Structure-based design, synthesis of novel probes for cytochrome P450 OleT

Article abstract of DOI:10.1016/j.cclet.2020.09.042

Cytochrome P450 OleT_SA, a new cytochrome P450 enzyme from Staphylococcus aureus, catalyzes the oxidative decarboxylation and hydroxylation of fatty acids to generate terminal alkenes and fatty alcohols. The mechanism of this bifurcative chemist

Full text of DOI:10.1016/j.cclet.2020.09.042

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Structure-based design, synthesis of novel probes for cytochrome P450
OleT
Dumei Ma, Libo Zhang, Yin Yingwu, Qian Wang
PII:
S1001-8417(20)30547-7
https://doi.org/10.1016/j.cclet.2020.09.042
CCLET 5871
DOI:
Reference:
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Chinese Chemical Letters
Received Date:
Revised Date:
Accepted Date:
31 May 2020
13 August 2020
23 September 2020
Please cite this article as: Ma D, Zhang L, Yingwu Y, Wang Q, Structure-based design,
synthesis of novel probes for cytochrome P450 OleT, Chinese Chemical Letters (2020),
doi: https://doi.org/10.1016/j.cclet.2020.09.042
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Chinese Chemical Letters
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Communication
Structure-based design, synthesis of novel probes for cytochrome P450 OleT
a,1
b,1
a,
b,


Dumei Ma , Libo Zhang , Yingwu Yin , Qian Wang
a Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China
b Department of Chemistry and Biochemistry, University of South Carolina, SC 29208, Columbia, United States
Graphical Abstract
Fine-tuned structure of substrates regulates OleT bufurcative pathway
Alcohol
n
o
i
t
a
l
y
x
Fatty acids with different tail
o
b
r
r
a
O
c
e
R
D
H
OH
y
d
o
x
y
l
a
t
i
o
n
Alkene
Substrate
COOH
OH
R
engineering
N
HO
O
O
N
O
S
O
N
O
H
N
N
O
N
H
H
N
H3C
S
O
N
O
O
H
S
O
N
N
H
N
N
N
N
H
H
S
H3C
10 different tails
O
N
S
N
N
N
N
N
H
H
H
O
A class of structurally diversified fatty acids were synthesized and analyzed aiming to explore the
decarboxylation/hydroxylation reaction of OleT.
ARTICLE INFO
ABSTRACT
Article history:
Cytochrome P450 OleTSA, a new cytochrome P450 enzyme from Staphylococcus aureus,
catalyzes the oxidative decarboxylation and hydroxylation of fatty acids to generate terminal
alkenes and fatty alcohols. The mechanism of this bifurcative chemistry remains largely
unknown. Herein, a class of derivatized fatty acids were synthesized as probes to investigate
the effects of substrate structure on the product type of P450 OleTSA. The results demonstrate
that the fine-tuned structure of substrates, even in a remote distance from the carboxyl group,
significantly regulates OleT catalyzed decarboxylation/hydroxylation reactions. Molecular
docking analysis indicated the potential interactions between the carboxylate groups of
different probes and the enzyme active center which was attributed to the bifurcative
chemistry.
Received 31 May 2020
Received in revised form 11 August 2020
Accepted 6 September 2020
Available online
Keywords:
Substrate engineering
Cytochrome P450 OleT
Decarboxylation
Hydroxylation
Fatty acid derivative
Cytochromes P450 (P450s) are a large family of heme-containing redox enzymes, playing important role in the synthesis of many
molecules such as steroid hormones, cholesterol and other fats and acids [1-4]. Among them, OleT, a new cytochrome P450s enzyme
firstly reported by Rude and coworkers in 2011 [5], has attracted great attentions because of its ability to utilize hydrogen peroxide
(H2O2) as cofactor to catalyze the decarboxylation and hydroxylation (mainly at β-position) chemistries on abundant and bioavailable
———
1These authors contributed equally to this work.
 Corresponding authors:
E-mail address: ywyin@xmu.edu.cn (Y. Yin), wang263@mailbox.sc.edu (Q. Wang).

2
fatty acids [6-7] to generate valuable chemicals. As shown in Fig. 1, both reactions are initiated from the abstraction of a substrate
hydrogen atom from the β-position of the free fatty acids by the high-valent iron (IV)-oxo heme π-cation radical intermediate
(Compound I) [8-9], followed by a competition between the OH rebound and carbon-carbon scission that deliver fatty alcohols and
terminal olefin products [10].
Many studies have reported that, with different substrates (e.g., different chain lengths of fatty acids), the product profile including
the ratio of terminal alkene and fatty alcohols produced by OleT catalysis was different. For eicosanoic acid, terminal alkene was
identified as the sole product; while for lauric acid, more than half of the products were identified as fatty alcohols [11-12]. However,
the specific determinants that regulate the hydroxylation and decarboxylation have not been clearly elucidated so far. Researchers
strived to explore the bifurcative mechanism mainly through enzyme engineering. For example, Reetz and coworkers reported some
OleT mutants (Phe294Ala, Phe79Leu) could induce some trans/cis-selectivity change on the aromatic carboxylic acids [13]. Bai et al.
constructed OleT-BM3R by genetic fusion and found that the ratio of hydroxylation/decarboxylation products was dependent on chain
length [14]. Makris group adopted small molecules (e.g., norcarane) to trigger the bifurcative pathway [15]. Moreover, they found that
the distal F-G loop, a structurally disordered region, was critical for mediating the product release as well as the shift of
decarboxylation/hydroxylation reactions [11].
Fig. 1. The reaction and proposed mechanism of P450 OleT catalysis.
In this works, a class of structurally diversified carboxylic acids with different end groups and anchors were synthesized and
analyzed as OleT non-natural substrates aiming to better understand OleT catalyzed decarboxylation and hydroxylation reaction.
First, 11-(7-hydroxycoumarintriazolo)undecanoic acid (HCTUDA) with a coumarin anchored by a triazole in the end of fatty acid
(Scheme S1 in Supporting information) and 11-(dansylamino)undecanoic acid (DAUDA) were employed to study the OleTSA based
enzymatic catalysis (Fig. 1). Both compounds could be accepted as substrates of OleT despite of the bulky group attached to the fatty
acid, and more than 99% of both probes were readily metabolized. More interestingly, significant different product profiles were
observed. As shown in Fig. 2A, DAUDA was converted to terminal alkene as a sole product via the decarboxylation reaction (Fig. S1 in
Supporting information), while HCTUDA was hydroxylated predominantly with different alcohols produced (Fig. S2 in Supporting
information). These results indicate that the changes in the remote group of the substrates have profound effects on the enzyme
catalyzed product distribution.
Fluorescent probes are useful to track enzymatic reactions without the need of additional protein labeling [16]. As shown in Fig. 2B,
increasing fluorescence intensity with a blue shift (17 nm) was observed for DAUDA with the addition of OleT and H2O2, which is
consistent with the previous study [11]. By contrast, the fluorescence of HCTUDA was significantly quenched during the enzymatic
reaction (Fig. 2C), while the enzyme only and H2O2 only had no such effect (Fig. S12 in Supporting information).

Fig. 2. (A) Molecular structures of DAUDA and HCTUDA and their metabolic profiles (For HCTUDA, alcohol products were > 95%; Fluorescence quenching
experiments of (B) DAUDA and (C) HCTUDA (OleTSA 5 μmol/L, DAUDA 35 μmol/L, HCTUDA 2 μmol/L).
Next, more non-natural substrates containing different end groups (phenyl, tolyl and naphthyl) and anchors (triazole, sulfonyl, urea,
thiourea) derivatized fatty acids were designed and synthesized to further investigate the relationship between the substrate structure
and the product profile of OleT (Table 1, Figs. S3-S9 in Supporting information). We found that, with the phenyl end group and
different anchors (1c-1f), all probes could be metabolized by OleT with the conversion rates of 42.1%-89.2%. Notably, the product
patterns of these substrates were dramatically different. For the molecule 1c, the ratio of hydroxylated and decarboxylated products
were 3:7, whereas substrate 1d mainly went hydroxylation with the only minor decarboxylated product (with 14:1 ratio). Probes 1e and
1f bearing urea and thiourea moieties exhibited similar products pattern. Interestingly, 1g and 1h with the addition of methyl group in
tails showed different alcohol/alkene product ratios compared to 1c and 1d without methyl group, indicating the importance of the
substrates’ remote end in OleT catalysis. The enzyme showed much low activity when the naphthyl group was attached in the fatty acid
tails (1i-1j).
It is well known that molecular size is important for enzymatic metabolism, as most physicochemical properties are strongly size-
related [16-18], which can be estimated from 3D molecular geometries using van der Waals radius [19]. Therefore, the structures of
DAUDA and HCTUDA were optimized by density-functional theory (DFT) to estimate the molecular parameters with the
consideration of solvent effect. The result was shown in Table S1 (Supporting information). The structures of these two molecules are
significantly different. DAUDA molecule displays twisted conformation providing a more concentrated molecule, while HCTUDA
molecule stretches out displaying longer conformation. While for eicosanoic acid and lauric acid that also have dramatically different
product profile, no such kind of conformational alterations were observed (Table S1). Other geometry variations (e.g., height, shape and
angle) were also observed among different fatty acid derivatives (Tables S1 and S2 in Supporting information), revealing no significant
correlations based on the DFT calculation. We therefore speculate that the substrate binding pocket of OleT provides unique
environment where the non-natural substrates’ geometry and conformation are stringently confined, which triggers the bifurcative
pathway.
Table 1 Metabolic profile of OleTSA with differnt fatty acid derivativesa.
Alcohol Alkene
product product
Conversion
Entry
Substrate
(%)
(%)
(%)
1c
29.8
70.2
89.2
1d
1e
1f
93.4
60.2
69.2
6.6
83.8
42.1
74.9
39.8
30.8

1g
1h
1i
44.7
70.6
55.3
29.4
94.8
81.7
Low activity
15.8
1j
84.2
18.6
a Reaction conditions: Solutions containing 5 μmol/L OleTSA, 0.5 mmol/L substrates, 0.3 μmol/L GOx, and 6.6 mmol/L of glucose in 150 μL were incubated at
room temperature for 60 min [12]. The reactions were quenched by addition of 1% acetic acid. After centrifuge at 12000 rpm for 20 min, the supernatants were
analyzed with LC-MS.
To further understand the bifurcative reaction for different probes, additional molecular docking studies were carried out. The
structure of OleTJE, a functional ortholog from Jeotgalicoccus sp. 8456 has been reported (PDB code of ligand free enzyme: 4L54; PDB
code of eicosanoic acid bound enzyme: 4L40) [7,20]. It shows high amino acid sequence identity (69%), and all secondary elements are
identity to OleTSA [21,22]. The ligand free OleTJE was used as template for docking [23]. OleT has an elongated hydrophobic fatty acid
binding pocket and a flexible F-G loop which regulates its catalysis [7,11]. As shown in Figs. 3A and B, the eicosanoid acid’s
orientation and geometry are maintained through hydrogen bonding with Arg245 and a series of hydrophobic contacts including Leu41,
Phe46, Phe294, Leu176 lining the entrance and Phe173, Pro296, Pro293, Val292, Phe291 locating at the pocket. Though there is no
clear evidence of His85 in the catalysis, collective studies indicate that this residual, as a potential proton donor to reactive iron-oxo
species, may play significant roles during substrate decarboxylation [20,24,25].
The predicted DAUDA bound OleT model reveals that the end group of DAUDA is sandwiched by Phe46 and Phe294, and the
contacts with the hydrophobic residues are largely maintained. The position of carboxylate group of DAUDA is similar to that of
eicosanoic acid. Hydrogen bonding with Arg245 is the only polar contacts (Figs. 3C and D). The distance between His85 and the
carboxylate of DAUDA is 5.1 Å. For comparison, a docking was done with the probe 1d which has fatty alcohols as main products
(Fig. S10 in Supporting information). The predicated model shows that there is a slight shift of the carboxylate group of 1d relative to
that of DAUDA, implying that the interactions between the bulky end group of the probe and the enzyme induce the position change of
the carboxylate, thus affect the reaction bifurcation dramatically.
HCTUDA bond OleT model reveals that the end group of this probe slides towards to the pocket, which can be attributed to the
strong polarity of the hydroxylated coumarin and the triazole anchor (Fig. 3E). Interestingly, a significant shift of the carboxylates in the
active site is induced with this structure of end group (Fig. 3F). The distance between the carboxylate group and His85 is 3.1 Å within
hydrogen bonding distance. This position of carboxylate is more like that of lauric acid (Fig. S11 in Supporting information). We
speculate that the hydrogen bonding between His85 and the carboxylates of the probes may interrupt its native function, thus lead to
substrate hydroxylation. Notably, compared to the short chain of fatty acid (i.e., lauric acid), the bulky end group of HCTUDA is more
closed to the F-G loop, which may be beneficial to stabilize this unique conformation. Consequently, HCTUDA predominantly goes to
the hydroxylation pathway.

Fig. 3. (A, B) Crystal structure of eicosanoic acid-bound OleT (PDB:4L40). (C, D) Proposed model of DAUDA bound OleT. (E, F) Proposed model of HCUDA
bound OleT. The substrates were shown in green. The hydrophobic residues along the substrate binding pocket and Arg245, His58 were shown in sticks. F-G
loop was highlighted in cyan. The heme was shown in red and the iron was in yellow sphere. The distance between carboxylate and Arg245 and His85 was
labled.
In summary, a series of novel fatty acid derivatives were designed and synthesized to probe the hydroxylation/decarboxylation
activity of P450 OleT. It demonstrated that most of the derivatives were well accepted as substrates of OleT, which extraordinarily
broadened the enzyme’s substrate scope. More importantly, diversified product profiles were observed based on the difference in end
groups, which indicated that the minor change in the remote site of substrates could regulate the hydroxylation/decarboxylation
reaction. Evidently the molecular docking study implies that the position shifts of the carboxylate group induced by the remote structure
contributes the bifurcative catalysis. This work sheds light on the understanding of the OleT catalysis and will benefit the application of
OleT enzymes in synthesis and biofuel industry [12].
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
Acknowledgments
We wish to acknowledge the support from the State Scholarship Fund of the China Scholarship Council (No. 201806310084). We
also wish to thank Dr. Thomas M. Makris for providing the OleTSA plasmid, thank Olivia Manley, Suman Das for their help on protein
purification. We also acknowledge technical supports from the staffs at the Mass Spectrometry at the University of South Carolina.
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