Unexpected Reactions of α,β-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases

Article abstract of DOI:10.1002/anie.202111163

CYP152 peroxygenases catalyze decarboxylation and hydroxylation of fatty acids using H₂O₂ as cofactor. To understand the molecular basis for the chemo- and regioselectivity of these unique P450 enzymes, we analyze the activities of three CYP152 peroxygenases (OleT_JE, P450_SPα, P450_BSβ) towards cis- and trans-dodecenoic acids as substrate probes. The unexpected 6S-hydroxylation of the trans-isomer and 4R-hydroxylation of the cis-isomer by OleT_JE, and molecular docking results suggest that the unprecedented selectivity is due to OleT_JE’s preference of C2?C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450_SPα and P450_BSβ regardless of the cis/trans-configuration of substrates. The combined H₂¹⁸O₂ tracing experiments, MD simulations, and QM/MM calculations unravel an unusual mechanism for Compound I-mediated aldehyde formation in which the active site water derived from H₂O₂ activation is involved in the generation of a four-membered ring lactone intermediate. These findings provide new insights into the unusual mechanisms of CYP152 peroxygenases.

Full text of DOI:10.1002/anie.202111163

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Unexpected Reactions of α,β-Unsaturated Fatty Acids
Provide Insight into the Mechanisms of CYP152 Peroxygenases
Authors: Yuanyuan Jiang, Wei Peng, Zhong Li, Cai You, Yue Zhao,
Dandan Tang, Binju Wang, and Shengying Li
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202111163
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10.1002/anie.202111163
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RESEARCH ARTICLE
Unexpected Reactions of α,β-Unsaturated Fatty Acids Provide
Insight into the Mechanisms of CYP152 Peroxygenases
Yuanyuan Jianga,b,c,+, Wei Pengd,+, Zhong Lia,b,c,+, Cai Youa, Yue Zhaoe, Dandan Tanga, Binju Wangd*
and Shengying Lia,f*
[a]
Dr. Y. Jiang, Dr. Z. Li, Dr. C. You, Dr. D. Tang, Prof. Dr. S. Li
State Key Laboratory of Microbial Technology
Shandong University
No. 72 Binhai Road, Qingdao, Shandong 266237, China
E-mail: lishengying@sdu.edu.cn
[b]
Dr. Y. Jiang, Dr. Z. Li
Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels
Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
No. 189 Songling Road, Qingdao, Shandong 266101, China
[c]
[d]
Dr. Y. Jiang, Dr. Z. Li
University of Chinese Academy of Sciences
Beijing 100049, China
W. Peng, Prof. Dr. B. Wang
State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of
Chemistry and Chemical Engineering, Xiamen University
Xiamen 361005, China
E-mail: wangbinju2018@xmu.edu.cn
[e]
Y. Zhao
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education
School of Pharmaceutical Sciences, Wuhan University
Wuhan 430071, China
[f]
Prof. Dr. S. Li
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology
Qingdao, Shandong 266237, China
These authors contributed equally.
[+]
Supporting information for this article is given via a link at the end of the document.
Abstract: CYP152 peroxygenases catalyze decarboxylation and
hydroxylation of fatty acids using H2O2 as cofactor. To understand the
molecular basis for the chemo- and regioselectivity of these unique
P450 enzymes, herein, we analyze the activities of three
redox partner(s) as the oxygen donor, electron source, and
electron shuttle(s), respectively. However, a handful of P450s
such as CYP152 peroxygenases have evolved the ability to
directly utilize H2O2 as the sole oxygen and electron donor to drive
the P450 catalysis via a so-called peroxide shunt pathway.[2]
The P450 peroxygenase OleTJE (CYP152L1) from
Jeotgalicoccus sp. ATCC 8456 has been attracting great attention
from the fields of both biofuels and biomaterials in the past decade
because it catalyzes a single-step oxidative decarboxylation of
fatty acids (Cn: n = 4 – 20) to form α-olefins (Cn-1) as value-added
products using H2O2 as cofactor (Figure 1a).[3] By contrast, the
representative CYP152 peroxygenases (OleTJE, P450SPα and P450BSβ
towards cis- and trans-dodecenoic acids as substrate probes. The
unexpected 6S-hydroxylation of the trans-isomer and 4R-
hydroxylation of the cis-isomer by OleTJE, and molecular docking
results suggest that the unprecedented selectivity is likely due to
OleTJE’s preference of C2-C3 cis-configuration. More surprisingly, in
addition to the common epoxide products, undecanal is the
unexpected major product of P450SPα and P450BSβ regardless the
cis/trans-configuration of substrates. The combined isotopically-
labeled H218O2 tracing experiments, MD simulations and QM/MM
calculations unravel an unusual mechanism for Compound I-
mediated aldehyde formation, in which the active site water derived
from H2O2 activation is involved in the generation of a four-membered
ring lactone intermediate. These findings provide new insights into the
unusual mechanisms of CYP152 peroxygenases.
)
fatty acid
α-hydroxylase
P450SPα
(CYP152B1)
from
Sphingomonas paucimobilis and the fatty acid β-hydroxylase
P450BSβ (CYP152A1) from Bacillus subtilis preferentially convert
fatty acids into α- and β-hydroxy fatty acids, respectively.[4]
Mechanistically, it has been widely accepted that the three most-
studied CYP152 enzymes activate H2O2 via the substrate-
assisted heterolytic cleavage of the peroxy bond to generate the
iron (IV) oxo π cation radical (Compound I, abbr. Cpd I) species,
which is responsible for abstracting the Cα- or Cβ-hydrogen from
the fatty acid substrates. Differentially, P450SP exclusively
α
abstracts Cα-H to generate the substrate radical at Cα position,
thereby yielding α-hydroxy fatty acid via a normal OH rebound
mechanism; whereas P450BSβ and OleTJE prefer Cβ-H abstraction
and the different fates of the Cβ radical give rise to α-olefin (OleTJE)
and β-hydroxy fatty acid (P450BSβ) as the major products (Figure
S1).[2a, 3b, 4c, 4d, 5] Of note, both P450BSβ and OleTJE can produce a
small amount of α-hydroxy fatty acid as side product, indicative of
Introduction
Cytochrome P450 enzymes (CYPs or P450s) catalyze diverse
oxidative reactions towards a myriad of natural and unnatural
substrates, thus being named the most versatile biocatalysts in
nature.[1]
monooxygenation mechanism that requires O2, NAD(P)H, and
A
vast majority of P450s share
a
common
their lower regioselectivity than P450SP
.
α
1
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10.1002/anie.202111163
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RESEARCH ARTICLE
Figure 1. Reactions catalyzed by CYP152 peroxygenases towards three different C12 fatty acids 1-3. The percentage ratios are shown for each individual products.
Reaction conditions: 1 μM P450, 500 μM substrate (1, 2 or 3), 5 μM AldO and 10% glycerol at 30 °C for 6 h. Unless otherwise specified, the product distribution of
the reaction system using exogenously added H2O2 as cofactor is similar to that of the corresponding AldO/glycerol system for in situ gradual H2O2 generation.
To understand the underlying mechanisms for the distinct
chemo- and regioselectivity of three representative CYP152
family members that share significant similarity in protein
sequences (Figure S2), a growing number of mechanistic studies
have been conducted. For example, structural analysis and
product profiling of abundant mutants indicated that accurate
positioning of substrate is essential for the selectivity of OleTJE.[3b,
6] Substrate kinetic isotope effect and QM/MM studies confirmed
that the decarboxylation results from substrate Cβ-H abstraction
by Cpd I.[5a, 5b] Furthermore, computational studies revealed that
the reactivity of OleTJE is mostly determined by the regioselective
H-abstraction from the substrate by Cpd I; that is, a weaker Cβ-H
bond initiates either β-hydroxylation or decarboxylation while a
weaker Cα-H bond leads to α-hydroxylation and the desaturation
of α-methylated fatty acids.[7]
Despite these significant progresses, it remains unclear how
the conformational and electronic properties of substrate would
affect the catalytic outcome of CYP152 peroxygenases because
all previous mechanistic studies unanimously utilized the
saturated fatty acids as testing substrates. To address these
issues, in this work, cis- and trans-2-dodecenoic acid (α,β-
unsaturated C12 fatty acids) were selected as substrate probes for
comparative catalytic analysis (chemoselectivity, product profile
and catalytic efficiency) of OleTJE, P450BSβ and P450SP with
α
complementary experimental and computational approaches.
Results and Discussion
Identification of distinct regioselective hydroxylation
activities of P450 fatty acid decarboxylase OleTJE. The
2
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10.1002/anie.202111163
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RESEARCH ARTICLE
oxidative transformations of lauric acid (1; the OleTJE-preferred
substrate was used as a reference), cis-2-dodecenoic acid (2),
and trans-2-dodecenoic acid (3) by OleTJE were first investigated
using either the exogenous H2O2 addition system or the
AldO/glycerol-based in situ H2O2 releasing system.[8] As a control
reaction (1 μM P450, 500 μM substrate, 5 μM AldO and 10%
glycerol at 30 °C for 6 h), OleTJE converted 100% (88.1% for the
exogenous H2O2 addition system) of 1 into a mixture of three
known products including 1-undecene (4; relative product ratio =
63.6%), α-hydroxy-lauric acid (5; 4.2%) and β-hydroxy-lauric acid
(6; 22.9%), and two newly identified minor products 2-
undecanone (7; 7.9%, presumably due to over-oxidation of 6) and
undecanal (8; 1.4%, see below for the putative mechanism)
(Figure 1a).
5.3 Å and 5.8 Å for the pro-5S and pro-6R C-H bonds, respectively)
away from the heme-iron reactive center, qualitatively explaining
the regio- and stereoselectivity (5R- and 6S-hydroxylation) of
OleTJE towards 3.
Towards the cis-α,β-unsaturated fatty acid substrate, with a
100% substrate conversion, OleTJE mainly catalyzed the γ-
hydroxylation of 2, giving rise to 4R-hydroxy-cis-2-dodecenoic
acid (10; 89.3%, Figure 1d), whose structure was determined by
mass spectrometry (MS) and nuclear magnetic resonance (NMR)
analyses (Table S2, Figures S3-S16). Further electronic circular
dichroism (ECD) analysis supported the R-configuration of C4-
hydroxy in 10 with a positive cotton effect at 228 nm (Figure S17).
In addition to a small amount of the aldehyde product 8 (1.8%),
OleTJE was also able to further oxidize 10 into 4-keto-cis-2-
dodecenoic acid (11; 8.9%) only in the AldO/glycerol-based H2O2
releasing system (Table S3, Figures S18-S26). In comparison, the
substrate conversion ratio of 3 (43.2%) was significantly lower
than that of 2 (100%). To our surprise, 6S-hydroxy-trans-2-
dodecenoic acid (12; 75.8%, Figure 1g) turned out to be the main
product of the trans-α,β-unsaturated fatty acid 3, demonstrating
an unprecedented regio- and stereoselectivity. The structure and
stereochemistry of the C6-hydroxyl of 12 was determined using
the Mosher ester method (Table S4, Figures S27-S40). OleTJE
also stereoselectively hydroxylated the C-H bond at C5, giving
rise to the minor product 5R-hydroxy-trans-2-dodecenoic acid (13;
22.7%; see Table S5 and Figures S41-S51 for structural
determination). The substantial difference in specific rotations of
12 ([α]2D0 = 3.21) and 13 ([α]2D0 = -5.03) further supports their
opposite configurations. Notably, no alkyne product (1-undecyne)
was detected in all reactions (Figure S52), indicating that no
typical decarboxylation occurred. Instead, a trace amount of 8
was observed. Collectively, 2 and 3, as a pair of cis-trans-isomers,
gave distinct conversion efficiencies and product profiles in the
OleTJE-catalyzed reactions (Figures 1d and 1g).
To rationalize the distinct regioselectivity of OleTJE towards 2
and 3, we performed molecular docking using Sybyl-X 2.0
software.[9] As shown in Figure 2a, the docking conformation of
the cis-isomer 2 in the active site of OleTJE is analogous to the
cis-like configuration seen both in the co-crystal structure of
OleTJE and the C20 substrate arachidic acid (PDB ID code:
4L40)[10] and the docking structure of 1 in OleTJE (Figure S53).
These results suggest that the C2-C3 cis-configuration might be
favored by the architecture of OleTJE active site. In this binding
mode, the shorter distance between the pro-4R C-H bond (relative
to the pro-4S C-H bond) and the heme-iron may also explain the
stereoselectivity of OleTJE towards 2 (Figure 2a). Interestingly, the
trans-isomer 3 binds to OleTJE in a “U-shape” with the turning
points at C5 and C6 (Figure 2b), which is similar to the reported
P450Biol-mediated hydroxylation of the central carbons of a fatty
acyl chain.[11] In this binding mode, the pro-5R and pro-6S C-H
bonds to be differentially hydroxylated are 4.1 Å and 5.0 Å (versus
Figure 2. Docking structures of (a) cis-2-dodecenoic acid (2) and (b) trans-2-
dodecenoic acid (3) in the active site of OleTJE (PDB ID code: 4L40). In the top
20 lowest energy docking solutions, a and b are found to adopt the ideal
catalytic conformations. The distances in angstrom are indicated by the dashed
lines.
Identification of the unexpected C-C bond cleavage
products of fatty acid hydroxylase P450BSβ and P450SPα
.
Motivated by the unexpected reactivity of OleTJE against the α,β-
unsaturated fatty acids, we further investigated the activities of
P450SPα and P450BSβ towards 1-3. Consistent with the previous
report,[4d] P450SPα almost exclusively catalyzed the α-
hydroxylation of 1 at a conversion of 100%, leading to the
predominant product 5 (94.6%). Unexpectedly, we also observed
two previously unreported aldehyde products 8 (2.5%) and
decanal (9; 2.9%) in trace amounts (Figure 1c). By contrast,
P450BSβ completely transformed 1 into a mixture of 4 (12.5%), 5
(38.6%), 6 (33.9%), 7 (10.5%), and 8 (4.5%) (Figure 1b).
Unlike OleTJE, both P450SPα and P450BSβ demonstrated
similar catalytic behaviors towards the cis-trans-isomers 2 and 3,
leading to the common main product of 8 and the side products
of 7 and 9 (Figures 1e, 1f, 1h and 1i, Figures S54-S61). Moreover,
the epoxide products 2R,3R-epoxy-dodecanoic acid (14, [α]2D0 = -
9.09) and 2S,3S-epoxy-dodecanoic acid (15) with opposite
stereochemistry were generated from 2 by P450BSβ and P450SPα
,
respectively (Figures 1e and 1f, Table S6, Figures S62-S76).
Interestingly, a favored formation of 2R,3S-epoxy-dodecanoic
acid (16, [α]2D0 = -6.56) by P450BSβ and P450SPα was observed
consistently for the trans-isomer 3. (Figures 1h and 1i, Table S7,
Figures S77-S86).
Of note, P450BSβ, but not P450SPα, was also capable of
hydroxylating 2 and 3 to 10 and 12 (also seen in the OleTJE-
mediated reactions, Figures 1d and 1g), respectively (Figure 1,
Figures S54 and S59), likely due to the differences in their
substrate binding pockets. Specifically, the active site cavity of
P450SPα is smaller than that of P450BSβ (similar to OleTJE), and
their hydrophobic channels adopt different orientations,[4c, 4d] thus
leading the substrates (1, 2 and 3) to be more perpendicular to
the heme plane of P450SPα (Figures S87-S88). Again, no alkyne
product (1-undecyne) was observed in the reactions catalyzed by
either P450BSβ or P450SPα (Figure S89).
Mechanistic analysis for undecanal formation. To explain
the intriguing mechanisms for the efficient carbon-carbon bond
cleavage of cis-2-dodecenoic acid (2) by P450SPα leading to the
unexpected products 7-9 (especially the main product 8, Figure
1f), we first confirmed that 7-9 and 2S,3S-epoxy-dodecanoic acid
3
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(15) were all stable end products (Figures S90-S93). Next, using
the 18O-labelled H218O2, we determined that the oxygen atom in
7-9 and 15 is unanimously derived from hydrogen peroxide since
the 18O-incorporation was observed by GC-MS analysis (Figures
S94-S97).
might involve the sequential Cα-hydroxylation, isomerization to α-
keto fatty acid, and oxidative decarboxylation (analogous to the
reported α-keto acid decarboxylase).[12] To test this hypothesis,
the tentative transformation of α-keto-dodecanoic acid (17) into 8
by P450SPα or P450BSβ was conducted. Briefly, 17 was prepared
from 1 by an enzymatic cascade in one pot: 1 was first α-
hydroxylated by P450SPα to 5, which was in turn oxidized to 17 by
the (S)-α-hydroxyacid oxidase from Aerococcus viridans[13] with
internal H2O2 recycling (Figure S98). However, no further
P450SPα-mediated decarboxylation of 17 to 8 was observed
(Figures S99-S100), indicating that the α-keto fatty acid is not a
precursor of 8.
To unravel the mechanism of P450SPα-catalyzed aldehyde (8)
formation from 2, the combined molecular dynamics (MD)
simulations and quantum mechanics/molecular mechanics
(QM/MM) calculations were conducted. In the initial reactant
complex of FeIII-H2O2 (RC, Figure 3), H2O2 forms a strong H-bond
with the carboxylate group of fatty acid, suggesting that the fatty
acid substrate plays a key role in stabilizing H2O2. Starting from
RC, we investigated two competing reaction pathways. In one
pathway that transpires via the heterolytic O−O cleavage (Figure
3), the proton transferred from proximal hydroxyl (-O1H1) of H2O2
to the O3 atom of the substrate, which is coupled with the H-bond
shift from O1 to O2, thereby generating the ferric hydroperoxide
species (Cpd 0, i.e., IC1). The subsequent O−O heterolysis in Cpd
0, which is triggered by the proton transfer from O3 to O2,
affording the active species of Cpd I (i.e., IC2).[14] As shown in
Figure 3, the Cpd I formation requires overcoming an energy
barrier of 15.2 kcal/mol (RC→TS2). In the alternative but
unfavorable mechanistic route, the formation of Cpd I is initiated
by the homolytic O−O cleavage mechanism,[15] which involves an
energy barrier of 21.6 kcal/mol (Figure S101). Obviously, the
heterolysis mechanism (Figure 3) is much favored over the
Figure 3. The calculated mechanism (with energies in kcal/mol) for the
formation of Cpd I by P450SPα in the presence of the substrate cis-2-dodecenoic
acid (2). The fatty acid carboxylate group interacting with the guanidyl group of
Arg241 is located above the heme. The key distances are given in angstrom (Å).
For the production of the aldehyde 8 from either 2 or 3 by
P450SPα and P450BSβ, we initially hypothesized that the reactions
Figure 4. QM/MM calculated mechanisms (with energies in kcal/mol) for the formation of four-membered ring lactone intermediate (IC4’’) and 2S,3S-epoxy-
dodecanoic acid (PC, 15) from cis-2-dodecenoic acid (2) starting from Cpd I of P450SPα, along with the QM/MM optimized structures of key species involved in the
reactions. The fatty acid carboxylate group interacting with the guanidyl group of Arg241 is located above the heme. The key distances are given in angstrom (Å).
4
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10.1002/anie.202111163
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homolysis mechanism (Figure S101) in Cpd I generation.
group, leading to the formation of enol IC6 and CO2. The final
enol-keto isomerization would afford the final product of aldehyde
PC’’ (i.e., 8) (see Figures S104-S105 for details). Thus, our
calculations support that the oxygen atoms in both 8 and epoxide
15 should be originated from H2O2, which is consistent with the
isotopically-labeled H218O2 tracing experiments (Figures S95 and
S97) as described above.
In addition, we attempted to explain the generation of minor
products 9 and 7 (Figures S106-S111). The formation of 7 is
similar to that of 8 with the departure of one molecular of CO2; the
difference is that the water OH derived from H2O2 activation
attacks substrate C3 site (other than C2 for 8) to afford the three-
Staring from the Cpd I/2 complex IC2’, we investigated two
competing pathways (Figure 4). The one on the right (in blue
profile) involves oxygen atom transfer from Cpd I to C2 of the
double bond in substrate, which involves a small barrier of 16.6
kcal/mol (IC2’→TS3), leading to the C3-centered substrate
radical intermediate IC3. Further C3-O1 coupling is quite facile,
which only overcomes a slight barrier of 1.9 kcal/mol (IC3→TS4),
leading to the formation of epoxidation product, 2S,3S-epoxy-
dodecanoic acid (PC, 15). In the other pathway on the left (the red
profile), the reaction is initiated by the H-abstraction from H2 of
the adjacent water molecule derived from H2O2 activation
(IC2’→TS3’), which is coupled with the attack of water O2 onto
the C2 site of substrate. The water molecule, derived from H2O2
activation, is stable during the long time MD simulation (Figure
S102). And this reaction pathway experiences a barrier of 17.8
kcal/mol (IC2’→TS3’), giving rise to the formation of Fe(IV)-OH
species (Cpd II) and the C2-hydroxylated substrate radical
intermediate IC3’. Stemming from IC3’, we considered two
competing pathways. The one via TS4’ involves the H1
abstraction from O2 by Cpd II, which is coupled with the O2-C3
bond formation. This reaction requires overcoming a barrier of
12.3 kcal/mol (IC3’→TS4’), thus forming the epoxide product
2R,3R-epoxy-dodecanoic acid (PC’, 14). In the alternative
pathway via TS4’’, the nucleophilic attack of the substrate O3 onto
the substrate C3 is coupled with the electron transfer from the
substrate to Cpd II, which involves a barrier of 9.4 kcal/mol and
affords the four-membered ring intermediate IC4’’, Obviously, the
formation of the four-membered ring intermediate is kinetically
favored over the epoxide formation pathway (IC3’→TS4’). Thus,
the reaction on the left (the red profile) would lead to the main
intermediate of four-membered ring IC4’’.
membered ring intermediate.
A mechanism involving diol
cleavage by Cpd I was proposed for the C10 aldehyde product 9,
which is similar to the C-C bond cleavage route of P450SCC and
[17]
P450BioI
.
To consolidate this mechanism, glyoxylic acid, the
predicted C-C bond cleaved product was successfully detected
by LC-MS analysis of the 2,4-dinitrophenylhydrazine-derivatized
sample (Figures S112-S120).[18]
Reactivity of CYP152 peroxygenases. It is interesting that
the overall substrate conversions of these CYP152
peroxygenases largely varied (Table 1). Of note, the catalytic
activities supported by the AldO/glycerol-based in situ H2O2
releasing system were unanimously higher than those of the
exogenous H2O2 addition system. In the case of OleTJE, the
highest turnover number (TON) was achieved by substrate 2
(TON = 990, conversion = 99%, Table 1), which is 21.5 times
higher than that of substrate 3 (TON = 44, 4.4%). Consistently,
the dissociation constant (KD) values determined by substrate
titration experiments[19] revealed tighter binding of 2 than both 1
and 3 (1.7 ± 0.1 μM versus 3.2 ± 0.2 μM and 4.1 ± 0.4 μM,
respectively; Table S8, Figure S121). Similarly, the preference of
the cis-isomer 2 over the trans-isomer 3 was shown as a common
phenomenon for P450SPα and P450BSβ (Table 1). It is worth noting
that P450SPα showed the highest TON of 14,381 towards 2 when
supported by the in situ H2O2-generating system at 30 °C for 24 h
(Table S9). Moreover, the KD values of 2 were similar to those of
1 for P450BSβ (1.9 ± 0.2 μM versus 1.8 ± 0.2 μM) and P450SPα (1.9
± 0.4 μM versus 1.8 ± 0.3 μM) (Table S8, Figures S122-S123),
which is consistent with the corresponding catalytic activities
(Table 1). Besides, P450SPα and P450BSβ could still maintain high
catalytic activity in the presence of H2O2 up to 10 mM with the
highest conversion of 23.1% (TON = 2,307), demonstrating
superior H2O2 tolerance far beyond OleTJE (Table 1, Table S9),
which is consistent with the previous studies.[20] As in situ H2O2-
generating system further increased the TON of P450SPα to 9,207
under the same reaction conditions, we reason that a CYP152
peroxygenase supported by the in situ H2O2-generating system is
a more productive and sustainable enzymatic system for the
future practical applications.
Figure 5. The proposed reaction mechanism (with energies in kcal/mol) from
the four-membered ring lactone intermediate (IC4’’-1) to aldehyde (8) in
aqueous solution.
We further investigated the transformation of 2-epoxide (14
and 15) and four-membered ring intermediate in water solution
with hybrid cluster-continuum (HCC) model calculations (see
Figures S103-S105 for details).[16] Our calculations (Figure S103)
showed that the acid-catalyzed ring opening of 2-epoxide needs
to overcome a very high energy barrier of 25.2 kcal/mol, thus
being infeasible in water solution, which is consistent with our
experimental result (Figure S93). Therefore, we proposed a new
mechanism for aldehyde formation from the four-membered ring
intermediate (Figure 5). Then, the transformation of IC4’’ could be
mediated by the acid-base catalysis of the carboxylic group from
either the substrate itself or the surface residues of enzyme (such
as Asp or Glu) in the reaction system. Indeed, our calculations
showed that the nucleophilic attack of carboxylate onto C3 of IC4’’,
which is coupled with the C3−O3 cleavage, generates the 3-
acyloxylated intermediate IC5. The following C1−C2 bond
cleavage in IC5 is coupled with the dissociation of the carboxylate
Conclusion
In summary, this study provides the first insights into the
reactivity of CYP152 peroxygenases towards the α,β-unsaturated
fatty acids. The presence of the Cα=Cβ double bond dramatically
changes the chemo- and regioselectivity of OleTJE, P450SPα and
P450BSβ, likely due to the shortened bond length, the different
electronic property, and the conformation-fixing effect of the
double bond, which would lead to the changed substrate
5
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Table 1. The conversions of substrates 1-3 to different products by the three representative CYP152 peroxygenases in the two different H2O2-supplying systems.
Enzyme
Substrate
mM
TON[a]
TON[b]
Conversion/%
Products
0.5
1
440 ± 3
102 ± 10
0
500
88.1a/100b
10.2a/18.9b
0a/5.7b
4-8
1
189 ± 30
113 ± 39
500
4
4
2
0.5
1
465 ± 17
704 ± 44
0
93.1a/100b
70.4a/99b
0a/3.6b
10, 11*, 8
10, 11*, 8
10
OleTJE
2
3
990 ± 3
71 ± 31
215 ± 26
44 ± 8
2
0.5
1
75 ± 7
0
15.2a/43.2b
0a/4.4b
12, 13, 8
12
0.5
1
317 ± 36
534 ± 25
3336 ± 273
2307 ± 247
283 ± 3
500
1000
63.4a/100b
53.4a/100b
66.7a/97.5b
23.1a/92.1b
56.6a/100b
41.6a/100b
10.9a/96.5b
17.1a/67.1b
2.8a/11.6b
2.7a/8.6b
5, 8, 9
5, 8, 9
5, 8, 9
5, 8, 9
7-9, 15
7-9, 15
7-9, 15
7-9, 15
7-9, 16
7-9, 16
1
5
4877 ± 22
9207 ± 247
500
10
0.5
1
P450SPα
416 ± 21
547 ± 213
1714 ± 122
14 ± 7
1000
2
3
5
4826 ± 8
6708 ± 25
58 ± 9
10
0.5
1
27 ± 12
86 ± 7
0.5
1
320 ± 4
201 ± 21
340 ± 84
816 ± 11
131 ± 2
227 ± 2
763 ± 241
557 ± 105
0
500
64.1a/100b
20.1a/98.9b
6.8a/88.1b
8.2a/51.8b
26.2a/100b
22.7a/99.6b
15.3a/75.3b
5.6a/11.8b
0a/57.8b
4-8
4-8
989 ± 1
1
5
4404 ± 142
5181 ± 281
500
4-8
10
0.5
1
4-8
7-10, 14
7-10, 14
7-10, 14
7-10, 14
7-9, 12, 16
7-9, 12, 16
7-9, 16
P450BSβ
996
2
3
5
3767 ± 77
1177 ± 319
289 ± 13
534 ± 26
187 ± 11
10
0.5
1
0
0a/53.4b
2
0
0a/9.4b
[a]Reaction conditions: 1 μM P450 supported by exogenous H2O2 with the same molar amount as the substrate at 30 °C for 6 h; [b]Reaction conditions: 1 μM P450
supported by the 5 μM AldO + 10% glycerol system, at 30 °C for 6 h. *A small amount of product 11 appeared only in the AldO/glycerol-based in situ H2O2
generating system.
positioning (relative to the saturated fatty acid with the same
carbon chain length) in the subtly different active sites of the three
P450 enzymes. The preference of the C2-C3 cis-configuration
over the trans-configuration is a common phenomenon for the
three studied CYP152 family members due to the cis-favoring
topology of their substrate binding pockets. The binding of the
unfavorable trans-isomer may require torsion of the fatty acyl
chain to some extent, thus leading to a larger entropy penalty of
substrate binding.
The combined isotopically-labeled H218O2 tracing experiments,
MD simulations and QM/MM calculations unravel a highly
uncommon mechanism for the Cpd I-mediated aldehyde
6
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10.1002/anie.202111163
Angewandte Chemie International Edition
RESEARCH ARTICLE
formation, which is initiated by the H-abstraction from the adjacent
water rather than commonly from the C-H bond of substrate. This
unusual event is coupled with the attack of water OH that is
derived from H2O2 activation onto the substrate C2 site, leading
to the formation of Cpd II and the key C2-hydroxylated substrate
radical. Then, Cpd II accepts a single electron transfer from the
substrate and triggers the formation of a four-membered ring
intermediate, which further loses CO2 by acid-base catalysis in
water solution and rearranges to form undecanal. This finding
expands our understanding of the mechanism of CYP152
peroxygenases for the C-C bond cleavage reactions. This
mechanism is totally different from the C-C scission reaction of
the saturated fatty acid by OleTJE, which is initiated by a typical
substrate Cβ-H abstraction, and followed by a similar single
electron transfer to the heme, thus leading to a carbocation poise
for later C-C bond cleavage to liberate CO2.[5a, 5b]
The present study not only proposes the key role of the active
site water derived from H2O2 activation in the catalysis of CYP152
peroxygenases for the first time, but also provides a new, simple
and more productive enzymatic system for aldehyde biosynthesis
compared with the reported biocatalytic aldehyde-producing
systems.[21] An important question to be answered is whether the
aldehyde-forming mechanism is also applicable for other α,β-
unsaturated fatty acids with different chain length. To our delight,
both P450SPα and P450BSβ indeed produced the aldehyde
products lauraldehyde (C12) and tridecanal (C13) as the main
products from trans-2-tridecenoic acid (C13) and trans-2-
tetradecenoic acid (C14), respectively (Figures S124-S125).
However, due to the commercial unavailability, we were unable to
test more substrates especially the cis-isomers. Thus, further
studies using synthesized substrates are required for a more
general conclusion.
Of note, a high TON of 14,381 was achieved for P450SPα
towards 2 with the in situ H2O2-generating system, showing great
potential as an applicable biocatalyst to synthesize fatty
aldehydes and other downstream products. Fatty aldehydes (C8-
C13) have long served as flavor and fragrance components; they
also represent the essential metabolic intermediates for microbial
synthesis of various industrially relevant oleochemicals.[22]
Furthermore, the unexpected products C6-hydroxy-2-dodecenoic
acid (12) and C5-hydroxy-2-dodecenoic acid (13) of OleTJE
towards the trans-isomer 3 can be saponified to obtain the
analogues of Massoia lactone,[23] which have anti-fungal, anti-
cancer and anti-virial activities, thus holding significant application
potentials in pharmaceutical and biomedical sectors.
Jingyao Qu (Shandong University) for their assistance in GC-MS
and LC-HRMS data collection.
Keywords: CYP152 peroxygenases • α,β-unsaturated fatty acids
• substrate probes • QM/MM calculations • molecular dynamics
simulations
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Science Foundation of Shandong Province, China (ZR2019ZD20),
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Entry for the Table of Contents
The presence of cis or trans Cα=Cβ double bond in the unsaturated fatty acid substrates dramatically changes the catalytic efficiency
and chemo-/regioselectivity of three representative CYP152 peroxygenases including OleTJE, P450SPα and P450BSβ, which provides
new insights into their unusual catalytic mechanisms.
9
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