The self-sufficient CYP102 family enzyme, Krac9955, from Ktedonobacter racemifer DSM44963 acts as an alkyl- and alkyloxy-benzoic acid hydroxylase

Article abstract of DOI:10.1016/j.abb.2016.12.014

A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the substrate range of this enzyme was hampered by low levels of production using E. coli. The yield and purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag and modification of the purification protocol. The heme domain was isolated and in?vitro analysis of substrate binding and turnover was performed. Krac9955 was found to preferentially bind alkyl- and alkyloxy-benzoic acids (≥95% high spin, K_d?a self-sufficient CYP102 family member Krac9955 showed low levels of NAD(P)H oxidation activity for all the substrates tested though product formation was observed for many. For nearly all substrates the preferred site of hydroxylation of Krac9955 was eight carbons away from the carboxylate group with certain reactions proceeding at ≥ 90% selectivity. Krac9955 differs from CYP102A1 (P450Bm3), and is the first self-sufficient member of the CYP102 family of P450 enzymes which is not optimised for fast fatty acid hydroxylation close to the ω-terminus.

Full text of DOI:10.1016/j.abb.2016.12.014

Archives of Biochemistry and Biophysics 615 (2017) 15e21
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
The self-sufficient CYP102 family enzyme, Krac9955, from
Ktedonobacter racemifer DSM44963 acts as an alkyl- and alkyloxy-
benzoic acid hydroxylase
Natasha K. Maddigan, Stephen G. Bell*
Department of Chemistry, University of Adelaide, SA 5005, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium
Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the
substrate range of this enzyme was hampered by low levels of production using E. coli. The yield and
purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag
and modification of the purification protocol. The heme domain was isolated and in vitro analysis of
substrate binding and turnover was performed. Krac9955 was found to preferentially bind alkyl- and
Received 23 November 2016
Received in revised form
30 December 2016
Accepted 30 December 2016
Available online 31 December 2016
alkyloxy-benzoic acids (ꢀ95% high spin, Kd < 3
mM) over saturated and unsaturated fatty acids. Unusually
Keywords:
for a self-sufficient CYP102 family member Krac9955 showed low levels of NAD(P)H oxidation activity for
all the substrates tested though product formation was observed for many. For nearly all substrates the
preferred site of hydroxylation of Krac9955 was eight carbons away from the carboxylate group with
certain reactions proceeding at ꢀ 90% selectivity. Krac9955 differs from CYP102A1 (P450Bm3), and is the
first self-sufficient member of the CYP102 family of P450 enzymes which is not optimised for fast fatty
Cytochrome P450 enzymes
Monooxygenases
Fatty acid oxidation
Ktedonobacter
Alkylbenzoic acids
Alkyloxybenzoic acids
acid hydroxylation close to the
u-terminus.
© 2016 Elsevier Inc. All rights reserved.
1. Introduction
CYP enzymes as biocatalyts for generating alcohols at unactivated
CeH bonds, especially when these reactions are carried out with
high regio- and stereoselectivity [5e10]. The isolation of new en-
zymes from microbes is an active area of research as these systems
are often involved in the synthesis of complex natural product such
as antibiotics as well as in the breakdown of available organic
molecules for use as a source of energy [11e14]. In addition bac-
terial enzymes have been reported to have the highest activities
among P450 enzymes when their electron transfer systems are
available [6,10,15e20].
The self-sufficient cytochrome P450 enzymes from the CYP102
family consist of a 55 kDa P450 heme domain fused to a 65 kDa
reductase domain and require only substrate, NADPH and oxygen
to function [6]. These systems exhibit the highest turnover fre-
quency (>1000 minꢁ1) of any CYP enzymes which overcomes one
of the major hurdles to their use as catalysts [6,21e23]. As such they
have been used as platforms for creating monooxygenase bio-
catalysts through protein engineering by both site-directed and
random mutagenesis [5,6,23e27]. The best studied member of this
family is CYP102A1 (P450Bm3) from Bacillus megaterium which
hydroxylates saturated and unsaturated fatty acids at sub-terminal
The P450 cytochrome (CYP) superfamily of b-type heme-
dependent monooxygenases catalyse the oxidation of organic
substrates inserting a single oxygen atom from molecular dioxygen
into a carbonehydrogen bond resulting in the corresponding
alcohol [1e4]. As such there is great interest in the application of
Abbreviations: BSTFA, N,O-Bis(trimethylsilyl)trifluoroacetamide; CYP, cyto-
chrome P450 enzyme; DEAE, diethylaminoethanol; DMSO, dimethyl sulfoxide;
EMM, E. coli minimal media; EtOH, ethanol; GB, gblock; GC, gas chromatography;
IPTG, isopropyl-b-D-1-thiogalactopyranoside; Krac0936, a second P450 enzyme
from the CYP102 family found in Ktedonobacter racemifer; Krac9955, the holoen-
zyme produced by the optimised gene; Krac9955HD, the heme domain of the
protein produced from the optimised gene; Krac9955NS, the holoenzyme with a
histidine tag incorporated by removal of the stop codon; Krac9955HDNS, the his-
tidine tagged heme domain; MS, mass spectrometry; NADH, reduced nicotinamide
adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phos-
phate; P450Bm3, the self-sufficient P450 enzyme CYP102A1 from Bacillus mega-
terium; PCR, Polymerase chain reaction; TMSCl, Trimethylsilyl chloride; Tris,
Tris(hydroxymethyl)aminomethane; 2xYT, Yeast Extract Tryptone.
* Corresponding author.
E-mail address: stephen.bell@adelaide.edu.au (S.G. Bell).
http://dx.doi.org/10.1016/j.abb.2016.12.014
0003-9861/© 2016 Elsevier Inc. All rights reserved.

16
N.K. Maddigan, S.G. Bell / Archives of Biochemistry and Biophysics 615 (2017) 15e21
positions, close to the
u
-terminus [6,28,29]. The optimal activity is
(Australian Genome Research Facility, Adelaide node) using the T7F
and T7T primers of the pET26 vector and additional sequencing
primers (Supporting Information).
for longer chain polyunsaturated fatty acids such as arachidonic
acid (5,8,11,14-all-cis-eicosatetraenoic acid) [30,31]. Other self-
supporting fatty acid oxidising P450s have been identified and
investigated including CYP102A family variants from Bacillus spe-
cies, CYP102D1 from Streptomyces avermitilis MA4680 and CYP505
(P450foxy) from the fungus Fusarium oxysporum [6,21,32e36].
Recently we identified two self-sufficient CYP102 family mem-
ber encoding genes (Krac_0936 and Krac_9955) in the bacterium
Ktedonobacter racemifer DSM44963, a species which is represen-
tative of a group of organisms which is genealogically highly iso-
lated [37,38]. Preliminary studies indicated that both could oxidise
fatty acid substrates in vivo but their substrate profiles were nar-
rower than CYP102A1. In vitro analysis showed that Krac0936 more
closely resembled CYP102A1 with a preference for pentadecanoic
acid and longer monounsaturated fatty acids such as oleic acid (cis-
9-octadecenoic acid). However, unlike CYP102A1, which hydrox-
The recombinant plasmids were transformed into Escherichia
coli strain BL21(DE3) and these cells were cultured in 2xYT medium
at 37 ꢂC with 30 g mLꢁ1 kanamycin. When the OD600 of the culture
m
reached 0.6e0.8 the temperature was reduced to 20 ꢂC and the
media was supplemented with 3 mL Lꢁ1 of trace elements solution
(0.74 g CaCl2$H2O, 0.18 g ZnSO4$7H2O, 0.132 g MnSO4$4H2O, 20.1 g
Na2EDTA, 16.7
g FeCl3$6H2O, 0.10 g CuSO4$5H2O, 0.25 g
CoCl2$6H2O), and 1 mM 5-aminolevulinic acid. Finally 0.1 mM IPTG
was added in order to induce the production of the protein. After
further growth for 18 h at 20 ꢂC, cells were harvested by centrifu-
gation, resuspended in 40 mM potassium phosphate, pH 7.4 (buffer
P), 1 mM in dithiothreitol and lysed by sonication on ice. The crude
extracts were then centrifuged at 37000 g for 25 min at 4 ꢂC to
remove the cell debris.
ylated linear saturated fatty acids at
apparent preference, Krac0936 showed a strong preference for the
-1 and -2 positions.
Initial studies on Krac9955 were hampered by low levels of
u
-1,
u
-2 and
u
-3 with no
For Krac9955HDNS the supernatant was loaded directly onto a
GE-Healthcare DEAE fast-flow Sepharose column (XK50,
200 ꢃ 50 mm). The protein was eluted using a linear gradient,
100e400 mM KCl in Tris buffer, pH 7.4. The P450 containing frac-
tions were combined and concentrated to a final volume of ~15 mL
by ultra-filtration using a Vivacell 100 (Sartorius Stedim, 10 kDa
membrane) aided by centrifugation (1500 g). The concentrated
u
u
protein production using E. coli resulting in turnover assays being
performed on extracts from cell lysates rather than purified
enzyme. Krac9955 showed optimal activity with the shorter tri-
decanoic acid and favoured oxidation at more sub-terminal posi-
tions with the preferred site of hydroxylation being eight carbons
from the carboxylate group. The gene encoding Krac9955 con-
tained more clusters of rare codons than that of CYP102A1 and here
we report the improved production of the holoenzyme using codon
optimised genes and the use of affinity tag purification protocols.
We also generated constructs to enable the production of the heme
domain of Krac9955 in order to assess substrate binding and
product formation for the first time.
protein was loaded onto a
5 mL nickel NTA column, pre-
equilibrated with buffer T1 (300 mM NaCl, 20 mM imidazole,
1 mM DTT in 50 mM Tris, pH 7.4). The protein was then washed
with buffer T1 and eluted with buffer T2 (300 mM NaCl, 300 mM
imidazole, 1 mM DTT in 50 mM Tris, pH 7.4). The eluted protein was
concentrated and buffer exchanged with 50 mM Tris, pH 7.4, before
being stored. Krac9955NS (holoprotein with 6 x His tag) was
extracted from E. coli by the same method described above. The
supernatant was subjected to an ammonium sulfate precipitation.
The 25e60% fraction was purified by the nickel NTA column, as
described for Krac9955HDNS. All the proteins were stored
at ꢁ20 ꢂC in 50% (v/v) glycerol.
2. Experimental section
2.1. Cloning, expression and purification
Glycerol and salts were removed from proteins immediately
prior to experiments using a GE Healthcare 5 mL PD-10 desalting
column pre-equilibrated with 50 mM Tris buffer, pH 7.4. The con-
centration of the proteins was calculated using the extinction co-
General DNA and microbiological experiments were carried out
using standard methods [39]. The KOD polymerase, used for the
PCR steps, and pET26a expression vectors were from Merck Bio-
sciences, UK. T4 DNA ligase and restriction enzymes for molecular
biology were from Lucigen and New England Biolabs. Codon opti-
mised DNA was purchased from Integrated DNA Technology (IDT,
USA). The Krac9955 gene was purchased as two gene blocks (GBs)
one of which (GB1) covered the P450 domain, whilst the other
(GB2) encompassed the remainder of the sequence, including the
reductase domain (Supporting Information). GB1 incorporated a 50
NdeI and 30 BamHI and HindIII restriction sites, and GB2 a 50 BamHI
and 30 HindIII site (Fig. S1). These were cloned together into the
vector pET26 using standard techniques to create the full length
optimised gene (Fig. S2). The heme domain (HD) Krac9955HD was
obtained via the polymerase chain reaction (PCR) using the
respective GB1 as a template and the primers listed in the Sup-
porting Information. Additionally a C-terminal 6 x histidine tag was
incorporated into the Krac9955 holoprotein and heme domains by
using PCR to remove the stop codon (Krac9955NS and
Krac9955HDNS, Supporting Information). This method extended
each protein sequence by 13 amino acids by integrating additional
codons from the pET26 vector including those which encode the 6 x
His tag (Supporting Information). PCR was performed using KOD
Hot Start DNA polymerase and the following method: 94 ꢂC, 30 s;
55 ꢂC, 45 s; 68 ꢂC, 205 s (or 100 s for the heme domains) for 30
cycles; 72 ꢂC final extension 10 min and 10 ꢂC final hold. The se-
quences of all clones were confirmed by DNA sequencing
efficient
of
the
CO
reduced
difference
spectrum
ε450 ¼ 91 mMꢁ1 cmꢁ1 [28,40]. Using this value, the extinction
coefficients for the ferric resting state of the heme domain and
holoprotein (Krac9955HDNS and Krac9955NS) were both esti-
mated to be approximately ε419 ¼ 105 mMꢁ1cmꢁ1
.
2.2. Substrate binding and turnover assays
For substrate binding assays the P450 heme domains were
diluted to ~1e2 mM using 50 mM Tris, pH 7.4 and aliquots of the
substrate were added from a 20 mM stock solution in DMSO until
the spectra stopped shifting. The high spin heme content was
estimated, to approximately 5%, by comparison with a set of nine
other spectra (10%e90% in increments of 10%) generated from the
sum of the weighted averages of the spectra of the substrate-free
form (>95% low spin, Soret maximum at 418 nm) and camphor-
bound form (>95% high spin, Soret maximum at 392 nm) of wild-
type CYP101A1 (see supporting information).
For dissociation constant determination the CYP199A4 enzyme
was diluted to 0.5e2.0
mM using 50 mM Tris, pH 7.4, in 2.5 mL and
0.5e2 L aliquots of the substrate were added using a Hamilton
m
syringe from 1, 10 or 20 mM stock solutions in ethanol or DMSO.
The maximum difference in the Soret peak-to-trough absorbance
(DA) was recorded between 700 nm and 250 nm. Further aliquots of
substrate were added until the peak-to-trough difference of the

N.K. Maddigan, S.G. Bell / Archives of Biochemistry and Biophysics 615 (2017) 15e21
17
Soret band did not change. The dissociation constants, Kd, were
obtained by fitting A against total substrate concentration [S] to a
hyperbolic function.
fragments (gblocks; GB1 and GB2), which incorporated the heme
and reductase domains, respectively, into the vector pET26 (Sup-
porting Information, Fig. S1). The portion of the gene which en-
codes the heme domain of Krac9955 (defined in this instance as the
first 473 amino acid residues) was isolated by PCR and cloned into
the same vector (Krac9955HD). Furthermore PCR was used to
remove the stop codon of each construct (Fig. S2). This incorporated
a 13 amino acid insertion at the C-terminus of each construct which
contained a 6 x His tag, which would allow more facile purification
of the proteins (Krac9955HDNS and Krac9955NS, Supporting In-
formation). Each gene sequence was confirmed by DNA sequencing
and all correctly aligned with the sequence of the purchased gene
blocks.
D
D
Amax ꢃ ½Sꢄ
DA ¼
Kd þ ½Sꢄ
All the alkyloxy and alkyl-benzoic acids exhibited tight binding,
with Kd < 5 M and their data were fitted to the tight binding
quadratic equation:
m
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
n
o
ð½Eꢄ þ ½Sꢄ þ KdÞ ꢁ
ð½Eꢄ þ ½Sꢄ þ KdÞ2 ꢁ 4½Eꢄ½Sꢄ
2½Eꢄ
D
A
¼
D
Amax
The level of protein production of each construct was tested in
the presence of the heme precursor,
d-aminolevulinic acid and
where
DAmax is the maximum absorbance difference and [E] is the
trace elements. The proteins could be produced using 2xYT media,
however, the improvement in protein production using the codon
optimised gene was not substantial for the holoenzyme and the
heme domain was not generated in high quantities. The holo-
protein Krac9955NS and the heme domain Krac9955HDNS, were
produced at low levels using the best conditions but the superna-
tant was visibly orange and the levels of protein that could be
purified was enhanced by using affinity chromatography resulting
in sufficient yield for further characterisation (Figs. S3 and S4).
Each purified Krac9555 sample was analysed by SDS-PAGE
analysis which showed that proteins of the correct size were be-
ing produced (Fig. S5). UV/Vis spectroscopy was also used to
monitor the ratio of the Soret peak at 419 nm with the peak at
280 nm (Fig. S4). After purification each protein was tested for the
characteristic Soret absorbance of the CO bound reduced P450.
Purified samples of Krac9955NS and Krac9955HDNS were both
almost completely shifted to 450 nm (ꢀ95%, Fig. S6).
enzyme concentration.
NADPH turnovers were run in 1200 mL of 50 mM oxygenated
Tris, pH 7.4 at 30 ꢂC, containing 0.2
mM enzyme and 120 mg bovine
liver catalase. Assays were held at 30 ꢂC for 1 min prior to addition
of 0.25e1 mM substrate from a 20 mM stock in DMSO. NADPH was
added from a 20 mg mLꢁ1 stock to a final concentration of ~320
mM.
For whole-cell oxidation reactions the protein was produced as
described above. The resulting cell pellet was resuspended in
double the volume of E. coli minimal media containing 2% DMSO;
(EMM; K2HPO4, 7 g, KH2PO4, 3 g, Na3citrate, 0.5 g, (NH4)2SO4,1 g,
MgSO4, 0.1 g, 20% glucose, 20 mL and glycerol, 1% v/v in one litre).
The substrate was added to a final concentration of 0.25e1 mM
before shaking at 180 rpm at 30 ꢂC. After 16 h the turnover was then
centrifuged (15 min, 5000g) and the supernatant isolated. Samples
(1 mL) of the turnover were taken for analysis via GC-MS.
2.3. Product analysis
3.2. Substrate binding
After the in vitro NADPH oxidation assays or whole-cell in-
cubations had finished 998
L of concentrated HCl. The mixture was extracted three times
with 400 L of ethyl acetate and the organic extracts were com-
bined and dried over MgSO4. Solvent was evaporated under a
stream of dinitrogen and the sample dissolved in 200 L anhydrous
acetonitrile. For fatty acids 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 reaction mixtures were used
directly for GC-MS analysis. For straight chain, saturated fatty acids
acid the oven temperature was held at 120 ꢂC for 3 min and then
increased at 7 ꢂC minꢁ1 up to 220 ꢂC were it was maintained for
7 min (total 24.3 min). For the longer benzoic acids (ꢀC15) better
separation of the products was obtained using the following
method; initial temperature of 120 ꢂC was held for 3 min before
being increased to 240 ꢂC at a rate of 7 ꢂC minꢁ1 where it was held
till the end of the run (time 35 min). The retention times for the
trimethylsilyl (TMS) derivatives are given in the Supporting Infor-
mation. Products were identified by matching the GC-MS retention
time and mass spectra fragmentation pattern to those expected for
mL of the reaction mixture was mixed
Previously no assessment of substrate binding could be per-
formed with Krac9955. The holoproteins have interfering absorp-
tion from the FAD and FMN cofactors in the reductase domain and
the heme domain, Krac9955HDNS, was used for binding analyses
with fatty acids. In addition, to determine whether the substrate
range was limited solely to fatty acids, a variety of related molecules
was tested including alkanes and alkenes, methyl esters, alcohols,
and amines (C12). Only the addition of the fatty acid substrate
resulted in a type I shift in the spin state (Fig. S7 and Fig. S8).
Addition of dodecylamine caused a type II shift to 427 nm in
Krac9955HDNS (Fig. S9). A range of straight chain, saturated fatty
acids (C10 to C18) and selected unsaturated fatty acids was therefore
tested for substrate binding to the heme domain of Krac9955.
Krac9955HDNS was observed to have a preference for the
shorter chain fatty acids. The percentage of high spin (HS) enzyme
was seen to increase with chain length from decanoic (<10% high
spin; HS), through to dodecanoic acid (40% HS) before reaching a
maximum at tridecanoic acid (45% HS, Table 1, Fig. 1 and Fig. S7).
The high spin content decreased to 30% HS with tetradecanoic and
20% HS for pentadecanoic, with hexadecanoic, heptadecanoic and
octadecanoic acids all giving shifts of 10% or less (Table 1 and
Fig. S7). In agreement with the preliminary whole-cell turnover
studies the binding data showed that Krac9955 appeared to have a
narrower substrate range than CYP102A1, which by way of com-
parison is shifted to 40%, 65% and 95% high spin by dodecanoic,
tetradecanoic and hexadecanoic acids [6]. In addition the substrate
preference between the Krac9955 and Krac0936 enzymes from
K. racemifer is significantly different [38].
2
m
m
m
m
u
-n trimethylsilyl derivatised alcohols.
3. Results
3.1. Protein production and characterisation
The gene Krac_9955 (Genbank: EFH88481.1) from Ktedonobacter
racemifer DSM44963 encodes
a protein which is similar to
CYP102A1 (41% sequence identity) [38]. Codon optimised genes for
the production of Krac9955 were generated by cloning two gene
Unsaturated fatty acids are known to be good substrates for
CYP102A1 and these were also tested with Krac9955HD. Shorter

18
N.K. Maddigan, S.G. Bell / Archives of Biochemistry and Biophysics 615 (2017) 15e21
Table 1
binding affinity. For example it can bind 4-n-hexylbenzoic acid
(Kd 2.6 0.1 M) and 4-n-nonyloxybenzoic acid
(Kd ¼ 1.9 0.2 M) though neither substrate induces a greater than
Spin state shifts for Krac9955HDNS with a range of saturated fatty acids. For com-
parison CYP102A1, gives spin state shifts of 40%, 65% and 95% for dodecanoic, tet-
radecanoic and hexadecanoic acid, respectively. The equivalent length of the benzoic
acid substrates is given for comparison with the fatty acid substrates.
¼
m
m
50% shift to the high spin form [41]. Given that none of the satu-
rated and unsaturated fatty acids tested thus far with
Krac9955HDNS resulted in a full spin state shift para-substituted
alkyl- and alkyloxy-benzoic acids were tested as potential sub-
strates. Addition of 4-n-heptylbenzoic acid to Krac9955HDNS
resulted in a 90% shift to the high spin form indicating a good
substrate/enzyme active site fit (Table 1 and Fig. S11). Further tests
of Krac9955 using para substituted benzoic acids with varying
chain lengths showed that 4-n-hexylbenzoic acid displayed a
reduced spin state shift (35% HS). The shorter alkyl chain substrates,
4-n-pentylbenzoic acid, 4-n-butylbenzoic acid and 4-n-pro-
pylbenzoic acids did not induce a significant shift on binding to
Krac9955HD (0e10% HS, Table 1 and Fig. S11). The shortest chain
alkyloxybenzoic acid substrate, 4-n-propoxybenzoic acid, did not
shift the spin state whilst 4-n-butoxybenzoic acid gave a slight shift
(<10%). Extending the chain length was found to increase the
extent of the spin state shift with 4-n-pentyloxybenzoic acid giving
a shift of 45% (Fig. S11). 4-n-Hexyloxybenzoic acid, 4-n-heptylox-
ybenzoic acid and 4-n-octyloxybenzoic acid all gave shifts of ꢀ95%
(Fig. 1 and Fig. S11) but 4-n-nonyloxybenzoic acid and 4-n-unde-
cylbenzoic acid resulted in lower shifts, 80% and 0%, respectively
(Fig. S11). Krac0936 was tested with each of these benzoic acid
substrates and no shift was observed.
Substrate
Length
Krac9955HDNS
HS%
decanoic acid
undecanoic acid
dodecanoic acid
tridecanoic acid
tetradecanoic acid
pentadecanoic acid
hexadecanoic acid
heptadecanoic acid
C10
C11
C12
C13
C14
C15
C16
C17
C18
C14
C16
C18
C18
C18
C18
C20
C22
<10
35
40
45
30
20
10
<10
<10
40
octadecanoic acid
cis-9-tetradecenoic acid
cis-9-hexadecenoic acid
cis-9-octadecenoic acid
trans-9-octadecenoic acid
cis,cis-9,12-octadecadienoic acid
cis,cis,cis-9,12,15-octadecatrienoic acid
5,8,11,14-all-cis-eicosatetraenoic acid
cis-13-docosenoic acid
30
<10
<10
10
50
40
0
4-n-butylbenzoic acid
4-n-pentylbenzoic acid
4-n-hexylbenzoic acid
4-n-heptylbenzoic acid
4-n-octylbenzoic acid
4-n-propoxybenzoic acid
4-n-butoxybenzoic acid
4-n-pentyloxybenzoic acid
4-n-hexyloxybenzoic acid
4-n-heptyloxybenzoic acid
4-n-octyloxybenzoic acid
4-n-nonyloxybenzoic acid
4-n-undecyloxybenzoic acid
9
0
<10
35
10
11
12
13
9
10
11
12
13
14
15
17
90
ꢀ95
0
Dissociation constants were determined for Krac9955HDNS
with the four substrates which gave the greatest spin state shift. 4-
<10
45
ꢀ95
ꢀ95
ꢀ95
80
n-Octyloxybenzoic acid (Kd ¼ 2.8
ybenzoic acid, Kd ¼ 2.8 0.2 M, bound more tightly than 4-n-
hexyloxybenzoic acid, Kd ¼ 8.5 1.5 M (Fig. 2), and 4-n-heptyl-
0.7 M, (Fig. S12). Weaker binding and low
0.1 mM) and 4-n-heptylox-
m
m
benzoic, Kd ¼ 11
m
0
substrate solubility prevented accurate determination of the
binding affinity for the fatty acid substrates. The Kd of tridecanoic
acid could be estimated, Kd ¼ 45
5 mM, indicating a significantly
monounsaturated fatty acids showed improved binding to
Krac9955 with cis-9-tetradecenoic (40% HS) and cis-9-
hexadecenoic (30% HS) both giving larger shifts than the equiva-
lent saturated acids (Table 1 and Fig. S10). However no visible
change was observed for the mono- and di-unsaturated C18 chain
acids. The polyunsaturated fatty acids cis,cis,cis-9,12,15-
octadecatrienoic and 5,8,11,14-all-cis-eicosatetraenoic acids
induced moderate spin state shifts with Krac9955HD (50% and 40%,
respectively, Table 1 and Fig. S10).
lower binding affinity with Krac9955 compared to the benzoic acid
derivatives. Overall these benzoic acid derivatives appear to be
more promising subtrates than regular fatty acids for Krac9955.
3.3. Activity assays and product formation
Using our assay conditions resulted in no increase in the rate of
NADPH (or NADH) oxidation for Krac9955NS above the leak rate
upon addition of the fatty and benzoic acid substrates (<10 nmol-
nmol P450ꢁ1-minꢁ1, Fig. S13). This was unexpected given that
CYP102A1 has also been reported to accept non-natural sub-
strates, such as alkylbenzoic and alkyloxybenzoic acids, with high
Fig. 1. Spin state shifts of tridecanoic acid and 4-n-hexyloxybenzoic acid with the heme domain of Krac9955 (Krac9955HDNS).

N.K. Maddigan, S.G. Bell / Archives of Biochemistry and Biophysics 615 (2017) 15e21
19
product at
also observed at
Fig. S14). Tridecanoic acid hydroxylation resulted in a shift in the
major product to -4 (62%) with a lower proportion of -2 and -3
products (16% and 20%) and -1, -5, -6 (all 1%) as minor products
u
-3 (68%) followed by
u
-2 (17%). Minor products were
u
-1 (8%),
u-4 (6%) and u-5 (1%), (Table 2 and
u
u
u
u
u
u
(Table 2 and Fig. 3). The systematic shift in the oxidation products to
more subterminal positions continued with tetradecanoic acid.
Two major products at
with tetradecanoic acid. Minor products at
u
-4 and
u
-5 were identified (33% and 38%)
-1, -2, -3 and -6
u
u
u
u
were also observed (Table 2 and Fig. S14). The oxidation of the
trans-9-octadecenoic unsaturated fatty acid generated low levels of
hydroxylated products at
u-1 and u-2 (Fig. S14). The turnovers of
the other unsaturated fatty acids resulted in little or no product
formation and were not analysed further.
In line with their greater spin state shifts and binding affinities
the turnovers of the benzoic acid derivatives with alkyl or alkyloxy
chains of six atoms or longer also generated hydroxylated products.
The products were identified in the same manner as the fatty acids
using the MS fragmentation patterns (Supporting Information). The
oxidation of 4-n-octylbenzoic acid was the most efficient gener-
Fig. 2. Dissociation constants of 4-n-hexyloxybenzoic acid with Krac9955HDNS. The
different symbols correspond to each of the three triplicate runs that were performed.
ating alcohol products at
product at -3 and -2 (Table 2 and Fig. 4). The turnovers of 4-n-
heptylbenzoic acid produced the -3 alcohol as the major product
(89%). Similarly, 4-n-hexylbenzoic acid produced a major oxidation
product at -2 (89%, Table 2, Fig. S15). No product was detected
u-4 (70%) and u-2 (20%) with minor
u
u
several of the alkyl- and alkyloxy-benzoic acid substrates induced a
ꢀ95% shift to the high spin form of the enzyme. CYP102A1 is known
to function as a dimer which is more prominent at higher con-
centrations (above 10 nM) [42,43]. Increasing the concentration of
u
u
after the in vitro turnovers of 4-n-pentyl- and 4-n-butyl-benzoic
acids with Krac9955NS but a small amount of product could be
observed using the whole-cell oxidation system (Table S1 and
Fig. S16).
enzyme from 0.2 mM to 0.8 mM in our assays did not result in any
increase in the rate of NADPH oxidation (data not shown).
GC-MS analysis of BSTFA/TMSCl derivatised turnover extracts
was used to investigate product formation and identification which
was based on the MS fragmentation patterns. For all of the products
there was a peak corresponding to that of the doubly derivatised
mass minus 15 (due to the cleavage of a methyl group). An increase
in the base peaks at m/z at 117.1, 131.1, 145.1, 159.1, 173.1 and 187.1
4-n-Octyloxybenzoic acid oxidation by Krac9955NS generated
five products with the
(71%). The -3 (20%) and the
lower quantities with -1 and
u
-5 alcohol being the major metabolite
-4 (8%) alcohols were formed in
-2 being formed in low yields (<1%
u
u
u
u
each, Table 2 and Fig. S15). Decreasing the chain length in 4-n-
heptyloxybenzoic acid saw a shift in the preferred position of hy-
droxylation to the u-4 hydroxyl group (71%) with u-2 (27%) making
up the majority of the remaining products (Table 2 and Fig. 4). The
trend towards more terminal oxidation continued with 4-n-hex-
yloxybenzoic acid and 4-n-pentyloxybenzoic acid whose turnovers
also showed increased selectivity (Table 2 and Fig. 4). The major
was used to identify the respective
u-1 to u-6 products for the
Krac9955 turnovers (Supporting Information). These peaks arise
from cleavage at positions adjacent to the OSiMe3 (CH3CHOSiMeþ3 ).
A secondary cleavage, also adjacent to the OSiMe3, often gave
confirmatory peaks at the derivatised product mass minus 15, 29,
43, 57, 71 and 85 (Supporting Information). Similar product dis-
tributions were observed for each substrate with the whole-cell
oxidations and the in vitro turnovers.
For saturated fatty acids the highest levels of product were
generated with tridecanoic acid [38]. The product distributions
were in broad agreement with those reported earlier using whole-
cell oxidations and crude cell lysates (Scheme 1) [38]. Oxidation of
dodecanoic acid produced five alcohol products with the major
product of 4-n-hexyloxybenzoic acid oxidation was the
alcohol while for 4-n-pentyloxybenzoic acid the alcohol at
(93%) was generated in the highest proportion (Fig. 4 and Fig. S16).
The alcohol at -1 (7%) was the most prominent minor product for
both substrates with low levels of the -2 alcohol (3%) being
u-3 (90%)
u
-2
u
u
observed in the turnover of 4-n-hexyloxybenzoic acid. Little or no
Table 2
Product distribution data from the in vitro turnover reactions of Krac9955 with
different fatty acid and benzoic acid substrates. The data are reported as the mean of
at least three experiments with standard deviations of <5%. Whole-cell turnovers
generated the products in the same ratios. The whole-cell turnovers generated
products with certain substrates which could not be detected after the in vitro
turnovers (Table S1).
Substrate
u
-1%
u
-2%
u
-3%
u
-4%
u
-5%
u
-6%
dodecanoic acid
tridecanoic acid
tetradecanoic acid
8
1
3
39
<1
<1
7
7
2
7
10
17
16
10
61
<1
27
3
93
20
4
68
20
14
6
62
33
1
1
38
1
2
trans-9-octadecenoic acid
4-n-octyloxybenzoic acid
4-n-heptyloxybenzoic acid
4-n-hexyloxybenzoic acid
4-n-pentyloxybenzoic acid
4-n-octylbenzoic acid
4-n-heptylbenzoic acid
4-n-hexylbenzoic acid
21
1
90
8
71
70
8
89
<1
70
Scheme 1. The site of the major hydroxylation product (38%e93%) in the Krac9955
turnovers of fatty acids and alkyl and alkyloxybenzoic acids.
89

20
N.K. Maddigan, S.G. Bell / Archives of Biochemistry and Biophysics 615 (2017) 15e21
nonyloxybenzoic acid (u-4 and u-6, Table S1 and Supporting In-
formation). There was no evidence of Krac9955 catalysed oxidative
dealkylation for any of the substrates tested.
The oxidation of the benzoic acid derivatives by Krac9955NS
occurs at increasingly sub-terminal positions as the chain is
lengthened (Scheme 1). The major site for each benzoic acid
consistently occurs five atoms from the aromatic ring, eight carbons
from the carboxylate, with the main minor product, where possible,
occurring two carbons further away towards the
u terminus. In
addition the selectivity became more pronounced as the chain
length decreased (Table 2).
4. Discussion
Krac9955 is a self-sufficient heme monooxygenase from the
CYP102 family. It was produced in greater quantities and higher
purities than previously by using codon optimised genes and a His
tagged purification strategy. The purified holoprotein and heme
domain were obtained which allowed more detailed characterisa-
tion of the substrate range. Krac9955 functions on a narrower range
of fatty acid substrates compared to CYP102A1 with the highest
levels of product formation observed with tridecanoic acid.
CYP102A1 is known to undergo ꢀ95% shifts to the high spin form
with several saturated and unsaturated fatty acids (e.g hex-
adecanoic acid, cis-9-hexadecenoic and 5,8,11,14-all-cis-eicosate-
traenoic acids) however Krac9955 did not oxidise these substrates
effectively [6,31,44]. Krac9955 was found to bind 4-n-alkyl and 4-n-
Fig. 3. Product distributions of in vitro turnovers of tridecanoic acid with Krac9955NS.
Impurities are labelled (*) as are the products.
detectable product was generated with 4-n-butyloxybenzoic acid
but a metabolite could be detected using the whole-cell oxidation
system (Table 1 and Fig. S16). No product was observed with the
longer chain 4-undecyloxybenzoic acid but trace amounts of two
products could be detected in the in vitro turnovers of 4-n-
Fig. 4. Product distributions of in vitro turnovers of 4-n-heptylbenzoic, 4-n-octylbenzoic acid, 4-n-hexyloxybenzoic and 4-n-heptyloxybenzoic acids with Krac9955NS. Impurities
are labelled (*) as are the products.

N.K. Maddigan, S.G. Bell / Archives of Biochemistry and Biophysics 615 (2017) 15e21
21
[5] S.G. Bell, N. Hoskins, C.J.C. Whitehouse, L.L. Wong, in: A. Sigel, H. Sigel,
R.K.O. Sigel (Eds.), Met. Ions Life Sci, John Wiley & Sons, Weimheim, 2007, pp.
437e476.
[6] C.J. Whitehouse, S.G. Bell, L.L. Wong, Chem. Soc. Rev. 41 (2012) 1218e1260.
[7] G.D. Roiban, M.T. Reetz, Chem. Commun. 51 (2015) 2208e2224.
[8] R. Fasan, ACS Catal. 2 (2012) 647e666.
[9] S.G. Bell, L.L. Wong, in: R.B. King (Ed.), Encyclopedia of Inorganic Chemistry,
Wiley, New York, 2005.
[10] V.B. Urlacher, S.G. Bell, L.L. Wong, in: R.D. Schmid, V.B. Urlacher (Eds.), Modern
Biooxidation, Wiley, New York, 2007, pp. 99e122.
[11] S. Janocha, D. Schmitz, R. Bernhardt, Adv. Biochem. Eng. Biotechnol. 148
(2015) 215e250.
[12] S.L. Kelly, D.E. Kelly, Philos. Trans. R. Soc. Lond. B Biol. Sci. 368 (2013)
20120476.
[13] K.R. Kim, D.K. Oh, Biotechnol. Adv. 31 (2013) 1473e1485.
[14] L.M. Podust, D.H. Sherman, Nat. Prod. Rep. 29 (2012) 1251e1266.
[15] S.G. Bell, A. Dale, N.H. Rees, L.L. Wong, Appl. Microbiol. Biotechnol. 86 (2010)
163e175.
[16] W. Yang, S.G. Bell, H. Wang, W. Zhou, N. Hoskins, A. Dale, M. Bartlam,
L.L. Wong, Z. Rao, J. Biol. Chem. 285 (2010) 27372e27384.
[17] D. Zehentgruber, F. Hannemann, S. Bleif, R. Bernhardt, S. Lutz, Chem. Biochem.
11 (2010) 713e721.
alkyloxybenzoic acids with higher affinity and oxidised these sub-
strates efficiently. In almost all cases Krac9955 favoured hydrox-
ylation at the CeH bonds of the fatty acids which were eight
carbons away from the carboxylic acid group in both the fatty acid
and benzoic acid substrates.
CYP102A1 has previously been investigated in detail with two
benzoic acid derivatives. It produced two major products after
oxidising 4-n-hexylbenzoic acid with the major and minor products
occurring at
acid produced both a singly hydroxylated
well as a dihydroxy -2, -4 (minor) product. With Krac9955 our
u
-2 and
u
-1 in a 7:1 ratio [41]. 4-n-Nonyloxybenzoic
u
-2 (major) alcohol as
u
u
data suggests that the benzoic acid substrates are positioned in a
way that aligns the fifth carbon from the benzene ring (eighth
carbon from the carboxylate group) most closely to the reactive
oxygen intermediate, however structural data would be needed to
confirm this. In contrast to CYP102A1, Krac9955 was not was active
towards longer polyunsaturated fatty acids such as (5Z,8Z,11Z,14Z)-
5,8,11,14-eicosatetraenoic acid (arachidonic acid). More detailed
characterisation of the structure of the heme domains of Krac9955
would enable insight into these differences between the CYP102
enzymes.
[18] S.G. Bell, J.H. McMillan, J.A. Yorke, E. Kavanagh, E.O. Johnson, L.L. Wong, Chem.
Commun. 48 (2012) 11692e11694.
[19] S.G. Bell, A.B. Tan, E.O. Johnson, L.L. Wong, Mol. Biosyst. 6 (2010) 206e214.
[20] D.B. Hawkes, G.W. Adams, A.L. Burlingame, P.R. Ortiz de Montellano, J.J. de
Voss, J. Biol. Chem. 277 (2002) 27725e27732.
[21] P.K. Chowdhary, M. Alemseghed, D.C. Haines, Arch. Biochem. Biophys. 468
(2007) 32e43.
The low NADPH oxidation activity of Krac9955 cannot be
ascribed to the lack of suitable substrate as several which bind with
high affinity and induce an almost complete shift have been
discovered. Nor can it be attributed to the low yield and purity of
the enzyme. This is the first example of a self-sufficient enzyme of
this family which does not have enhanced activity with high af-
finity substrates. The enzyme does generate product in good yield
and the Soret band of the protein suggests the heme domain is
intact. CYP102A1 is known to function as a dimer and to have very
low activity due to intramoleculer electron transfer. One potential
explanation for the slow NADPH oxidation rate of Krac9955 is that
it does not dimerise and that the electron transfer was purely
intramolecular and inefficient. Alternatively the rate determining
step in the P450 catalytic cycle of this enzyme may not be related to
electron transfer. Krac9955 may have a role in its host bacterium,
which in contrast to the other CYP102 family members, requires
only low activity. Further investigation is necessary to confirm this
hypothesis but the results show that the fusion protein nature of
P450-electron transfer partner proteins does not necessarily guar-
antee high activity.
[22] B. Rowlatt, J.A. Yorke, A.J. Strong, C.J. Whitehouse, S.G. Bell, L.L. Wong, Protein
Cell 2 (2011) 656e671.
[23] M.A. Noble, C.S. Miles, S.K. Chapman, D.A. Lysek, A.C. MacKay, G.A. Reid,
R.P. Hanzlik, A.W. Munro, Biochem. J. 339 (Pt 2) (1999) 371e379.
[24] C.J. Whitehouse, S.G. Bell, H.G. Tufton, R.J. Kenny, L.C. Ogilvie, L.L. Wong, Chem.
Commun. (2008) 966e968.
[25] A. Seifert, S. Vomund, K. Grohmann, S. Kriening, V.B. Urlacher, S. Laschat,
J. Pleiss, Chem. Biochem. 10 (2009) 853e861.
[26] J.A. McIntosh, P.S. Coelho, C.C. Farwell, Z.J. Wang, J.C. Lewis, T.R. Brown,
F.H. Arnold, Angew. Chem. Int. Ed. Engl. 52 (2013) 9309e9312.
[27] S. Kille, F.E. Zilly, J.P. Acevedo, M.T. Reetz, Nat. Chem. 3 (2011) 738e743.
[28] S.S. Boddupalli, R.W. Estabrook, J.A. Peterson, J. Biol. Chem. 265 (1990)
4233e4239.
[29] M.J. Cryle, R.D. Espinoza, S.J. Smith, N.J. Matovic, J.J. De Voss, Chem. Commun.
(2006) 2353e2355.
[30] J.F. Buchanan, A.J. Fulco, Biochem. Biophys. Res. Commun. 85 (1978)
1254e1260.
[31] J.H. Capdevila, S. Wei, C. Helvig, J.R. Falck, Y. Belosludtsev, G. Truan,
S.E. Graham-Lorence, J.A. Peterson, J. Biol. Chem. 271 (1996) 22663e22671.
[32] K.Y. Choi, E. Jung, D.H. Jung, B.P. Pandey, H. Yun, H.Y. Park, R.J. Kazlauskas,
B.G. Kim, FEBS J. 279 (2012) 1650e1662.
[33] M. Dietrich, S. Eiben, C. Asta, T.A. Do, J. Pleiss, V.B. Urlacher, Appl. Microbiol.
Biotechnol. 79 (2008) 931e940.
[34] M.C. Gustafsson, O. Roitel, K.R. Marshall, M.A. Noble, S.K. Chapman,
A. Pessegueiro, A.J. Fulco, M.R. Cheesman, C. von Wachenfeldt, A.W. Munro,
Biochemistry 43 (2004) 5474e5487.
[35] T. Kitazume, A. Tanaka, N. Takaya, A. Nakamura, S. Matsuyama, T. Suzuki,
H. Shoun, Eur. J. Biochem. 269 (2002) 2075e2082.
Acknowledgements
[36] N. Nakayama, A. Takemae, H. Shoun, J. Biochem. 119 (1996) 435e440.
[37] Y.J. Chang, M. Land, L. Hauser, O. Chertkov, T.G. Del Rio, M. Nolan, A. Copeland,
H. Tice, J.F. Cheng, S. Lucas, C. Han, L. Goodwin, S. Pitluck, N. Ivanova,
G. Ovchinikova, A. Pati, A. Chen, K. Palaniappan, K. Mavromatis, K. Liolios,
T. Brettin, A. Fiebig, M. Rohde, B. Abt, M. Goker, J.C. Detter, T. Woyke,
J. Bristow, J.A. Eisen, V. Markowitz, P. Hugenholtz, N.C. Kyrpides, H.P. Klenk,
A. Lapidus, Stand. Genomic Sci. 5 (2011) 97e111.
SGB thanks the Australian Research Council for a Future
Fellowship (FT140100355).
Appendix A. Supplementary data
[38] S.D. Munday, N.K. Maddigan, R.J. Young, S.G. Bell, Biochim. Biophys. Acta 1860
(2016) 1149e1162.
[39] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, New York, 1989.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.abb.2016.12.014.
[40] T. Omura, R. Sato, J. Biol. Chem. 239 (1964) 2370e2378.
[41] R.K. Gudiminchi, M.S. Smit, Appl. Microbiol. Biotechnol. 90 (2011) 117e126.
[42] T. Kitazume, D.C. Haines, R.W. Estabrook, B. Chen, J.A. Peterson, Biochemistry
46 (2007) 11892e11901.
[43] R. Neeli, H.M. Girvan, A. Lawrence, M.J. Warren, D. Leys, N.S. Scrutton,
A.W. Munro, FEBS Lett. 579 (2005) 5582e5588.
References
[1] P.R. Ortiz de Montellano, Cytochrome P450: Structure, Mechanism and
Biochemistry, Springer International Publishing, Cham, 2015.
[2] A. Sigel, H. Sigel, R. Sigel, The Ubiquitous Roles of Cytochrome P450 Proteins,
John Wiley & Sons, Weinheim, 2007.
[3] F.P. Guengerich, Chem. Res. Toxicol. 14 (2001) 611e650.
[4] P.R. Ortiz de Montellano, Chem. Rev. 110 (2010) 932e948.
[44] A.W. Munro, S. Daff, J.R. Coggins, J.G. Lindsay, S.K. Chapman, Eur. J. Biochem.
239 (1996) 403e409.