methoxybenzoic acid
derivatives. A methoxy group at the para position is essential for optimal activity.
Substitutions at other positions or the insertion of a heteroatom in the ring can be tolerated
but the activity is reduced. CYP199A4 does not catalyse methylenedioxy ring formation with
3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid but does catalyse oxidative demethenylation of 3,4ꢀ
(methylenedioxy)benzoic acid. As a result the biocatalytic scope of CYP199A4 has been
determined and this knowledge can be used to improve substrate binding and turnover of
CYP199A4 with nonꢀnatural substrates through protein engineering. Probes, based on the
core benzoic acid structure, which complement the size and shape of the substrate binding
pocket of CYP199A4 can be designed to investigate different aspects of P450 mechanism.
These will minimise competing uncoupling pathways and allow the tight control of the site
on the probe which is attacked by the enzyme, which will be at the para substituent.
Acknowledgment
This work was supported by ARC grant DP140103229 (to JJDV and SGB). The authors also
acknowledge the award of an Australian Postgraduate Award (to TC) and thank the
University of Adelaide for the award of a M. Phil Scholarship (to RRC).
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Table 1 Substrate binding parameters and catalytic turnover activity data for CYP199A4 with various substrates. The data are given as mean ±
S.D. with n ≥ 3. The reaction mixtures (50 mM Tris, pH 7.4) contained 0.5 ꢁM P450, 5 ꢁM HaPux and 0.5 ꢁM HaPuR. Rates are given as nmol
(nmolꢀCYP)−1 min−1.
Substrate
% HS
Kd (µM)
N[a]
PFR[b]
Coupling (%)[c]
4ꢀmethoxybenzoic acid
≥95%
10%
40%
70%
90%
10%
<10%
90%
0.28 ± 0.01
n.m.d
1340 ± 28
ꢀe
1219 ± 120
91 ± 2
ꢀf
2ꢀmethoxybenzoic acid
ꢀf
3ꢀmethoxybenzoic acid
ꢀf
ꢀf, g
69 ± 2.0
498 ± 5.0
807 ± 35
664 ± 32
49 ± 2.0
ꢀe
3,4ꢀdimethoxybenzoic acid
2,4ꢀdimethoxybenzoic acid
3,5ꢀdimethoxybenzoic acid
2,5ꢀdimethoxybenzoic acid
3ꢀhydroxyꢀ4ꢀmethoxybenzoic acid
2ꢀhydroxyꢀ4ꢀmethoxybenzoic acid
6ꢀmethoxynicotinic acid
3,4ꢀ(methylenedioxy)benzoic acid
benzoic acid
29.5 ± 3.1
47 ± 1.0
626 ± 100
384 ± 21
7 ± 2
77 ± 9
58 ± 1
14 ± 4
ꢀf
1500 ± 200
n.m.d
ꢀf
1.7 ± 0.09
0.16 ± 0.01
0.46± 0.05
0.17 ± 0.05
13 ± 0.2
847 ± 60
483 ± 32
353 ± 6.0
265 ± 7.2
257 ± 2.0
663 ± 75
271 ± 20
286 ± 32
158 ± 19
ꢀf
75 ± 8
56 ± 4
81 ± 8
59 ± 7
ꢀf, h
≥95%
≥95%
70%
30%
[a] N: NADH turnover rate. [b] PFR: product formation rate. [c] Coupling is the percentage of NADH consumed in the reaction that led to the
formation of product arising from substrate oxidation. [d] Not reported due to low levels spin state shift preventing accurate determination (> 1.5
mM) . [e] No significant NADH oxidation activity above the leak rate. [f] no detectable levels of product formation. [g] Hydrogen peroxide
levels were only 8.5 ± 1.4 ꢀM (2.2 ± 0.7%) of the turnover. By way of comparison 4ꢀmethoxybenzoic acid turnover generated 5.5 ± 0.5 ꢀM
H2O2. [h] Hydrogen peroxide levels were only 18 ± 0.6 ꢀM (4.7 ± 0.3%) of the turnover.
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Figures
Scheme 1 The products formed from the CYP199A4 enzyme turnovers of the benzoic acid
substrates (abbreviations; BA, benzoic acid; MBA, methoxybenzoic acid and NA, nicotinic acid).
.
COOH
COOH
R1
COOH
R1
COOH
CYP199A4
CYP199A4
X
X
R2
R2
OH
O
O
OH
OH
O
3,4-dihydroxy
-benzoic acid
3,4-(methylene-
dioxy)-benzoic acid
Substrate
R1
H
R2
X
1
4-methoxyBA
H
C
COOH
COOH
3,4-dimethoxyBA
2,4-dimethoxyBA
3-hydroxy-4-MBA
2-hydroxy-4-MBA
6-methoxyNA
H
OMe C
OMe H
OH C
C
CYP199A4
H
OH H
C
N
O
O
O
OH
H
-
3,5-dimethoxy-
benzoic acid
3-hydroxy-5-
methoxy-benzoic
acid
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DOI: 10.1039/C5RA08730A
Scheme 2 The proposed mechanism of methylenedioxy ring formation catalysed by the plant P450
CYP719A family.
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Scheme 3 Proposed reaction mechanism for methylenedioxy deprotection by mammalian P450
enzymes. Included is the proposed formation of the carbene intermediate which can act as an
inhibitor of P450 enzymes by binding to the heme iron.
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DOI: 10.1039/C5RA08730A
Figure 1a The substrates tested for binding and activity with CYP199A4.
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