Cytochrome P450 CYP199A4 from Rhodopseudomonas palustris Catalyzes Heteroatom Dealkylations, Sulfoxidation, and Amide and Cyclic Hemiacetal Formation

Article abstract of DOI:10.1021/acscatal.8b00909

The cytochrome P450 enzymes execute a range of selective oxidative biotransformations across many biological systems. The bacterial enzyme CYP199A4 catalyzes the oxidative demethylation of 4-methoxybenzoic acid. The benzoic acid moiety of the molecule binds in the active site of the enzyme such that the functional group at the para-position is held close to the heme iron. Therefore, CYP199A4 has the potential to catalyze alternative monooxygenase reactions with different para-substituted benzoic acid substrates such as thioethers and alkylamines. The oxidation of 4-methyl- and 4-ethyl-thiobenzoic acids by CYP199A4 resulted in sulfur oxidation. 4-Ethylthiobenzoic acid sulfoxidation and 4-ethylbenzoic acid hydroxylation by CYP199A4 occurred with high enantioselectivity (>74% enantiomeric excess). By way of contrast, CYP199A4 catalyzed exclusive oxidative N-demethylation over N-oxide formation with 4-methyl- and 4-dimethylaminobenzoic acids. Unexpectedly acetamide formation by CYP199A4 competes with dealkylation in the turnover of 4-ethyl- and diethyl-aminobenzoic acids. No oxidative dealkylation was observed with 3,4-ethylenedioxybenzoic with only hydroxylation to form a cyclic hemiacetal being detected. The X-ray crystal structures of four substrate-bound forms of the enzyme were solved and revealed subtle changes in the location of the para substituent which, when combined with the reactivity of the substituents, provided a basis for understanding the changes in selectivity. Furthermore, in the 4-ethylthiobenzoic acid-bound structure, the active site residue Phe298 moves to accommodate the substituent which points away from the heme iron. As such, the CYP199A4 enzyme provides ready access to a combination of structural, binding, and activity data with which to study a variety of reactions which are catalyzed by the P450 superfamily of enzymes.

Full text of DOI:10.1021/acscatal.8b00909

Subscriber access provided by Kaohsiung Medical University
Article
The Cytochrome P450 CYP199A4 from Rhodopseudomonas
palustris catalyses heteroatom dealkylations,
sulfoxidation and amide and cyclic hemiacetal formation
Tom Coleman, Siew Hoon Wong, Matthew N Podgorski, John
B Bruning, James Joseph De Voss, and Stephen G. Bell
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00909 • Publication Date (Web): 17 May 2018
Downloaded from http://pubs.acs.org on May 17, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
The Cytochrome P450 CYP199A4 from
Rhodopseudomonas palustris catalyses heteroatom
dealkylations, sulfoxidation and amide and cyclic
hemiacetal formation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
1
3
Tom Coleman, Siew Hoon Wong, Matthew N. Podgorski, John B. Bruning, James J. De
*
2
,
*1
Voss and Stephen G. Bell
1
2
Department of Chemistry, University of Adelaide, SA 5005, Australia;
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Qld,
4072, Australia;
3
*
School of Biological Sciences, University of Adelaide, SA 5005, Australia
Corresponding authors: Stephen Bell, stephen.bell@adelaide.edu.au; James De Voss
j.devoss@uq.edu.au
1
ACS Paragon Plus Environment

ACS Catalysis
Page 2 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT. The cytochrome P450 enzymes execute a range of selective oxidative
biotransformations across many biological systems. The bacterial enzyme CYP199A4 catalyses
the oxidative demethylation of 4ꢀmethoxybenzoic acid. The benzoic acid moiety of the molecule
binds in the active site of the enzyme such that the functional group at the paraꢀposition is held
close to the heme iron. Therefore, CYP199A4 has the potential to catalyse alternative
monooxygenase reactions with different paraꢀsubstituted benzoic acid substrates such as
thioethers and alkylamines. The oxidation of 4ꢀmethylꢀ and 4ꢀethylꢀthiobenzoic acids by
CYP199A4 resulted in sulfur oxidation. 4ꢀEthylthiobenzoic acid sulfoxidation and 4ꢀ
ethylbenzoic acid hydroxylation by CYP199A4 occurred with high enantioselectivity (>74%
enantiomeric excess). By way of contrast, CYP199A4 catalysed exclusive oxidative Nꢀ
demethylation over Nꢀoxide formation with 4ꢀmethylꢀ and 4ꢀdimethylꢀaminobenzoic acids.
Unexpectedly acetamide formation by CYP199A4 competes with dealkylation in the turnover of
4ꢀethylꢀ and diethylꢀaminobenzoic acids. No oxidative dealkylation was observed with 3,4ꢀ
ethylenedioxybenzoic with only hydroxylation to form a cyclic hemiacetal being detected. The
Xꢀray crystal structures of four substrateꢀbound forms of the enzyme were solved and revealed
subtle changes in the location of the para substituent which, when combined with the reactivity
of the substituents, provided a basis for understanding the changes in selectivity. Furthermore, in
the 4ꢀethylthiobenzoic acid bound structure the active site residue Phe298 moves to
accommodate the substituent which points away from the heme iron. As such the CYP199A4
enzyme provides ready access to a combination of structural, binding and activity data with
which to study a variety of reactions which are catalysed by the P450 superfamily of enzymes.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
KEYWORDS biocatalysis, cytochrome P450 enzymes, dealkylation, heteroatom oxidation,
crystal structures, C−H bond oxidation, enzyme mechanism.
2
ACS Paragon Plus Environment

Page 3 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
Introduction
The hemeꢀdependent cytochrome P450 (CYP) monooxygenase enzymes typically catalyse the
1
ꢀ6
insertion of an oxygen atom derived from dioxygen into unactivated carbonꢀhydrogen bonds.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
There is strong experimental and theoretical evidence that the ubiquitous hydroxylation reaction
7
, 8
of P450s proceeds via the electrophilic ferryl intermediate (Cpd I).
However, while theory
favours Cpd I involvement in the majority of P450 reactions, experimental studies have
suggested that earlier intermediates of the catalytic cycle, including the electrophilic ferricꢀ
hydroperoxide (Cpd 0) and the nucleophilic ferricꢀperoxo anion, could also have in role in other
P450ꢀcatalysed monooxygenase activity (Scheme 1). These intermediates have been proposed to
play a part in epoxidation, sulfoxidation and other less common transformations such as Baeyer
9ꢀ16
Villigerꢀlike oxidations and carbonꢀcarbon bond cleavage.
Scheme 1 The potential active oxidants of cytochrome P450 which could lead to product
formation and the main uncoupling pathways arising from them.
CYP199A4, from Rhodopseudomonas palustris strain HaA2, is able to demethylate 4ꢀ
methoxybenzoic
acid
and
4ꢀmethoxycinnamic acid,
and
demethenylate 3,4ꢀ
3
ACS Paragon Plus Environment

ACS Catalysis
Page 4 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
17ꢀ19
methylenedioxybenzoic acid with complete selectivity.
It reacts with paraꢀsubstituted
alkylbenzoic acids, such as 4ꢀethylꢀ and 4ꢀisopropylꢀbenzoic acids, to form a mixture of alkene
1
7, 20
and alcohol products through discrete hydroxylation and desaturation pathways.
These
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
activities are supported by a ferredoxin (HaPux; [2Feꢀ2S]) and a ferredoxin reductase (HaPuR;
FADꢀdependent) both of which are also from the R. palustris HaA2 bacterium. This class I
electron transfer chain mediates heme reduction and uses NADH as the source of the reducing
equivalents. When CYP199A4 was paired with these electron transfer partners, the activities of
oxidative Oꢀdemethylation, hydroxylation and desaturation of paraꢀsubstituted benzoic acids
were high and the loss of reducing equivalents due to uncoupling side reactions (i.e. NADH
1
7, 21ꢀ23
consumption without organic product formation) was low (Scheme 1).
The high activity and ease of use of the CYP199A4 enzyme means it is eminently
suitable for biocatalytic applications involving C–H bond activation as well as studies on the
1
7, 20, 24ꢀ26
mechanism of action of cytochrome P450 enzymes.
Adding to its desirability,
CYP199A4 has high chemoꢀ and substrate specificity for oxidation of the para substituent of
methoxyꢀsubstituted benzoic acids. Alterations to the benzene ring and carboxylate group or
removal of the paraꢀsubstituent all reduced the substrate binding and monooxygenase activity of
17, 20, 27
CYP199A4.
However, replacement of the methoxy group with alternate substituents is
1
7, 20, 27
tolerated.
This high substrate regioselectivity can be related to how the substrate is bound
in the enzyme active site. The crystal structure of several substrateꢀbound forms of CYP199A4
have been solved including those of 4ꢀmethoxybenzoic (PDB: 4DO1), 4ꢀethylbenzoic (PDB:
EGM) and 3,4ꢀdimethoxybenzoic (PDB: 4EGN) acids. These crystal structures reveal that the
carboxylate group forms direct and indirect hydrogen bonds with hydrophilic arginine and serine
residues. The benzene ring interacts hydrophobically with the side chains of phenylalanine and
4
ACS Paragon Plus Environment

Page 5 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
other hydrophobic residues (e.g. Phe182 and Phe185). These binding interactions hold the
substrate in such an orientation that the para substituent is held over the heme iron and
1
8, 20
substituents at the ortho and meta positions point away from the heme.
This accounts for the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
high level of selectivity for the para position in, for example, the oxidative demethylation of 3,4ꢀ
dimethoxybenzoic acid which generated 4ꢀhydroxyꢀ3ꢀmethoxybenzoic acid as the sole
1
7, 20
product.
As CYP199A4 would bind different paraꢀsubstituted benzoic acids with the altered
substituent held over the heme iron it is potentially an ideal enzyme with which to study and
compare alternative P450 monooxygenase activities. Suitably substituted benzoic acids could be
used to investigate and compare the different monooxygenase reactions of this enzyme. Here we
report the binding, turnover kinetics and the substrateꢀbound crystal structures of a range of
paraꢀsubstituted benzoic acids in which the substituent is an oxygen, nitrogen or sulfur
containing moiety. For example, we compare the reactivity of CYP199A4 with 4ꢀmethoxyꢀ, 4ꢀ
methylthioꢀ, and 4ꢀmethylaminoꢀbenzoic acids and differently substituted variants. This enables
a comparison of heteroatom (O, N and S) dealkylation versus oxidation in which the minimal
changes to the substrate have been made. We also show that this enzyme is able to efficiently
catalyse a range of common and more unusual P450 supported biotransformations, generating
the metabolites in good yields. The new structures provide valuable information on the positions
of the sites of monooxygenase action in relation to the heme iron.
5
ACS Paragon Plus Environment

ACS Catalysis
Page 6 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Experimental Section
General
General reagents and organic substrates were from SigmaꢀAldrich, TCI, Fluorochem or VWR.
N,OꢀBis(trimethylsilyl)trifluoroacetamide with trimethylsilyl chloride (BSTFA + TMSCl, 99:1)
was from SigmaꢀAldrich. Buffer components, NADH and isopropylꢀβꢀDꢀthiogalactopyranoside
(IPTG) were from Astral Scientific, Australia. 3ꢀ(Methylsulfinyl)benzoic acid was obtained from
Enamine Ltd. UV/Vis spectra and spectroscopic activity assays were recorded on an Agilent
CARYꢀ60 or Varian CARYꢀ5000 spectrophotometer with temperature control (30 ± 0.5 °C).
High Performance Liquid Chromatography (HPLC) analysis was performed on an Agilent 1260
Infinity Pump equipped with an autoinjector connected using an Agilent Eclipse Plus C18
column (250 mm x 4.6 mm, 5 ꢀm). Gradients of 20 ꢀ 95%, 0 ꢀ 80% or 0 ꢀ 50% acetonitrile in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
−
1
water (0.1% trifluoroacetic acid, TFA) at a flow rate of 1 mL min over 30 minutes were used.
Gas ChromatographyꢀMass Spectrometry (GCꢀMS) was undertaken on a Shimadzu GCꢀ17A
using a QP5050A GCꢀMS detector equipped with a DBꢀ5 MS fused silica column (30 m x 0.25
mm, 0.25 ꢀm). The interface and injector were held at 280 °C and 250 °C, respectively. The
−1
oven temperature was held at 100 ºC for 1 min and then increased at 15 ºC min up to 220 ºC.
The chromatography retention times for the substrates and products (trimethylsilyl (TMS)
derivatives for GCꢀMS) are given in the Supporting Information. Enantioselective
chromatography of methyl 4ꢀ(1′ꢀhydroxyethyl)ꢀbenzoate was performed using
a
CHIRALCEL®OJ column (Analytical: 250 mm, 4.6 mm, 5 µm, Daicel; Preparative: 250 mm, 20
mm, 5 µm, Daicel) using an isocratic run of 5% isopropanol in hexane (flow rate: Analytical 0.8
−1
−1
ml min ; Preparative 16 ml min ) and monitoring at 254 nm. For methyl 4ꢀ
methylsulfinylbenzoate, analysis was performed with a CHIRALCEL®OD Analytical column
6
ACS Paragon Plus Environment

Page 7 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
(
250 mm, 4.6 mm, 5 µm, Daicel) eluting isocratically 5% isopropanol in hexane at a flow rate of
−1
0
.8 ml min and monitoring at 270 nm.
The expression, purification and characterisation of CYP199A4, HaPux and HaPuR were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
17, 18, 20
undertaken as described previously.
The CYP199A4 protein concentration was calculated
−1
−1 17, 18
using ε419 = 119 mM cm .
Substrate binding: spin state determination and binding titrations
The spinꢀstate shift induced upon substrate binding was determined using a CYP199A4
concentration of 1.5 – 3.3 µM in 50 mM Tris, pH 7.4. Addition of 1ꢀL aliquots of substrate
(from a 100 mM stock) were performed until the spectrum did not change further. The
percentage spin state shift was compared with a set of spectra calculated from the sum of the
appropriate percentages of the spectra of the substrateꢀfree (>95% lowꢀspin) and camphorꢀbound
(>95% highꢀspin) forms of wildꢀtype CYP101A1 (P450cam) and estimated to approximately
±5%.
The binding affinity was determined by measuring the dissociation constant. The enzyme
was diluted to 0.4 – 1.7 µM in 2.5 mL of 50 mM Tris, pH 7.4 and this was used to baseline the
spectrophotometer. Aliquots of the substrate (0.5 – 2 µL) were added from 1, 10 or 100 mM
stock solutions in ethanol or DMSO using a Hamilton syringe until the peakꢀtoꢀtrough difference
of the Soret band did not change. The maximum Soret band peakꢀtoꢀtrough absorbance
difference (ꢁA) was recorded. The dissociation constants, K , were obtained by fitting ꢁA against
d
the added substrate concentration [S] using a hyperbolic function:
ꢁAmax ×[S]
ꢁA =
K +[S]
d
7
ACS Paragon Plus Environment

ACS Catalysis
Page 8 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢁ
Amax is the maximum absorbance difference. All the paraꢀsubstituted alkyloxy, alkylamino and
alkylthioꢀbenzoic acids exhibited a K of < 5 µM. Therefore this data were fitted to the Morrison
d
28
tight binding quadratic equation:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
(
[E]+[S]+ K ) − {([E]+[S]+ K ) − 4[E][S]}
ꢁ
ꢁAmax
A
d d
=
2[E]
ꢁAmax is the maximum absorbance difference and [E] is the enzyme concentration.
Activity assays
In vitro NADH turnover rate assays were performed in a total volume of 1.2 mL containing 50
mM Tris, pH 7.4, 0.5 ꢂM CYP199A4, 5 ꢂM HaPux and 0.5 ꢂM HaPuR. The buffer solution was
oxygenated and the enzymes were then added before equilibration at 30 °C for 2 min. NADH
was added to a final A340 of ∼ 2.00 (∼ 320 ꢀM concentration) and this absorbance was monitored
over time. Substrates were added from a 100 mM stock solution in ethanol or DMSO to a
concentration of 1 mM to initiate the reaction. The NADH oxidation rate was calculated using
–
1
–1
ε340 = 6.22 mM cm . The product formation rate and coupling efficiency were calculated by
quantitating the amount of product in the turnover as described below.
The concentration of hydrogen peroxide generated during the NADH turnovers was
29
assayed using a horseradish peroxidase (HRP)/phenol/4ꢀaminoantipyrine (4ꢀAP) method. To
4
00 µL of the turnover mixture 200 µL of a solution of 50 mM phenol (pH 7.4, Tris buffer) and
200 µL of 5 mM 4ꢀAP (pH 7.4, Tris buffer) were added. The absorbance of the resulting mixture
at 510 nm was set to zero, and 1 µL of a 20 mg mL−1 solution of HRP was added. The
absorbance at this wavelength was recorded again and used to calculate the concentration of
−
1
−1
hydrogen peroxide (ε510 = 6.58 mM cm ).
8
ACS Paragon Plus Environment

Page 9 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
Analysis of metabolites
The products arising from enzyme turnover were mostly identified via coꢀelution of chemical
1
7
standards using HPLC or GCꢀMS of the trimethylsilyl chloride (TMSCl) derivatised forms. For
HPLC analysis 150 ꢂL of the final reaction mixture was combined with 2 ꢂL of an internal
standard solution (20 mM 9ꢀhydroxyflourene in ethanol) and 50 ꢂL of acetonitrile.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For GCꢀMS analysis, 990 ꢂL of the final reaction mixture was mixed with 10 ꢂL of
internal standard (10 mM 9ꢀhydroxyfluorene) and 3 ꢂL of 3 M HCl. These samples were
extracted with ethyl acetate (3 x 400 ꢂL) and the organic extracts combined. The extract was
dried over MgSO and the solvent was evaporated under a stream of dinitrogen. This dried
4
sample was resuspended in 150 ꢂL anhydrous acetonitrile. Excess (35 ꢂL) BSTFA + TMCS
(99:1) was added and the mixture left for at 2 hours to produce the derivatised carboxylic acid
and the alcohol or amine groups, if formed. These reaction mixtures were injected directly into
the GCꢀMS. Calibrations were performed using fixed amounts of these standards to quantitate the
level of product in the turnovers, either by HPLC or GCꢀMS.
The turnovers of 4ꢀdimethylaminoꢀ, 4ꢀethylaminoꢀ and 4ꢀdiethylaminoꢀbenzoic acids
contained products which required two P450 catalytic cycles to be completed. These were 4ꢀ
aminobenzoic acid produced by double dealkylation in the cases of 4ꢀdimethylaminoꢀ and 4ꢀ
diethylaminoꢀbenzoic acids, and 4ꢀacetamidobenzoic acid in the cases of 4ꢀethylaminoꢀ and 4ꢀ
diethylaminoꢀbenzoic acids. In determining the total coupling efficiency of these turnovers,
double the molar amount of NADH was assumed to be consumed during formation these
products.
Synthesis, isolation and purification of products
9
ACS Paragon Plus Environment

ACS Catalysis
Page 10 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To isolate the products of 3,4ꢀethylenedioxybenzoic and 4ꢀethylaminobenzoic acids for
characterisation we utilised a wholeꢀcell oxidation system comprising the plasmids pETDuet and
1
7
pRSFDuet, as has been described previously. The plasmids were transformed into BL21(DE3)
−
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
E. coli cells and grown on LB plates containing the antibiotics ampicillin, 100 ꢂg mL , and
kanamycin, 30 ꢂg mL− (LBamp/kan). A colony was added to 500 mL broth (2xYTamp/kan) and
grown at 37 ºC overnight at 150 rpm. The temperature was reduced to 25 °C, the shaker speed
reduced to 120 rpm and protein production was started by the addition of 100 ꢂM IPTG (from a
1
0
.5 M stock in H O). The growths were continued for 24 hours before the cell pellet was
2
30
−1
harvested by centrifugation. The cell pellet (~ 6 g cell wet weight L ) was resuspended in
30
double the volume of E. coli minimal media (EMM) and split into 200 mL aliquots in 2 L
baffled flasks. The substrate was added to these cells to a final concentration of 2 mM and the
reactions were then shaken at 160 rpm and 30 °C for 20 hours.
The cells were removed by centrifugation (as described above) and the supernatant was
acidified before extraction with ethyl acetate (3 x 100 mL), washing with brine (100 mL) and
drying over MgSO . The majority of solvent was removed using a rotary evaporator before using
4
a stream of nitrogen. The products separated using an Agilent 1100 HPLC equipped with semiꢀ
prep Supelcosil LCꢀ18 column (5 ꢂm particle size, 25 cm × 10 mm). A gradient of 20 ꢀ 50%
acetonitrile (containing trifluoroacetic acid, 0.1%) in water (TFA, 0.1%) was used. UV detection
at 240, 254 and 280 nm was used and the product containing fractions were combined. The
acetonitrile and water were removed by lyophilisation. The extract dissolved in deuterated
1
13
DMSO and a combination of H and C NMR experiments were used to identify the product
(Supporting Information). These were acquired on an Agilent DD2 spectrometer operating at 500
1
13
MHz for H and 126 MHz for C.
10
ACS Paragon Plus Environment

Page 11 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
The different enantiomers of methyl 4ꢀ(1′ꢀhydroxyethyl)ꢀbenzoate were obtained by
separating the racemic mixture of the enantiomers using enantioselective HPLC. The
enantiomers were identified based upon comparison of their elution order and optical rotation
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
31, 32
with literature reports.
However, these assignments were apparently based upon catalyst
1
specificity and so formation of and H NMR analysis of the corresponding (R)ꢀαꢀmethoxyꢀαꢀ
trifluoromethylphenylacetic acid esters (Mosher’s Ester) of each enantiomer was undertaken to
confirm its configuration.
(
R) enantiomer: R 22.8 min (analytical conditions); [α] 38° (c 0.1, CHCl ) Mosher’s
T
D
3
1
Ester H NMR (400 MHz, CDCl ) δ 1.56 (d, J = 7 Hz, 3H), 3.54 (brd s, 3H), 3.90 (s, 3H), 6.13
3
(
q, J = 7 Hz, 1H) 7.31ꢀ7.43 and 7.99ꢀ8.02 (m, 9H).
(
S) enantiomer: R 20.3 min (analytical conditions); [α] ꢀ37° (c 0.1, CHCl ) Mosher’s
T
D
3
1
Ester H NMR (400 MHz, CDCl ) δ 1.62 (d, J = 7 Hz, 3H), 3.45 (brd s, 3H), 3.90 (s, 3H), 6.09
3
(
q, J = 7 Hz, 1H) 7.26ꢀ7.41 and 7.94ꢀ7.96 (m, 9H).
The racemic mixtures of the 4ꢀalkylthiobenzoic acids were generated by reacting them
with hydrogen peroxide before derivatisation to the methyl esters.
Protein crystallisation and X-ray crystallography
Prior to use, glycerol was removed from CYP199A4 using a 5 mL gel filtration column (PDꢀ10,
−
1
GE Healthcare). For crystallisation, CYP199A4 was concentrated to 30 ꢀ 35 mg mL in 50 mM
Tris, pH 7.4. Substrate was added to the protein from a 100 mM stock solution in EtOH/DMSO
to a final concentration of 1 mM.
11
ACS Paragon Plus Environment

ACS Catalysis
Page 12 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
Crystals were obtained using the hangingꢀdrop vapor diffusion method at 16 C using 1
µL of protein with 1 µL of reservoir solution and equilibrated with 500 µL of the same reservoir
solution. Rectangular plateꢀshaped crystals of approx. 300 ꢂm by 150 ꢂm by 20 ꢂm were
obtained in 1 week from 30 ꢀ 35 mg mL− CYP199A4 (containing 1 mM substrate) with a
reservoir solution containing 0.2 M magnesium acetate tetrahydrate, and 20 ꢀ 23% w/v PEGꢀ
3,350 and 0.1 M BisꢀTris pH 5.25 ꢀ 5.5. Crystals were harvested using a Microloop or
Micromount (MiTeGen), then cryoprotected by immersion in Parabar 10312 (ParatoneꢀN,
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hampton Research) and flash cooled in liq. N . Xꢀray diffraction data was collected at 100 K on
2
3
3, 34
the MX1 beamline at the Australian Synchrotron.
3
5
All diffraction data were indexed and integrated using iMosflm; scaled, merged and Rꢀ
3
6
37
free flags were added using Aimless, both available in the CCP4 suite of programs. The phase
38
problem was solved using the molecular replacement method with Phaser in CCP4, using 4ꢀ
methoxybenzoic acidꢀbound CYP199A4 (PDB: 4DO1, with the 4ꢀmethoxybenzoic acid and
heme ligands removed) as the initial search model. Electron density maps were obtained after
39
initial model building and the model was rebuilt using Coot. Structural refinements were
performed over multiple iterations using Phenix Refine available in the Phenix suite of
4
0
programs. Composite Omit maps were generated using the appropriate program in Phenix. In
the case of 4ꢀmethylaminobenzoic acid, a FeatureꢀEnhanced Map (FEM) was generated to
4
1
reduce model bias rather than a Composite Omit Map. Detailed data collection and structural
refinement statistics for the data sets are summarised in Table 1.
The coordinates for the crystal structures of the substrateꢀbound CYP199A4
combinations studied have been deposited in the Protein Data Bank (accession codes presented
in Table 1).
12
ACS Paragon Plus Environment

Page 13 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
Docking Studies
ꢀMethylaminobenzoic acid was docked into the active site of CYP199A4 (PDB: 5U6W) using
3
4
2
the ICMꢀPro software. The ligand bound in the active site (4ꢀmethylaminobenzoic acid) was
first modified to 3ꢀmethylaminobenzoic acid using the ICM 3D Ligand Editor. The 3ꢀ
methylamino moiety was placed pointing away from the substrate as per the crystal structure of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
,4ꢀdimethoxybenzoic acid bound CYP199A4 (PDB: 4EGN). Docking was performed using the
docking module of ICMꢀPro (Molsoft). The molecule reorientated into a lower energy
configuration with this substituent pointing towards the heme. A higher energy (lower scoring)
orientation had the 3ꢀmethylamino moiety pointing away the heme.
13
ACS Paragon Plus Environment

ACS Catalysis
Page 14 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1 Data collection and refinement statistics for the substrate bound CYP199A4 crystal
structures: Where multiple values are presented, the overall value is presented first, while the
value for the highest resolution (outer) shell in is parentheses.
Statistic
4ꢀMethylthioBA 4ꢀEthylthioBA
4ꢀMethylaminoBA
4ꢀEthoxyBA
PDB code
5KT1
5U6U
5U6W
5U6T
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Xꢀray wavelength
0.9537
0.9537
0.9537
0.9537
a = 44.07,
b = 51.28,
c = 79.98,
α = 90.00,
β = 91.82,
γ = 90.00
a = 44.18,
b = 51.35,
c = 79.91,
α = 90.00,
β = 92.11,
γ = 90.00
a = 44.37,
b = 51.15,
c = 78.92,
α = 90.00,
β = 91.98,
γ = 90.00
a = 44.44,
b = 51.42,
c = 79.12,
α = 90.00,
β = 91.95,
γ = 90.00
Unit cell
parameters
Space group
P12
1
1
P12
1
1
P12
1
1
P12
1
1
Molecules in
asymmetric unit
1
1
1
1
4
(
4.05 – 2.01
2.07 – 2.01)
7.2 (2.1)
44.15 – 1.79
(1.82 – 1.79)
44.34 – 2.63
(2.76 – 2.63)
44.42 – 1.94
(1.98 – 1.94)
Resolution range
<
I/σ(I)>
12.0 (2.1)
33670
6.9 (1.8)
10517
8.4 (2.1)
26777
Unique reflections 23479
Completeness of
data
Redundancy /
multiplicity
9
7
9.7 (97.2)
.1 (6.7)
99.8 (97.7)
7.4 (7.1)
98.6 (89.4)
7.4 (7.0)
99.8 (96.9)
7.4 (7.2)
Rmerge (%)
Rpim (%)
29.7 (89.4)
11.9 (36.7)
98.4 (82.8)
0.1555
10.7 (81.8)
4.2 (32.5)
99.8 (97.7)
0.1523
25.0 (88.6)
9.9 (35.7)
99 (77.9)
0.2243
16.8 (78.8)
6.6 (31.2)
99.5 (77.4)
0.1331
CC1/2 (%)
Rwork
Rfree (5% held)
0.2234
0.2021
0.2605
0.1842
Ramachandran
favored (%)
Ramachandran
outliers (%)
9
0
7.2
97.2
0.3
97.7
0
98.0
0
Rotamer outliers
%)
1.6
0.9
0.3
0.6
(
Composite Omit
Map 0.8 sigma, Map 0.8 sigma, 1.6 Map
Composite Omit
FeatureꢀEnhanced
Composite Omit
Map 0.8 sigma,
Omit map
generation method
43
43
41
43
1
.6 Å carve
Å carve
1 sigma, 1.6 Å carve 1.6 Å carve
14
ACS Paragon Plus Environment

Page 15 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
Results
Substrate binding and product formation
An array of benzoic acids with oxygen, nitrogen and sulfur containing substituents at the paraꢀ
position were screened for binding to and oxidation by CYP199A4 (Figure 1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1 Substrates of CYP199A4 investigated in this study
Oxygenꢀcontaining substituents: As with 4ꢀmethoxybenzoic acid, 4ꢀethoxybenzoic acid bound
tightly to CYP199A4 and induced a ≥95% shift of the enzyme spin state to the highꢀspin (HS)
form, indicative of binding in the active site (Table 2, Figure S1, Figure S2). It was oxidatively
demethylated to yield 4ꢀhydroxybenzoic acid as the sole product but the activity, as measured by
−
1
−1
−1
product formation rate (PFR, 527 nmol.nmolꢀCYP .min , henceforth abbreviated to min ),
was lower than observed for 4ꢀmethoxybenzoic acid (Scheme 2, Table 2, Figure S3. Figure S4,
Figure S5).
1
5
ACS Paragon Plus Environment

ACS Catalysis
Page 16 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2 Substrate binding parameters and catalytic turnover activity data for CYP199A4 with
various substrates.
[a]
[b]
[c]
Substrate
% HS
K (ꢁM)
d
N
PFR
C (%)
H O (%)
2 2
para- O, N, S and alkyl substituted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
g]
4
4
4
3
3
ꢀmethoxybenzoic acid
≥95% 0.22 ± 0.02 1340 ± 28 1220 ± 120
95% 0.17 ± 0.02 527 ± 10 527 ± 10
91 ± 2
2 ± 0.2
≤ 1
ꢀethoxybenzoic acid
100 ± 8
[
d]
[d]
ꢀtrifluoromethoxybenzoic acid
,4ꢀmethylenedioxybenzoic acid
,4ꢀethylenedioxybenzoic acid
≥95% 0.43 ± 0.05
70% 0.17 ± 0.05
294 ± 8
ꢀ
ꢀ
4 ± 2
ꢀ
[
g]
265 ± 7
158 ± 19
59 ± 7
70%
50 ± 1.5
437 ± 7
332 ± 7
76 ± 2
< 2
4
4
4
4
4
4
4
ꢀmethylaminobenzoic acid
ꢀethylaminobenzoic acid
ꢀdimethylaminobenzoic acid
ꢀdiethylaminobenzoic acid
ꢀmethylthiobenzoic acid
ꢀethylthiobenzoic acid
70%
1.6 ± 0.07 923 ± 200
669 ± 15
64 ± 2
3 ± 0.6
[f]
[f]
40% 0.92 ± 0.02
197 ± 8
284 ± 4
332 ± 17
112 ± 10
57 ± 3
ꢀ
[
e]
[e]
50%
70%
70%
20 ± 2
11 ± 1
239 ± 16
84 ± 4
ꢀ
[
f]
[f]
298 ± 8
90 ± 4
ꢀ
2.3 ± 0.3 1430 ± 180 1180 ± 130
83 ± 3
50 ± 3
63 ± 10
2 ± 0.4
ꢀ
[
h]
10%
0.99 ± 0.05
132 ± 6
64 ± 6
[
g]
ꢀethylbenzoic acid
≥95% 0.34 ± 0.02
812 ± 7
515 ± 88
2 ± 0.1
meta- O, N and S substituted
ꢀmethylaminobenzoic acid
3ꢀmethylthiobenzoic acid
3
10%
30%
40%
31 ± 1
33 ± 0.5
69 ± 2
255 ± 2
56 ± 0.2
498 ± 5
175 ± 1
37 ± 1
69 ± 1
66 ± 2
4 ± 0.2
∼1%
[
g]
[d]
[d]
3ꢀmethoxybenzoic acid
ꢀ
ꢀ
2 ± 0.7
The data are presented as a mean ± S.D. with n ≥ 3 and the rates are given as nmol.nmolꢀ
−1
−1
CYP .min . The reaction mixtures contained 0.5 ꢂM P450, 5 ꢂM HaPux and 0.5 ꢂM HaPuR.
−
1
−1
The average leak rate was 9.0 nmol.nmolꢀCYP .min . [a] NADH oxidation activity. [b] PFR:
product formation rate defines the number of product forming catalytic cycles completed over
the time period. [c] C is the coupling efficiency which is the proportion of NADH consumed in
the reaction that led to the formation of products expressed as a percentage. H O is the
2
2
percentage of NADH converted to hydrogen peroxide via this uncoupling pathway. [d] no
detectable product observed. [e] these coupling efficiencies were calculated on the basis that two
P450 catalytic cycles are required for the double dealkylation of these substrates. [f] these
coupling efficiencies were calculated on the basis that two P450 catalytic cycles are required for
acetamide formation (and double dealkylation when relevant) of these substrates. (–) indicates
1
7, 20, 27
that this value was not determined. [g] Data published previously.
[h] Note the Soret
16
ACS Paragon Plus Environment

Page 17 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
maxima shifted from 418 to 419 nm for 4ꢀethylthiobenzoic acid (a type I difference spectrum is
still obtained – Figure S2).
The coupling efficiency, which is the measure of the reducing equivalents that are channeled into
organic substrate oxidation rather than unproductive oxygen reduction reactions, approached
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7, 18, 20
100% (Table 2).
4ꢀTrifluoromethoxybenzoic acid bound to CYP199A4 with high affinity
(
K = 0.43 µM) and engendered a ≥95% shift to the HS state. The NADH oxidation activity (294
d
−
1
min ) was lower than that observed for both alkyloxybenzoic acids and, as would be expected
due to the strong C−F bonds, no product was formed. Additionally only low quantities of
hydrogen peroxide could be detected in these turnovers (3% of the total reducing equivalents
added) suggesting that the oxidase uncoupling pathway (four electron reduction of O to water)
2
5
, 21
was the major pathway in catalytic consumption of the reducing equivalents (Scheme 1).
Scheme 2 The products formed from the CYP199A4 catalysed oxidation of different substrates.
CYP199A4 can catalyse the efficient oxidative demethenylation of 3,4ꢀ
27
methylenedioxybenzoic acid producing formic acid as a product. The larger substrate 3,4ꢀ
ethylenedioxybenzoic acid bound to CYP199A4 with a much lower affinity (250ꢀfold less
tightly) but induced a similar spin state shift (70%, Table 2). The CYP199A4ꢀcatalysed oxidation
17
ACS Paragon Plus Environment

ACS Catalysis
Page 18 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of the ethylendioxy substrate was efficient (Table 2) and generated a single product, which was
isolated and identified by NMR as a cyclic hemiacetal, 6ꢀcarboxyꢀ2,3ꢀdihydroꢀ2ꢀhydroxyꢀ1,4ꢀ
benzodioxin (Scheme 3, Table 2, Figure S3, Figure S4, Figure S6).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3 The products formed from the CYP199A4 catalysed oxidation of 3,4ꢀmethylenedioxyꢀ
2
7
and 3,4ꢀethylenedioxyꢀbenzoic acid.
Nitrogenꢀcontaining substituents: 4ꢀMethylaminobenzoic acid bound tightly to CYP199A4
inducing a 70% type I HS shift but with lower affinity (K 1.6 µM) than 4ꢀmethoxybenzoic acid
d
(Table 2, Figure S1, Figure S2). As expected 3ꢀmethylaminobenzoic acid bound to CYP199A4
with lower affinity (31 ± 1 ꢀM) and induced a decreased spin state shift (20%, Table 2). The
addition of 4ꢀethylaminobenzoic acid resulted in a reduced modification of the spin state (40%
HS) but bound with higher affinity than the 4ꢀmethylamino analogue (K 0.92 µM; Table 2,
d
Figure S1, Figure S2). The observed oxidation activity of CYP199A4 toward paraꢀsubstituted
alkylamino benzoic acids was lower than toward the equivalent alkoxybenzoic acids (Table 2).
4
ꢀMethylaminobenzoic acid was dealkylated by CYP199A4 generating 4ꢀaminobenzoic
acid as the sole product with reasonable coupling efficiency (Table 2, Scheme 2, Figure S4). 3ꢀ
Methylaminobenzoic acid was also oxidised by CYP199A4 to generate significant quantities of
18
ACS Paragon Plus Environment

Page 19 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
the demethylation metabolite as the sole product (Figure S4). While this occurred with lower
activity relative to the paraꢀsubstituted isomer the coupling efficiency and product formation rate
were higher than expected, when compared to the metaꢀsubstituted methoxybenzoic acid (Table
2
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
). When this substrate was docked into CYP199A4 it preferred to bind with the 3ꢀ
methylamino group oriented towards the heme, rationalising the observed levels of product
formation (Figure S7).
The turnover of 4ꢀethylaminobenzoic was slower and less efficient than 4ꢀ
methylaminobenzoic acid (Table 2). Two products were generated in roughly equal amounts, one
coeluting with 4ꢀaminobenzoic acid while the other, which was formed in a slight excess (53%
of product), was isolated by semiꢀprep HPLC purification and assigned by NMR and MS as 4ꢀ
acetamidobenzoic acid. (Scheme 2, Figure S4, Figure S5, Figure S6). This assignment was
subsequently confirmed by HPLC coelution with an authentic product standard (Figure S4).
4
ꢀDimethylꢀ and 4ꢀdiethylꢀaminobenzoic acids bound to CYP199A4 with a 10ꢀfold lower
affinity than the 4ꢀmethylamino and 4ꢀethylamino equivalents (Table 2, Figure S2). The type I
spin state shift of CYP199A4 induced by 4ꢀdiethylaminobenzoic acid (70% HS) was greater than
observed with 4ꢀethylaminobenzoic acid but that of 4ꢀdimethylaminobenzoic acid (50% HS) was
lower than that of 4ꢀmethylaminobenzoic acid (Table 2, Figure S1). The changes in NADH
oxidation activity and product formation rate of the tertiary amine derivatives compared to their
secondary amine equivalents were in line with the modification in the spin state. As such, the
NADH oxidation activity of CYP199A4ꢀcatalysed oxidation of the 4ꢀdiethylamino species was
greater than that of 4ꢀethylaminobenzoic acid while the rate of oxidation of the 4ꢀdimethylamino
compound was lower than 4ꢀmethylaminobenzoic acid (Table 2).
19
ACS Paragon Plus Environment

ACS Catalysis
Page 20 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Only products arising from dealkylation of 4ꢀdimethylaminobenzoic acid were identified.
The double dealkylation product, 4ꢀaminobenzoic acid, was the major metabolite with only a
small amount of the intermediate secondary amine, 4ꢀmethylbenzoic acid, observed (∼8% of the
product) despite an excess of the starting material still remaining (Table 2 and Figure S4). In the
turnover of diethylaminobenzoic acid, 4ꢀaminobenzoic acid and 4ꢀacetamidobenzoic acid were
the two major products (Scheme 2). No significant levels of 4ꢀethylaminobenzoic acid, or a peak
that could be assigned to 4ꢀ(Nꢀethyl)ꢀacetamidobenzoic acid, was detected in the HPLC or GC
analysis (Figure S4). There was also no evidence of any product arising from Nꢀoxidation in any
of the turnovers of the alkylaminobenzoic acids.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4ꢀAminomethylbenzoic acid induced a very small type I shift to the highꢀspin form and a
binding affinity more than four orders of magnitude weaker than 4ꢀmethylaminobenzoic acid
(Table S1, Figure S1, Figure S2). Similar results were obtained with 4ꢀ
(dimethylamino)methylbenzoic and 4ꢀ(ethylamino)methylbenzoic acids, while the addition of 4ꢀ
(
methylamino)methylbenzoic acid resulted in a small red shift in the Soret band to 420 nm
Figure S1). Little catalytic activity was observed in the turnovers with these substrates (Table
(
S1). Terephthalic acid and 4ꢀformylbenzoic acid were detected by HPLC and GCꢀMS analysis
(Figure S4 and S5) and the latter could arise from the hydrolysis of a carbinolamine intermediate
arising from benzylic CꢀH bond hydroxylation, and the former by further oxidation of the formyl
product (Table S1, Figure S3, Figure S4). No other products which could be conclusively
assigned as arising from CYP199A4 activity were observed.
Sulfurꢀcontaining substituents: 4ꢀMethylꢀ and 4ꢀethylꢀthiobenzoic acids both induced a type I
spin state shift upon addition to CYP199A4 but the magnitude of the shifts, 70% and 10%
respectively, were lower than the equivalent alkoxybenzoic acids (Table 2, Figure S1). The spin
20
ACS Paragon Plus Environment

Page 21 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
state shifts and binding affinities (K 2.3 µM, methylthio and 1.0 µM, ethylthio) were similar to
d
those of the equivalent 4ꢀalkylamino benzoic acids (Table 2, Figure S2). 4ꢀEthylthiobenzoic acid
bound with higher affinity than 4ꢀmethylthiobenzoic acid despite the significantly lower shift in
the spin state observed (Table 2).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The rate of NADH oxidation with 4ꢀmethylthiobenzoic acid was higher than that with 4ꢀ
methoxybenzoic acid. In contrast, that found for 4ꢀethylthiobenzoic acid was lower than those of
4ꢀethoxyꢀ and 4ꢀethylaminoꢀbenzoic acids (Table 2). The major products from both 4ꢀ
alkylthiobenzoic acids arose from Sꢀoxidation, initially identified by GCꢀMS analysis (m/z =
2
4
56.05 versus substrate 240.05 for 4ꢀmethylthiobenzoic acid (Scheme 2, Figure S5)). Little or no
ꢀmercaptobenzoic acid, arising from P450ꢀcatalysed dealkylation, was detected with either
thioether (∼1% of the dealkylation product was detected in each turnover and in controls lacking
the P450 enzyme; Figure S4). The product formation rate of Sꢀoxidation with 4ꢀ
methylthiobenzoic acid was comparable to that of demethylation of 4ꢀmethoxybenzoic acid, both
ꢀ1
being in excess of 1000 min , due to a slightly lower coupling efficiency (Table 2). There was a
sharp drop in the activity of CYP199A4 with 4ꢀethylthiobenzoic acid predominantly due to the
reduced NADH oxidation rate (Table 2).
3ꢀMethylthiobenzoic acid bound to CYP199A4 with lower affinity (33 ± 0.5 ꢀM) and
induced a reduced spin state shift (30%, Table 2) than the corresponding paraꢀsubstituted
isomer. It was oxidised by CYP199A4 to 3ꢀmethylsulfinylbenzoic acid with a reasonably high
−
1
coupling efficiency (66%) but the rate of sulfoxidation was substantially reduced (37 ± 1 min ,
Table 2 and Figure S4).
21
ACS Paragon Plus Environment

ACS Catalysis
Page 22 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The alkylsulfinylbenzoic acid products arising from Sꢀoxidation are chiral. We therefore
set out to determine the enantioselectivity of the reactions. The methyl esters of both paraꢀ
substituted alkylsulfinylbenzoic acids were synthesised for enantioselective HPLC analysis. The
enantiomers of the methylsulfinyl metabolites could not be separated preventing further analysis
but they did confirm the identity of the turnover product. The ethylsulfinyl benzoic acids were
resolved by enantioselective HPLC. Analysis of the methyl esters of the products from
CYP199A4 catalysed oxidation revealed that there was a significant excess of one enantiomer
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(91:9, Figure S8). In order to compare the enantioselectivity of the sulfoxidation reaction to
hydroxylation we determined the enantioselectivity of the known CYP199A4ꢀcatalysed
1
3,14
oxidation of 4ꢀethylbenzoic acid to 4ꢀ(1′ꢀhydroxyethyl)ꢀbenzoic acid.
Enantioselective HPLC
demonstrated that the αꢀhydroxylated metabolite produced was predominantly one enantiomer
(87:13, Figure S8). This was shown to be the Sꢀenantiomer by comparison with literature and by
de novo analysis of the Mosher’s esters of chromatographically resolved enantiomers of methyl
4
ꢀ(1′ꢀhydroxyethyl)ꢀbenzoate (see experimental section for the experimental details and results).
Crystal structure analysis
The binding and oxidative activity of CYP199A4 with paraꢀsubstituted benzoic acids with
oxygen, nitrogen and sulfur containing moieties and the switch from Oꢀ and Nꢀdemethylation to
Sꢀoxidation provides an opportunity to understand how P450ꢀsubstrate interactions may control
these reactions. The orientation of the substrate in the active site and the position of the
substituents in relation to the reactive ironꢀoxo intermediate would be expected to impact the
formation of products which arise from different monooxygenase activities (heteroatom
oxidation versus dealkylation pathways). Xꢀray crystal structures were solved for the substrateꢀ
bound forms of CYP199A4 with 4ꢀethoxyꢀ (PDB: 5U6T), 4ꢀmethylaminoꢀ (PDB: 5U6W), 4ꢀ
22
ACS Paragon Plus Environment

Page 23 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
methylthioꢀ (PDB: 5KT1) and 4ꢀethylthioꢀbenzoic acid (PDB: 5U6U) to complement the
available structural data on the 4ꢀmethoxyꢀ (PDB: 4DO1) and 4ꢀethylꢀbenzoic acid (PDB:
4EGM) bound forms of CYP199A4 (Figure 2). The data processing and refinement statistics are
provided in Table 1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
(c)
(d)
Figure 2: Omit maps for the active site of various substrateꢀbound CYP199A4 crystal structures.
In each, the 2mFo – DFc density of the substrate is shown contoured as a grey mesh. The heme,
substrate and the active site residues are shown in green. The water molecule involved in the
hydrogen bonding network is shown as a red sphere. The contour level and carve radius of each
structure is displayed in Table 1. Shown are (a) 4ꢀethoxybenzoic acid, (b) 4ꢀmethylaminobenzoic
acid, (c) 4ꢀmethylthiobenzoic acid and (d) 4ꢀethylthiobenzoic acid.
For all the crystal structures of the CYP199A4–substrate complexes determined here, the
asymmetric unit contained a single molecule, which could be traced from residues 17 to 409. The
23
ACS Paragon Plus Environment

ACS Catalysis
Page 24 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
structures of these substrateꢀbound complexes of CYP199A4 displayed a high similarity to that
of CYP199A4 bound with 4ꢀmethoxybenzoic acid (Figure S9). All displayed a closed
conformation and contained a bound ion, modelled as chloride, close to the enzyme surface,
1
8, 44
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
which shields the active site from the external solvent.
The tertiary structures of the enzyme
in the different substrateꢀbound complexes are superimposable on one another. The r.m.s.d. for
α
all resolved C atoms between these molecules is <0.60 Å (measured over 393 atoms).
(a)
(b)
(c)
(d)
Figure 3: Overlay of the structures of 4ꢀmethoxybenzoic acid (green) with determined structures
(
yellow) of (a) 4ꢀethoxybenzoic acid, (b) 4ꢀmethylaminobenzoic acid, (c) 4ꢀmethylthiobenzoic
acid and (d) 4ꢀethylthiobenzoic acid. Water molecules in the active site are shown as spheres in
the structure’s respective color.
24
ACS Paragon Plus Environment

Page 25 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
In all the new structures the water molecule that is part of the hydrogen bonding network
to the carboxylic acid group of the substrate is found in an almost identical position to that in the
4ꢀmethoxybenzoic acid structure (Figure 3). In addition, for all structures except 4ꢀ
ethylthiobenzoic acid, the positions of all the active site residues are virtually unchanged (Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
). These crystal structures should therefore provide insight into the observed monooxygenase
activity and regioselectivity of the oxidations catalysed by CYP199A4 with the different
substrates.
4
ꢀEthoxybenzoic acid, which undergoes deethylation, is oriented similarly in the active
site to 4ꢀmethoxybenzoic acid (Figure 3a). The methylene of the ethyl substituent is the closest
substrate atom to the heme iron at a comparable distance to the methyl of 4ꢀmethoxybenzoic acid
(4.2 Å versus 4.1 Å). The methyl carbon points towards Val295 and is further from the heme
iron (4.3 Å, Table S2). When the position of the oxygen of the Cpd I intermediate was
4
5, 46
considered (modelled 1.6 Å from the Fe)
the methyl group was 0.2 Å further away than the
more reactive methylene group (Table S2). No products were observed that arise from H
abstraction from the stronger C−H bonds of the methyl group.
4
ꢀMethylaminobenzoic acid undergoes exclusive Nꢀdealkylation despite the observation
that Nꢀoxides are common products arising from P450 oxidations. The 4ꢀmethylamino moiety of
the substrate is orientated differently in relation to the heme iron compared to 4ꢀmethoxybenzoic
acid (the dihedral angle is 50.0° versus 2.1° and the angle in relation to Fe=O bond of Cpd I was
133.0° versus 140.7°; Figure 3b and Table S2). The distance from the methyl group to the heme
iron is almost identical to that of 4ꢀmethoxybenzoic acid (4.1 Å, Figure 3b). However, the
nitrogen is significantly closer to the heme and the Cpd I oxygen atom than the oxygen in 4ꢀ
methoxybenzoic acid (4.2 Å versus 5.2 Å and 3.0 versus 3.6 Å, respectively; Table S2). Despite
25
ACS Paragon Plus Environment

ACS Catalysis
Page 26 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
this change, there was no observed Nꢀoxidation product in the turnovers nor was there any
spectroscopic evidence that the nitrogen atom was interacting with the heme iron.
4
ꢀMethylthiobenzoic acid undergoes Sꢀoxidation. Compared to bound 4ꢀmethoxybenzoic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
acid in the enzymeꢀsubstrate crystal structure, the 4ꢀmethylthio moiety is rotated in a manner
more similar to that of the 4ꢀmethylaminobenzoic acid substrate (dihedral angle 33.9°, angle to
Cpd I Fe=O 131.9°; Figure 3c and Table S2). This orientation results in the methyl group being
further from the heme iron and therefore the oxygen of Cpd I than in the 4ꢀmethoxybenzoic acid
structure (4.4 Å versus 4.1 Å and 3.3 Å versus 2.7 Å, respectively; Table S2). However, the
reactive sulfur atom is closer to the heme iron and the ironꢀoxo moiety of Cpd I than the oxygen
in the 4ꢀmethoxybenzoic acid structure (4.9 Å versus 5.2 Å and 3.4 Å versus 3.6 Å, respectively;
Table S2). The sulfur atom is almost the same distance from the oxygen of Cpd I as the methyl
group (3.4 versus 3.3 Å and at an angle of 162.5° compared to 131.9°, Table S2). This coupled
with the increased orbital size, reactivity and electron density of sulfur must account for the
change in the CYP catalysed reaction.
In the active site of 4ꢀethylthiobenzoic acidꢀbound CYP199A4, residue Phe298 moves to
accommodate the ethylthio moiety (Figure 4). The side chain of the phenylalanine has rotated so
that it no longer points towards the substrate but is now oriented towards the plane of the heme
group (Figure 3d, Figure 4). This enables the methyl group of the ethylthio moiety to occupy the
space vacated by the aromatic ring. The 4ꢀethylthio moiety is twisted (dihedral angle 55.2°) so
that the sulfur is closer to the heme iron than the oxygen in 4ꢀmethoxybenzoic acid (4.7 Å versus
5
.2 Å, 3.3 Å from Cpd I oxygen, Figure 3d, Table S2). The methylene carbon of the ethyl group
is further from the heme iron and the Cpd I oxygen atom than the methyl of 4ꢀmethoxybenzoic
26
ACS Paragon Plus Environment

Page 27 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
acid (5.2 Å versus 4.1 Å and 4.0 Å versus 2.7 Å, respectively, Table S2) and of
methylthiobenzoic acid (5.2 Å versus 4.5 Å and 4.0 Å versus 3.3 Å, Table S2). The methyl group
is even further from the heme iron and Cpd I oxygen compared to the equivalent group in the 4ꢀ
ethoxybenzoic acid bound structure (Figure 4a, 6.7 Å versus 4.3 Å and 5.5 Å versus 3.0 Å,
respectively; Table S2).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(a)
(b)
Figure 4: Overlays showing the movement of Phe298 residue in 4ꢀethylthiobenzoic acid
structure. Shown are (a) comparison of 4ꢀethoxybenzoic acid structure (yellow) and 4ꢀ
ethylthiobenzoic acid structure (blue), and (b) comparison of 4ꢀmethylthiobenzoic acid structure
(
yellow) and 4ꢀethylthiobenzoic acid structure (blue). Arrow (green) shows movement of the
Phe298 residue between the structures.
The increased distance of 4ꢀethylthiobenzoic acid from the heme iron compared to the
other substrates in the substrate bound crystal structures may increase the solvent accessibility
accounting for the lower spin state shift, although there was no evidence of any water molecules
interacting with the heme iron in the crystal structure. The orientation of the substrate shows that
one face of the sulfur would be significantly closer to the heme iron than the other which could
explain the high enantioselectivity of sulfoxidation.
27
ACS Paragon Plus Environment

ACS Catalysis
Page 28 of 46
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Discussion
CYP199A4 binds and selectively oxidises paraꢀmethoxyꢀsubstituted benzoic acids and closely
1
7, 20, 27
related substrates.
The current work confirms that a range of paraꢀsubstituted benzoic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
acids can be accommodated within the active site of CYP199A4, and the nature of the
substituent influences the binding affinity of the enzyme. Within the sets of Oꢀ, Nꢀ and Sꢀ
containing paraꢀsubstituted benzoic acids the rate of NADH oxidation, and as a consequence the
product formation rate, tended to follow the trend seen in the shift to the high spin state upon
substrate binding. This shift is often used as an indicator of displacement of the hemeꢀbound
water ligand, a required step early in the catalytic cycle. The relative rates of NADH oxidation
4
7, 48
and product formation suggest that the reactions are gated by substrate binding.
Nꢀ
dealkylation, Sꢀoxidation and acetamide and cyclic hemiacetal formation can be added to this
CYP enzyme's known hydroxylation, Oꢀdealkylation and desaturation activities. Oxidative Oꢀ
demethylation is the most rapid reaction catalysed by CYP199A4, followed by sulfoxidation,
hydroxylation and then Nꢀdemethylation.
Two main mechanistic pathways have been proposed for heteroatom dealkylation by
P450s. NꢀDealkylation reactions can proceed in the same way as hydroxylation and Oꢀ
dealkylations, with hydrogen abstraction followed by radical rebound at the carbon alpha to the
4
9
heteroatom (HAT). This yields an unstable, hydroxylated product which then decomposes to
49
give the dealkylated product. It has been proposed that Nꢀdealkylation reactions catalysed by
heme proteins may also occur via an initial single electron transfer (SET) from the heteroatom to
5
0
compound I (Cpd I) to give a cation radical. Subsequent proton abstraction and radical rebound
11, 49, 51
can give the dealkylated product.
The current view, which is supported by large
intramolecular isotope effects for amide Nꢀdealkylations (> 13), DFT studies and the lack of ring
28
ACS Paragon Plus Environment

Page 29 of 46
ACS Catalysis
1
2
3
4
5
6
7
8
9
opening products with Nꢀcyclopropyl substrates, is that a HAT mechanism occurs for Nꢀ
5
2ꢀ56
dealkylation.
DFT calculations on selected paraꢀsubstituted N,Nꢀdimethylanilines showed
that the radical cation arising from the first single electron transfer on the SET pathway was
5
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
significantly higher in energy than its radical counterpart on the HAT pathway and that
dealkylation products can arise from either of the two spinꢀstates of Cpd I depending on the
51, 57, 58
nature of the substrate's substituent.
Some unusual major metabolites were observed during this work, including the isolable,
stable cyclic hemiacetal from 3,4ꢀdiethylenedioxy benzoic acid. The isolation of the cyclic
hemiacetal is probably due to the fact it is more stable than its nonꢀcyclic counterparts that
decompose to yield the dealkylated ether; this stability is analogous to that seen for the 6ꢀ
membered cyclic hemiacetal form of sugars. The observation of high levels of an acetamide
product with the Nꢀethylbenzoic acids, though not the Nꢀmethyl equivalents, was also
unexpected. To the best of our knowledge this is the first report of an acetamide being formed
from an ethylamine moiety as a result of P450 catalysed oxidation, although this transformation
has been observed in wholeꢀcell biotransformations of cyclic amines and Nꢀmethylanilines by a
5
9
nonꢀP450 enzyme. Hemiacetals and carbinolamine (hemiaminal) intermediates normally react
to form the aldehyde and alcohol/amine. Carbinolamine metabolites have been observed
60ꢀ63
previously, for example, with cyclophosphamide and Nꢀmethylcarbazole.
It has been
hypothesised that carbinolamine formation may be stabilised when delocalisation of the nitrogen
6
3
lone pair to an adjacent carbonyl or aromatic group is possible, which would be the case here.
Acetamide formation could arise from further oxidation of the carbinolamine intermediate if it
64
remained in the CYP199A4 active site. By analogy 4ꢀketocyclophosphamide has been
observed as
a
metabolite from P450 catalysed oxidation (arising from 4ꢀ
29
ACS Paragon Plus Environment