Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system

Article abstract of DOI:10.1073/pnas.1702317114

Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum. The system consisting of CreHI (for installation of a phosphate directing/ anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p-And m-Alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.

Full text of DOI:10.1073/pnas.1702317114

Selective oxidation of aliphatic C–H bonds in
alkylphenols by a chemomimetic biocatalytic system
Lei Dua,b,c,1, Sheng Donga,b,1, Xingwang Zhanga,b, Chengying Jiangd,e, Jingfei Chena,b, Lishan Yaoa,b, Xiao Wangf,
Xiaobo Wanf, Xi Liug,h,i, Xinquan Wangg,h,i, Shaohua Huanga,b, Qiu Cuia,b, Yingang Fenga,b,2, Shuang-Jiang Liud,e,2
and Shengying Lia,b,2
,
aShandong Provincial Key Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong
266101, China; bKey Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong
266101, China; cUniversity of Chinese Academy of Sciences, Beijing 100049, China; dState Key Laboratory of Microbial Resources, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101, China; eEnvironmental Microbiology and Biotechnology Research Center, Institute of Microbiology, Chinese
f
Academy of Sciences, Beijing 100101, China; Key Laboratory of Bio-based Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese
Academy of Sciences, Qingdao, Shandong 266101, China; gMinistry of Education Key Laboratory of Protein Science, School of Life Sciences, Tsinghua
University, Beijing 100084, China; hBeijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084,
i
China; and Collaborative Innovation Center for Biotherapy, School of Life Sciences, Tsinghua University, Beijing 100084, China
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved May 18, 2017 (received for review February 9, 2017)
Selective oxidation of aliphatic C–H bonds in alkylphenols serves
significant roles not only in generation of functionalized intermedi-
ates that can be used to synthesize diverse downstream chemical
products, but also in biological degradation of these environmentally
hazardous compounds. Chemo-, regio-, and stereoselectivity; control-
lability; and environmental impact represent the major challenges
for chemical oxidation of alkylphenols. Here, we report the develop-
ment of a unique chemomimetic biocatalytic system originated from
the Gram-positive bacterium Corynebacterium glutamicum. The sys-
tem consisting of CreHI (for installation of a phosphate directing/
anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols),
and CreD (for directing/anchoring group offloading) is able to selec-
tively oxidize the aliphatic C–H bonds of p- and m-alkylated phenols
in a controllable manner. Moreover, the crystal structures of the
central P450 biocatalyst CreJ in complex with two representative
substrates provide significant structural insights into its substrate
flexibility and reaction selectivity.
catalysts (9–11) and in biomimetic supramolecular assemblies (12–
14); and (v) chemical oxidants could be associated with significant
environmental concerns (2, 3). Given these issues, oxidative en-
zymes with inherent catalytic selectivity and reduced environmen-
tal impact may be developed into alternative catalysts for selective
oxidation of organic compounds including alkylphenols (15–18).
To date, a majority of efforts have focused on engineering of
P450 monooxygenases, which are believed to be the most versatile
biocatalysts in nature (19), using either directed evolution and/or
rational design (20–22). Recently, a new strategy of substrate en-
gineering has been explored (23, 24), wherein anchoring/directing
groups are chemically linked to nonsubstrate compounds. Initial
studies demonstrated that the addition of these anchoring groups
facilitates enzyme–substrate interactions and enables selective
oxidation of C–H bonds in previously inaccessible substrates.
Using the P450 monooxygenase PikC as a model system (25, 26),
chemically diverse substrates tethered to synthetic anchoring
groups containing the N,N-dimethylamine moiety were hydroxylated
alkylphenol selective oxidation chemomimetic biocatalysis cytochrome
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P450 enzymes Corynebacterium glutamicum
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Significance
lkylphenols, p-, m-, and o-alkylated phenols, e.g., 1–10, (Fig.
1A) are among the most important synthetic precursors for
Selective oxidation of aliphatic C–H bonds in alkylphenols is
important for both structural derivatization and biological deg-
radation of these fundamental chemicals. However, significant
problems are persistently associated with the chemical methods
for this oxofunctionalization. In this study, we developed a
unique chemomimetic biocatalytic system that is capable of se-
lectively oxidizing p- and m-alkylated phenols in a controllable
manner, overcoming the challenges faced by similar chemical
oxidation. The structural and bioinformatics analyses of the
central P450 biocatalyst CreJ suggest that its substrate flexibility
and reaction selectivity could be further leveraged. This novel
alkylphenol biooxidation system may hold great potential for
application in pharmaceutical, biomanufacturing, and environ-
mental industries once upscaled systems can be further de-
veloped in the future.
A
manufacturing a vast variety of chemical products including deter-
gents, polymers, lubricants, antioxidants, emulsifiers, pesticides, and
pharmaceuticals (1). Alkylphenols are also priority environmental
pollutants because they are toxic, xenoestrogenic, or carcinogenic to
wildlife and humans (2, 3). The selective oxidation of the aliphatic
C–H bonds in alkylphenols serves significant roles not only in gen-
eration of functionalized intermediates for synthesizing more
downstream products (4), but also in biological degradation of these
environmentally hazardous compounds (5). In particular, some oxi-
dized alkylphenols are direct structural motifs in drugs (e.g., meto-
prolol) (6) and bioactive ingredients of medicinal plants (Fig. 1B).
Chemically, these oxidative transformations have been practiced
for a long time by using superoxides, organocatalyst, high valence
transition metals, or strong oxidants such as nitric acid, chromic
acid, and potassium permanganate (4, 7, 8). However, a number of
problems are persistently associated with the chemical oxidation of
aliphatic C–H bonds in alkylphenols: (i) the requirement of pro-
tection/deprotection of the phenolic hydroxyl group under reaction
conditions that are distinct from those for oxidation complicates
the synthetic process; (ii) it is difficult to delicately control the
extent of oxidations (i.e., alcohol vs. aldehyde/ketone vs. carboxylic
acid); (iii) the oxidation of an aromatic C–H bond may sometimes
occur when using strong oxidants; (iv) the regio- and stereo-
selective oxidation of an unactivated sp3 C–H bond (in alkylphe-
nols) remains a central challenge despite recent advances in
combined use of specialized directing groups with transition metal
Author contributions: L.D., L.Y., X. Wan, Xinquan Wang, Q.C., Y.F., S.-J.L., and S.L. de-
signed research; L.D., S.D., X.Z., C.J., J.C., Xiao Wang, X.L., and S.H. performed research;
L.D., S.D., X.Z., C.J., J.C., L.Y., Xiao Wang, X. Wan, S.H., Y.F., S.-J.L., and S.L. analyzed data;
and L.D., S.D., Y.F., S.-J.L., and S.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID code 5GWE and 5XJN).
1L.D. and S.D. contributed equally to this work.
2To whom correspondence may be addressed. Email: lishengying@qibebt.ac.cn, fengyg@
qibebt.ac.cn, or liusj@im.ac.cn.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1702317114/-/DCSupplemental.
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oxidative transformations of both phosphorylated (1′a and 1′b) and
nonphosphorylated (1a and 1b) intermediates.
It is striking that this unique catabolic pathway, featuring
protection/direction, oxidation, and deprotection steps, mimics
the process of classical chemical oxidations (Fig. 2B). To the best
of our knowledge, it is rare, if not unprecedented, that a natural
biooxidation pathway contains the final deprotection step. As
such, this pathway represents a good initial starting point for the
development of an entirely biocatalytic system that can achieve
selective oxidation of aliphatic C–H bonds in a spectrum of
alkylphenols. To achieve this ambitious goal, we first solved the
X-ray crystal structure of the central P450 biocatalyst CreJ in
complex with its substrate 1′. With understanding of the struc-
tural basis for substrate recognition of this P450 enzyme, we next
successfully developed a biocatalytic system comprising CreHI,
CreJEF/CreG/CreC, and CreD, which is able to selectively oxi-
dize a suite of p- and m-alkylated phenols. Further elucidation of
the cocrystal structure of CreJ and p-ethylphenyl phosphate (2′)
revealed the molecular mechanism for the regio- and stereo-
selectivity of CreJ. In sum, the high selectivity, controllable extent
of oxidation, generality, and the “green” nature of this alkylphenol
biooxidation strategy may find potential application in pharma-
ceutical, biomanufacturing, and environmental industries upon
further development of upscaled systems in the future.
Results
Structural Basis for the Substrate Recognition of CreJ. The P450 en-
zyme CreJ (also named CYP288A2 by Nelson et al.) (31) catalyzes
the successive oxidations from 1′→1′a→1′b→1′c in the presence of
redox partners CreE and CreF, and the electron donor NADPH (30).
Although different oxidative modifications of the distal methyl group
are well tolerated by CreJ, the tethered phosphate group is required
for the activity of this P450 enzyme because the nonphosphorylated
substrate cannot be recognized (30). To understand this unique sub-
strate recognition mechanism, the X-ray crystal structure of CreJ in
complex with 1′ was solved at 2.0 Å (PDB ID code: 5GWE).
In the cocrystal structure of CreJ and 1′, four molecules that
are essentially identical (except some slight variations in flexible
loops) in an asymmetric unit (Fig. 3A) were observed (data
collection and refinement statistics are available in SI Appendix,
Table S1). Each monomer of CreJ adopts a typical triangular
P450 fold with a long I helix (Fig. 3B), where the highly con-
served acid/alcohol (E268/T269) residues reside (Fig. 3C).
Mechanistically, these two amino acids are involved in the re-
quired protonation steps to generate the highly reactive heme
iron(III)-peroxy (compound 0) and iron(IV)-oxo species (com-
pound I) during a P450 catalytic cycle (SI Appendix, Fig. S1). The
absolutely conserved proximal heme thiolate ligand C376 is lo-
cated at a loop before the L helix (Fig. 3C) (32).
Fig. 1. Alkylphenol structures. (A) Representative alkylphenols 1–10. (B) The
drug and medicinal plants derived bioactive ingredients that are structurally
related to oxidized alkylphenols. The oxidized alkylphenol motifs are high-
lighted in blue.
The substrate binding pocket of CreJ is formed with the I helix
as the wall and is covered by the BB2 loop, the B2C loop and the
C terminus of F helix (Fig. 3 B and C). These regions (FG helices
and BC loop) are generally flexible in P450 enzymes and readily
reposition in response to substrate binding (33, 34). The struc-
ture adopts a closed conformation in which the putative substrate
channel is blocked by the BB2 and B2C loops. The closed cavity
that accommodates 1′ has a volume of ∼260 Å3 (11.5 Å × 4.5 Å ×
5.0 Å), representing a relatively small substrate-binding pocket
(Fig. 3D). The substrate 1′ is anchored and orientated mainly
through a complex network of hydrogen bonds that exist between
the phosphate moiety and a number of protein backbone amides,
as well as hydroxyl and amine groups of amino acid side chains
from the BB2/B2C loops and F/I helices (Fig. 3E and SI Appendix,
Fig. S2A). Specifically, the 106SGLS109 motif of the B2C loop
contributes the majority of hydrogen bond contacts with the
phosphate oxygen atoms of the substrate, suggesting its crucial
role for substrate anchoring. The positively charged guanidine
of R194 from the F helix likely forms a strong electrostatic
in regio- or stereoselective manners (27–29). However, these ap-
proaches are not completely biological because the generation, in-
stallation, and offloading of anchoring groups still rely on chemical
synthesis.
Recently, our laboratories have identified a p-cresol, 1, bio-
degradation pathway (Fig. 2A) in the Gram-positive bacterium
Corynebacterium glutamicum (30). Initially, 1 is phosphorylated
to 1′ by a novel 4-methylphenyl phosphate synthase, CreHI, with
consumption of ATP. Next, a unique P450 monooxygenase CreJ,
when partnered with the native redox partner proteins including
ferredoxin CreF and ferredoxin reductase CreE, specifically
recognizes phosphorylated intermediates (1′, 1′a, and 1′b) and
successively oxidizes the aromatic methyl group into carboxylic acid
(1′c) via alcohol (1′a) and aldehyde (1′b) intermediates. Finally, the
phosphohydrolase CreD is responsible for removal of the phosphate
group to afford 1a–1c. In addition, the alcohol dehydrogenase CreG
and the aldehyde dehydrogenase CreC are recruited to reinforce the
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Fig. 2. The chemomimetic biooxidation pathway and the classical chemical oxidation route. (A) The chemomimetic p-cresol biodegradation pathway in
C. glutamicum. Phosphate anchoring groups are shown in blue. (B) The classical chemical oxidation route. DG, directing group; PG, protecting group. Oxi-
dative functional groups are highlighted in red.
interaction with the negatively charged phosphate group. In
addition, S261 from the I helix and Q83 from the BB2 loop also
form hydrogen bonds with 1′.
I312, or F416 displayed unsubstantially negative effects on CreJ
activity (Fig. 4 and SI Appendix, Figs. S12–S14). By contrast, the
mutation on F264 or W315 abolished the detectable substrate
binding (no valid KD could be deduced, SI Appendix, Figs. S15 and
S16), accompanied by no or extremely low enzymatic activity (Fig.
4). As shown in Fig. 3E, F264 and W315 are closer to the aromatic
ring of 1′ than other hydrophobic residues. More importantly,
F264 likely plays an important role in maintaining the proper
conformation of R194 via Van der Waals forces, and W315 forms
water-mediated H bonds with S109 and heme, contributing to the
proper conformation of the B2C loop (SI Appendix, Fig. S2B).
The distance between the methyl carbon atom of 1′ and the
heme ion was measured to be 4.7 Å, which is well within the re-
active range seen for other P450s (27, 36). Notably, there is small
additional space around the methyl group in the catalytic cavity,
which is partially occupied by a water molecule. Upon displace-
ment of this water molecule, this space should be sufficient to
accommodate oxidized intermediates, including the alcohol 1′a
and the aldehyde 1′b that can undergo further oxidation to gen-
erate the final product 1′c. Furthermore, this additional space
within the catalytic cavity could be leveraged to broaden the sub-
strate specificity of CreJ.
To evaluate whether these active site residues are essential for
CreJ catalysis, a series of mutants were constructed, expressed,
and purified (SI Appendix, Fig. S3A). The mutant enzymes were
then evaluated for their ability, relative to wild-type CreJ, to oxi-
dize substrate 1′ (Fig. 4). Remarkably, the replacement of R194 by
either an alanine (R194A), a lysine (R194K), or a glutamine
(R194E) residue completely abolished the enzyme activity (Fig. 4).
Consistently, no substrate binding could be detected based on the
low-to-high spin iron spectral shift (35) of the three R194 mutants
(SI Appendix, Figs. S4–S6), whereas the dissociation constant (KD)
of wild-type CreJ toward 1′ was determined to be 62 6 μM (SI
Appendix, Fig. S7). These results clearly indicate the necessity of
the electrostatic interaction between the guanidyl base and the
phosphate group for productive substrate binding. The mutants
S106A and S261A retained marginal activity (9.4% and 9.7%,
respectively) and showed significantly reduced substrate binding
affinities (KD = 431 147 μM and 459 109 μM, respectively)
compared with wild-type CreJ (Fig. 4 and SI Appendix, Figs.
S8 and S9), suggesting that they are also critical for the productive
substrate–protein interactions. In contrast, residues Q83 and
S109 appear to be less important, as their substitution with alanine
had unsubstantial effect on both substrate binding and enzyme
activity (Fig. 4 and SI Appendix, Figs. S10 and S11).
Selective Oxidation of Diverse Alkylphenols by the Chemomimetic
Biocatalytic System Composed of CreHI/CreJEF/CreGC/CreD. The
cocrystal structure of CreJ in complex with 1′ suggests that this
P450 enzyme might be capable of accepting a broader range of
phosphorylated alkylphenols as substrates. If this is true, CreJ
could be used to selectively oxidize aliphatic C–H bonds in diverse
alkylphenols. However, CreJ’s requirement of a phosphate an-
choring group would limit the application of this strategy. To
overcome this limitation, we envisioned that CreHI would be able
to phosphorylate variant alkylphenols, thereby providing potential
properly decorated substrates for CreJ oxidation. Furthermore, if
CreD could dephosphorylate diverse oxidized products produced
by CreJ, a useful multienzyme system could be established as an
Overall, the phosphate moiety of the substrate appears to be
gripped by CreJ, whereas the nonpolar portion of 1′ is recognized
predominantly by hydrophobic interactions between the benzyl
moiety and its surrounding residues including W199, F264, I312,
W315, and F416 (Fig. 3E and SI Appendix, Fig. S2B). These res-
idues constitute a hydrophobic pocket that extends beyond the
heme, which serves to position the methyl group to be oxidized
close to the heme iron reactive center. To examine the function of
these hydrophobic residues, five mutants including W199A,
F264A, I312A, W315A, and F416A were prepared (SI Appendix,
Fig. S3B) and their catalytic activities toward substrate 1′ were
evaluated (Fig. 4). As expected, alanine substitution of W199, alternate to chemical oxidations.
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Fig. 3. Structural analysis of CreJ. (A) An crystal asymmetric unit consisting of four molecules of CreJ. The molecules were traced from T29 to A433 except for
chain B, which has gaps due to unresolved electron density between T133 and E141. (B) Monomeric structure of CreJ. (C) Analysis of the amino acid residues in
the active site of CreJ. The 2Fo–Fc densities for 1′, heme, and C376 are shown as mesh at 2.0-σ level, and the heme iron is shown as Van der Waals sphere.
(D) Cross-sectional view of the catalytic cavity containing the heme group (HEM) and the substrate (1′). (E) Schematic diagram of the interactions between
CreJ and 1′ in the active site. Dashed lines denote the electrostatic and hydrophobic interactions with the distance between involved atoms shown in
angstroms. (F) Cross-sectional view of the CreJ in complex with 2′. The ethyl group of 2′ enters a cavity, by which the pro-S hydrogen would point to the heme
iron and be oxidized to S-2′a during the P450 catalysis.
To test these hypotheses, we selected 9 representative alkylphe- oxidizes 7′ rather than its structural analog 8′. We hypothesize
nols, 2–10 (Fig. 1A), namely, p-ethylphenol, 2; p-vinylphenol, 3;
p-propylphenol, 4; p-isopropylphenol, 5; m-cresol, 6; m-ethylphenol,
7; m-vinylphenol, 8; o-cresol, 9; and o-ethylphenol, 10 to examine
the activity of CreHI, CreJEF, and CreD. As expected, all of these
alkylphenols, with the exception of 10, were recognized by CreHI
that this might be the result of the intrinsic difference between the
sp3 and sp2 C–H bonds.
To establish the “chemomimetic” alkylphenol oxidation path-
way, we carried out a series of one-pot enzymatic reactions using
2–7 as individual substrates, into which CreHI/CreJEF (alterna-
and transformed into corresponding phosphorylated products tively with supplementation of CreG/CreC as helper enzymes),
2′–9′ (Fig. 5A and SI Appendix, Table S2 and Figs. S17–S25), thus and CreD were sequentially added. In this way, the ability of
demonstrating the broad substrate specificity of CreHI. The in- CreD to hydrolyze diverse phosphorylated compounds was
ability of CreHI to phosphorylate 10 might be due to the steric readily evaluated. More importantly, the hydrolysis of phosphoryl
interference of substrate binding or phosphate transfer by the esters would enable the structural assignments of oxidized prod-
adjacent ethyl group. To evaluate the activity of CreJEF toward ucts, by which the efficiency and selectivity of oxidation could
2′–9′, we elected to sequentially add CreHI (with necessary co- be ascertained.
factors, including Mg2+, Mn2+, and ATP) and CreJEF (with
When 2 was used as the substrate, CreJ mainly (as reflected by
the 82.1% yield of 2a, which was quantified based on HPLC peak
NADPH) into each reaction mixtures containing an individual
alkylphenol substrate. This strategy was pursued due to the dif- area integration) hydroxylates the C–H bond at the benzylic po-
ficulty in purifying highly hydrophilic and unstable phosphorylated sition of 2′ (Fig. 5B). Chiral HPLC analysis, using a manually
intermediates. In principle, we reasoned that the phosphorylated prepared bis(3-chlorophenylcarbamate)-(cyclohexylamide) chiral
intermediates (2′–9′) generated by CreHI catalysis, without iso- column (37), showed only a single enantiomer 2a [>99% enan-
lation and purification, would be recognized by CreJ and con- tiomeric excess (ee)] was produced, thus demonstrating the
verted into the corresponding oxidized phosphoryl products. In achievement of absolute stereoselectivity during catalysis. Re-
these multienzyme reactions, significant substrate consumption
and/or new product generation was detected when 2′–7′ were
substrates (SI Appendix, Table S2 and Figs. S17–S22). By contrast,
m-vinylphenyl phosphate (8′) and o-methylphenyl phosphate (9′)
were not oxidized. It is possible that the difficulty in oxidizing
compound 9′ is due to the methyl group at ortho position pointing
tention time comparison of the product to the enantiomerically
pure authentic standard established the S configuration at the
benzylic chiral center of 2a (SI Appendix, Fig. S26A). Based on the
detection of 2b (4.9% yield) and 2c (13.0% yield), the successive
oxidation steps 2′a→2′b→2′c can be readily deduced (Fig. 5B).
To elucidate the structural basis for the 2S stereoselectivity,
away from the P450 heme-iron reactive center. Surprisingly, CreJ the crystal structure of CreJ in complex with 2′ was solved at
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that both 2b and 2c were derived from 2′b upon the hydroxylation
of the benzylic position of 3′. The major product 3a could be de-
rived from the hydroxylation of the terminal sp2 C–H bond in the
vinyl group of 3 (compared with the hydroxylation of the benzylic
methylene in 2), and followed by isomerization (to 3′a) and hy-
drolysis (to 3a). We observed that compound 3′a was further oxi-
dized to 3′b, whose subsequent hydrolysis gave rise to 3b. The
different hydroxylation site selectivity of CreJ toward 3 and 2 may
be due to the presence of the vinyl group in 3. Its double bond
could form π–π or electrostatic interactions with neighboring aro-
matic residues, by which the sp2 C–H bond might adopt a con-
formation favoring the terminal hydroxylation.
According to the measurement of the substrate binding pocket
of CreJ (Fig. 3D), 4′ seems to be too large to be accommodated by
this P450 enzyme. However, the detection of multiple oxidative
products (4a–d, Fig. 5D) during the transformation of 4 suggests
that a catalytic cavity expansion in CreJ could be induced. Nota-
bly, the absolute configuration of 4a, 4c, and 4d were not identi-
fied due to lack of authentic standards. The low conversion
indicates that the size of 4′ might have reached the space limit of
the catalytic cavity. When 5 was used as a substrate, in addition to
the direct hydroxylation product 5a, three unexpected products
including 5b, 2b, and 2c were observed (Fig. 5E). Mechanistically,
5b could result from a direct desaturation of the isopropyl group.
A similar mechanism was previously proposed from the oxidation
process of ethyl carbamate by CYP2E1 (38). The products 2b and
2c might be derived from an oxidative cleavage event. Putatively,
5′a could be oxidized in one of the methyl groups to generate a
diol intermediate (5′c), which could then lose a single carbon unit
by the C–C bond cleavage during further oxidation, generating 2′b
and a molecule of formaldehyde. This mechanism is not un-
common in the reactions of cholesterol side chain cleavage (39,
40). For example, the transformation from cholesterol to preg-
nenolone depends on the cleavage of C20,C22 carbon–carbon
bond catalyzed by CYP11A1 (41).
Aside from p-alkylphenols, the two m-alkylphenols 6 and 7 were
also transformed by CreHI/CreJEF/CreD to different products
6a–c, and 7a–c plus 8, respectively, albeit with lower observed
conversion ratios (Fig. 5 F and G). Like S-2a, S-7a turned out to
be the only enantiomer (>99% ee) (SI Appendix, Fig. S26B),
demonstrating the perfect stereoselectivity of CreJ against 7. The
oxidation patterns of 6 and 7 are similar to those of 1 and 2, re-
spectively. The only exception is the production of 8, which is
likely attributed to the direct desaturation of the ethyl group.
We reasoned that the addition of CreG (alcohol dehydroge-
nase) and CreC (aldehyde dehydrogenase) could alter the ratios of
oxidative products, provided that the intermediate alcohols or al-
dehydes could be accepted by CreG or CreC as substrates, re-
spectively (Fig. 5 B–G). For example, in the presence of CreHI and
CreJEF, the alcohol 6a, the aldehyde 6b, and the carboxylic acid 6c
were all detected (Fig. 5F, trace ii), whereas the addition of CreG
and CreC led to the most oxidized compound 6c as the only de-
tectable product (Fig. 5F, trace iii). The improved oxidation could
be due to not only the catalytic activity of CreG (for both 6a→6b
and 6a′→6b′) and CreC (for both 6b→6c and 6b′→6c′), but also
the NAD(P)H recycling effects (Fig. 2A). By contrast, the addition
of CreG and CreC showed no positive impact on oxidative reac-
tions of 5 because none of the intermediates or products could be
recognized by CreG or CreC (Fig. 5E).
Fig. 4. The relative enzymatic activity and dissociation constant (KD) of CreJ
mutants. A total of 1 μM of CreJ was used to transform 1 mM substrate 1′ for
20 min. KD values are shown in brackets. “−” denotes that no valid KD could
be deduced. Substrate binding curves used for determining KD are shown in
SI Appendix.
1.7 Å (PDB ID code: 5XJN). This time, only one P450 molecule
existed in one asymmetric unit. The CreJ molecules in CreJ-2′
and CreJ-1′ have ∼0.17 Å rmsd, indicating the essentially iden-
tical CreJ structures in both complexes (SI Appendix, Fig. S27A).
The phosphate and benzyl moieties in both substrates are almost
identically positioned, and only slight conformational changes
were observed for the peripheral residues surrounding the ali-
phatic group of substrates. The terminal methyl group of 2′ in
the CreJ-2′ structure occupies the partial cavity space of a water
molecule in the CreJ-1′ structure, and the water molecule was
not observed in CreJ-2′ (SI Appendix, Fig. S27B).
In the substrate binding pocket of the 5XJN structure (Fig. 3F),
the ethyl group of 2′ enters a cavity that is shaped by the heme
group and a number of hydrophobic residues, including F264,
I312, F416, and T269, in which the pro-S hydrogen atom (4.2 Å) is
closer to the heme-iron reactive center than the pro-R hydrogen
atom (5.4 Å) of the methylene group in 2′. Although the C–Hpro-S
bond somehow points away from the heme iron, the oxygen atom
in the genuine ferryl-oxo catalytic species (compound I, SI Ap-
pendix, Fig. S1) would likely push away the terminal methyl group,
thus counterclockwise rotating the C–C bond that connects the
aromatic ring and the ethyl group, to project the pro-S hydrogen
atom toward the heme iron. Notably, there is indeed a cavity near
F264 that could accommodate the repelled methyl group. These
analyses, together with the fact that the benzylic C–H bond is
much more reactive than a primary C–H bond, can well explain
why S-2′a is exclusively formed. The keto group of 2′b likely results
from the second benzylic C–H hydroxylation, which is presumably
followed by a spontaneous dehydration. Interestingly, once the
benzylic position is fully oxidized, CreJ is able to hydroxylate the
primary C–H bond with much higher bond dissociation energy.
Sterically, 3 is highly analogous to 2. However, CreJ oxidized 3 in
a pattern that is distinct from 2. Four products including 2b (0.2%
yield), 2c (9.1% yield), 3a (62.1% yield), and 3b (12.9% yield) were
generated in reactions using 3 as the substrate (Fig. 5C). It appears
Controlled Oxidation of p-Ethylphenol, 2. It is challenging to pre-
cisely control the extent of oxidation of the aliphatic C–H bonds
in an alkylphenol by chemical methods (4). In the majority of our
multienzyme reactions (Fig. 5 B–G), a mixture of products was
observed. Although the product diversity showcases the ability of
this unique biocatalytic system to generate various oxidized
products, a single oxidized product is most often preferred in
practical application. Accordingly, we chose the substrate 2 as an
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Fig. 5. Chemomimetic oxidation of alkylphenols. (A) The tested phosphorylated alkylphenols. (B–G) HPLC analyses (Upper) and putative mechanisms (Lower)
of multienzyme reactions using p-ethylphenol (B), p-vinylphenol (C), p-propylphenol (D), p-isopropylphenol (E), m-cresol (F), and m-ethylphenol (G) as
substrates. Trace i, negative controls; trace ii, reactions sequentially catalyzed by CreHI/CreJEF (10 μM of each enzyme, 30 °C, 2 h) and CreD (10 μM, 30 °C, 2 h);
and trace iii, reactions sequentially catalyzed by CreHI/CreJEF/CreG/CreC (10 μM of each enzyme, 30 °C, 2 h) and CreD (10 μM, 30 °C, 2 h). The initial con-
centration of substrate was 1 mM in all reactions. The percentage numbers in black indicate the yield (conversion ratio) of a specific product. The percentage
numbers in green represent the ratio of unreacted substrates. Due to distinct extinction coefficients, the peak intensity of compounds do not necessarily
reflect relative amounts shown in percentage. ◆ indicates that the product was structurally assigned based on HRMS analysis and retention time comparison
with the corresponding authentic standard. ▲ indicates that the product was identified by NMR and HRMS. ● indicates that the product was structurally
deduced from HRMS and putative mechanism. nd, the specific percentage yield is not determined. Details for product structural assignments are shown in
SI Appendix.
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example to demonstrate that oxidation levels can be deliberately
and delicately controlled by careful management of the multi-
enzyme oxidation system.
To gain more general application of this multienzymatic oxi-
dation system, the substrate specificity of the central catalyst
P450 CreJ requires further broadening. Aside from the hydro-
philic portion of the substrate binding pocket that serves to
tightly anchor the phosphate group, the hydrophobic cavity oc-
cupied by the alkyl benzyl moiety is relatively small. This limited
space apparently prevents CreJ from accepting bulkier sub-
strates. Thus, protein engineering based on rational design and
combinatorial saturation mutagenesis could be used in the future
to achieve cavity expansion/reshaping of CreJ, as has been done
for P450 BM3 (16, 44).
The phosphate anchoring motif in CreJ is reminiscent of the
carboxylate-mediated substrate–enzyme interactions observed in
the fatty acid hydroxylase P450BSβ (45) and the P450 fatty acid
decarboxylase OleTJE (46). Unlike the phosphate docking site
remote from the heme iron in CreJ, the salt bridge for substrate
anchoring of the latter two P450 peroxygenases is formed between
the substrate carboxylate and a conserved arginine residue at a
location proximate to the heme iron. Interestingly, Watanabe and
colleagues revealed that nonnative substrates could be recognized
by P450BSβ using a free decoy molecule, such as short chain fatty
acids (47). This inspired us to test whether the free phosphate
group could enable the binding of the free alkylphenol 1. How-
ever, no oxidation of 1 by CreJEF was detected, even in the
presence of 100 mM inorganic phosphate, which indicates that
the covalently bound phosphate group is required for productive
substrate binding.
Notably, the hydrophobic pocket of CreJ is mainly shaped by a
number of aromatic amino acids, including W199, F264, W315,
and F416. It could be speculated that some of these aromatic
residues might be involved in π–π stacking interactions with the
benzene ring of substrate. If these specific interactions are es-
sential for productive substrate binding, this strategy would be
limited to the selective oxidation of alkylphenols. To evaluate this
possibility, we preliminarily assessed the reactivity of nonaromatic
substrates 1-hexanol phosphate (11′) and 4-methyl cyclohexanol
phosphate (12′) with CreJ. It is worth noting that these two
phosphorylated compounds were prepared chemically (SI Ap-
pendix for synthetic details) because neither compound could
be enzymatically prepared by CreHI from 1-hexanol, 11, and
4-methyl cyclohexanol, 12 (SI Appendix, Fig. S28). According
to LC/high-resolution mass spectrometry (HRMS) analysis,
11′ and 12′ were both oxidized to corresponding monohydroxylated
products, respectively (SI Appendix, Figs. S29 and S30). Although the
structures of products have yet to be determined, the substantial
conversion of 11′ and 12′ indicate that the chemomimetic oxidation
strategy developed in this work could be further extended toward the
selective oxidation of aliphatic alcohols, which is currently ongoing in
our laboratories.
In the sequential reactions mediated by CreHI/CreJEF and
CreD (Fig. 5B, trace ii), the enantiomer S-2a accounted for 82.1%
of total products. To further increase this percentage, we simply
lowered the concentration of NADPH from 3 mM to 2 mM at the
oxidation stage, by which further oxidation of S-2a to 2b and 2c
was stalled due to the limited reducing equivalents, thus leading to
a 92.7% yield of S-2a (Fig. 6, trace ii). To improve the production
of p-acetyl phenol (2b), CreG (10 μM) together with its cofactor
NAD+ (2 mM) were added to the reaction mixture upon com-
pletion of sequential catalysis by CreHI/CreJEF and CreD. As a
result, 94.5% of total detected products were 2b (Fig. 6, trace iii).
Finally, oxidation of 2 to 2c was driven to near completion by ti-
tration of fresh CreJEF and NADPH in the reaction comediated
by CreHI/CreJEF/CreG. Remarkably, final hydrolysis by CreD,
produced a 98.6% yield of 2c (Fig. 6, trace iv).
Discussion
In this study, the recently discovered p-cresol biodegradation
pathway from C. glutamicum (30) comediated by CreHI (for an-
choring/directing group installation), CreJEF (for selective oxi-
dation), CreG and CreC (as auxiliary catalysts), and CreD (for
anchoring/directing group offloading) was used to construct gen-
uine biosynthetic routes for production of various oxidized alkyl-
phenols. By harnessing the broad substrate specificity and
combined activities of CreHI, CreJ, and CreD, selective oxidation
of the aliphatic C–H bonds in a representative group of p- and
m-alkylphenols was successfully achieved. In particular, this bio-
catalytic system showed perfect stereoselectivity when hydroxyl-
ating p-ethylphenol, 2, and m-ethylphenol, 7 (Fig. 5 B and G),
which is highly challenging for chemical oxidation. However, this
biocatalytic system was not able to oxidize o-alkylphenols. In-
terestingly, recent work has demonstrated that P450 BM3 can
be engineered to hydroxylate alkylbenzenes to corresponding
alkylphenols (42, 43). Should these biocatalysts be coupled to
the current alkylphenol oxidation system, an extended bio-
chemical route from alkylbenzenes to oxidized alkylphenols could
be created.
Compared with other alkyl aromatics biooxidation pathways in
nature (5, 48), such as xylene monooxygenase (49–51) and p-cresol
methylhydroxylase (52), this unique chemomimetic system fea-
tures a greater generality because the key P450 enzyme CreJ
adopts the phosphate anchoring strategy for substrate recognition
and binding. Protein BLAST searches reveal a large number of
CreJ analogs (>40% sequence identity) from different genera of
microorganisms. Sequence alignment of these homologous pro-
teins demonstrates that the residues involved in phosphate an-
choring and hydrophobic interactions with substrates are highly
conserved (SI Appendix, Fig. S31), suggesting a new family of
P450 enzymes (SI Appendix, Fig. S32). Interestingly, a majority of
these P450 genes are clustered with groups of genes analogous to
creHI, creEF, as well as creD (SI Appendix, Fig. S33). The wide-
spread creJ-like P450 genes and cre-like gene clusters suggest that
similar oxidation systems might be broadly used by diverse mi-
croorganisms, from which analogous oxidative biocatalysts with
different substrate preference, improved oxidation selectivity, and
higher catalytic efficiency could be identified.
Fig. 6. Controlled oxidation of p-ethylphenol, 2. Trace i, negative control;
trace ii, the reaction was sequentially catalyzed by CreHI/CreJEF (10 μM of
each enzyme, 2 mM NADPH, 30 °C, 2 h) and CreD (10 μM, 30 °C, 2 h); trace
iii, the reaction mixture of trace ii was supplemented with CreG (10 μM,
30 °C, 2 h) and 2 mM NAD+; trace iv, the reaction was first comediated by
CreHI/CreJEF/CreG (10 μM of each enzyme, 30 °C, 2 h), followed by adding an
additional batch of CreJEF (10 μM of each enzyme, 30 °C, 2 h) and NADPH
(3 mM), and finally catalyzed by CreD (10 μM, 30 °C, 2 h). Due to distinct
extinction coefficients, the peak intensity of compounds do not necessarily
reflect relative amounts shown in percentage.
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For controlled oxidation of 2, the reactions were performed as follows: To
obtain the predominant product S-2a, the reaction mixture containing 10 μM
CreHI, 10 μM CreJEF, 20 mM MgCl2, 2 mM MnCl2, 2 mM ATP, and 2 mM
NADPH was first incubated at 30 °C for 2 h. Following this initial incubation,
CreD (10 μM) was added into the mixture and allowed to incubate for an
additional 2 h. To produce 2b as the major product, CreG (10 μM) with its
cofactor NAD+ (2 mM) was added into the above post-CreD reaction mix-
ture, followed by another 2-h incubation. To predominantly produce 2c, the
reaction mixture containing 10 μM CreHI, 10 μM CreJEF, 10 μM CreG, 20 mM
MgCl2, 2 mM MnCl2, 2 mM ATP, 2 mM NAD+, and 3 mM NADPH was first
incubated at 30 °C for 2 h. Subsequently, an additional batch of CreJEF (final
concentration of 20 μM, including the first addition) and NADPH (final
concentration of 6 mM, including the first addition) were added into the
above mixture, followed by another 2-h incubation. Finally, CreD (10 μM)
was added into the mixture and followed by incubation at 30 °C for 2 h.
Methods
Crystallization, Data Collection, and Structure Determination. Before crystal-
lization, the protein was diluted to 0.3 mM by mixing with 10 mM Tris·HCl
(pH 8.0), supplemented with 12 mM substrate 1′ or 2′. Crystallization conditions
were determined using commercial high throughput screening kits pur-
chased from Hampton Research and a nanoliter drop-setting Mosquito
robot (TTP Labtech) operating with 96-well plates. The crystallization
conditions amenable to crystal generations were further optimized in
24-well crystallization plates. The crystals of CreJ in complex with 1′ used for
X-ray data collection were obtained in hanging drops containing 0.2 M
ammonium sulfate, 0.1 M sodium cacodylate trihydrate, pH 6.4, and 27%
PEG8000, whereas the crystals of CreJ in complex with 2′ were obtained in
0.2 M sodium iodide and 20% PEG3350. The data were collected at the
Shanghai Synchrotron Radiation Facility, Beamline BL17U (53), in a 100-K
nitrogen stream. Data indexing, integration, and scaling were conducted
using MOSFLM (54). The CreJ structure was determined by molecular re-
placement using CCP4 program suite (55) and the PDB file 4RM4 (56) as a
search model. The structure was automatically built by ARP/wARP in the
CCP4i program package (57). Refinement of the structure was performed
using the programs COOT (58) and PHENIX (59). The final model was eval-
uated using PROCHECK (60). All molecular graphics were created using
PyMOL v1.8 (Schrödinger).
Analytical Methods. Samples were analyzed on an Agilent 1260 infinity HPLC
system (Agilent Technologies) with a photodiode array detector. Reaction
mixtures were separated on a ZORBAX SB-C18 column (Agilent Technologies)
by using a linear mobile phase gradient ranging from 2% (vol/vol) acetonitrile
in 0.1% (vol/vol) TFA aqueous solution to 80% (vol/vol) acetonitrile in 0.1%
(vol/vol) TFA aqueous solution over 25 min. The flow rate was set to 1 mL/min
and the injection volume was 20 μL. The detection wavelength was set to
275 nm. Substrate consumption and product formation were quantified by
HPLC peak area integration using corresponding authentic compounds as
standards. Product structural assignments were performed by comparison of
a detected compound with the corresponding authentic standard regarding
retention time and coinjection confirmation, HRMS, and/or NMR. HRMS data
were recorded in the negative and/or positive ionization mode on a MaXis
Impact UHR-qTOF System (Bruker).
Enzymatic Assays. Unless specified otherwise, all enzymatic assays were car-
ried out in 100 μL of 50 mM Tris·HCl buffer (pH 8.0) at 30 °C for 2 h, and the
final concentration of each enzyme was 10 μM. Methanol (3× volume) was
added to quench reactions. Precipitated proteins were removed by centri-
fugation at 20,000 × g for 10 min and the supernatants were used as LC-MS
samples. For reactions catalyzed by CreHI, the reaction mixture contained
1 mM substrate, 20 mM MgCl2, 2 mM MnCl2, 2 mM ATP, and purified CreH
and CreI. For CreJEF activity toward phosphorylated compounds (2′–9′), the
CreHI reactions were used to generate the phosphorylated substrates. Next,
CreJ, CreE, and CreF together with 3 mM NADPH were added into the in-
dividual post-CreHI reaction mixture.
ACKNOWLEDGMENTS. We thank the staff of BL17U Beamline at the Shanghai
Synchrotron Radiation Facility and Dr. Shilong Fan (Tsinghua University)
for assistance during X-ray diffraction data collection and Dr. Jeffrey D.
Kittendorf (Alluvium Biosciences) for helpful discussion and proofreading
of the manuscript. This work was supported by the National Natural Science
Foundation of China under Grant NSFC 31422002, Shandong Provincial Nat-
ural Science Foundation Grant JQ201407, the Key Frontier Project of the
Chinese Academy of Sciences, QYZDB-SSW-SMC042 (to S.L.), the 973 Project
from Ministry of Science and Technology Grant 2012CB7211-04 (to S.-J.L.),
and Grant NSFC 31270784 from the National Natural Science Foundation of
China (to Y.F.).
For one-pot chemomimetic alkylphenol oxidation reactions, the reaction
mixtures contained CreHI, CreJEF, and the two optional enzymes CreG (with
2 mM NAD+) and CreC (with 2 mM NADP+), 1 mM substrate 2–7, and nec-
essary cofactors, including 20 mM MgCl2, 2 mM MnCl2, 2 mM ATP, and 3 mM
NADPH. After incubation at 30 °C for 2 h, CreD was added into each of the
one-pot mixtures, followed by another 2-h incubation at 30 °C.
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