The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Article abstract of DOI:10.1016/j.phytochem.2020.112512

The CYP74 family of cytochromes P450 includes four enzymes of fatty acid hydroperoxide metabolism: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and epoxyalcohol synthase (EAS). The present work is concerned with catalytic specificities of three recombinant DESs, namely, the 9-DES (LeDES, CYP74D1) of tomato (Solanum lycopersicum), 9-DES (NtDES, CYP74D3) of tobacco (Nicotiana tabacum), and 13-DES (LuDES, CYP74B16) of flax (Linum usitatissimum), as well as their alterations upon the site-directed mutagenesis. Both LeDES and NtDES converted 9-hydroperoxides of linoleic and α?linolenic acids to divinyl ethers colneleic and colnelenic acids (respectively) with only minorities of HPL and EAS products. In contrast, LeDES and NtDES showed low efficiency towards the linoleate 13-hydroperoxide, affording only the low yield of epoxyalcohols. LuDES exhibited mainly the DES activity towards α?linolenate 13-hydroperoxide (preferred substrate), and HPL activity towards linoleate 13-hydroperoxide, respectively. In contrast, LuDES converted 9-hydroperoxides primarily to the epoxyalcohols. The F291V and A287G mutations within the I-helix groove region (SRS-4) of LuDES resulted in the loss of DES activity and the acquirement of the epoxyalcohol synthase activity. Thus, the studied enzymes exhibited the versatility of catalysis and its qualitative alterations upon the site-directed mutagenesis.

Full text of DOI:10.1016/j.phytochem.2020.112512

Phytochemistry 179 (2020) 112512
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
The CYP74B and CYP74D divinyl ether synthases possess a side
hydroperoxide lyase and epoxyalcohol synthase activities that are
enhanced by the site-directed mutagenesis
Yana Y. Toporkova*, Elena O. Smirnova, Tatiana M. Iljina, Lucia S. Mukhtarova,
Svetlana S. Gorina, Alexander N. Grechkin**
Kazan Institute of Biochemistry and Biophysics, FRC Kazan Scientific Center, Russian Academy of Sciences, P.O. Box 30, Kazan, 420111, Russia
A R T I C L E I N F O
A B S T R A C T
Keywords:
The CYP74 family of cytochromes P450 includes four enzymes of fatty acid hydroperoxide metabolism: allene
oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and epoxyalcohol synthase
(EAS). The present work is concerned with catalytic specificities of three recombinant DESs, namely, the 9-DES
(LeDES, CYP74D1) of tomato (Solanum lycopersicum), 9-DES (NtDES, CYP74D3) of tobacco (Nicotiana tabacum),
and 13-DES (LuDES, CYP74B16) of flax (Linum usitatissimum), as well as their alterations upon the site-directed
Cytochromes P450
CYP74 family
Divinyl ether synthase
Hydroperoxide lyase
Epoxyalcohol synthase
Oxylipins
mutagenesis. Both LeDES and NtDES converted 9-hydroperoxides of linoleic and αꢀ linolenic acids to divinyl
ethers colneleic and colnelenic acids (respectively) with only minorities of HPL and EAS products. In contrast,
LeDES and NtDES showed low efficiency towards the linoleate 13-hydroperoxide, affording only the low yield of
epoxyalcohols. LuDES exhibited mainly the DES activity towards
αꢀ linolenate 13-hydroperoxide (preferred
substrate), and HPL activity towards linoleate 13-hydroperoxide, respectively. In contrast, LuDES converted 9-
hydroperoxides primarily to the epoxyalcohols. The F291V and A287G mutations within the I-helix groove re-
gion (SRS-4) of LuDES resulted in the loss of DES activity and the acquirement of the epoxyalcohol synthase
activity. Thus, the studied enzymes exhibited the versatility of catalysis and its qualitative alterations upon the
site-directed mutagenesis.
1. Introduction
hemiacetal synthase; Mukhtarova et al., 2018) and the epoxyalcohol
synthase (EAS) (Toporkova et al., 2017a). Until recently, the CYP74
Plant oxylipins are bioactive derivatives of fatty acids that are
involved in the regulation of growth and development and stress
adaptation (Blee, 1998; Grechkin, 1998; Savchenko et al., 2014). A
major source of oxylipins is the lipoxygenase pathway (Grechkin, 1998;
Feussner and Wasternack, 2002; Stumpe and Feussner, 2006; Griffiths,
2015). In plants, the key enzymes of this pathway are lipoxygenases and
nonclassical cytochromes P450 of the CYP74 family. This family in-
cludes two dehydrases, namely the allene oxide synthase (AOS, Tijet and
Brash, 2002) and the divinyl ether synthase (DES) (Grechkin, 2002), as
well as two isomerases, i.e. the hydroperoxide lyase (HPL, synonym
enzymes were believed to occur only in flowering plants. However, the
CYP74 and related enzymes were detected in mosses (Bandara et al.,
2009; Scholz et al., 2012), spikemoss Selaginella moellendorffii (Gorina
et al., 2016, Toporkova et al., 2018а), proteobacteria Methylobacterium
(Lee et al., 2008), brown alga Ectocarpus siliculosus (Toporkova et al.,
2017a), as well as several Metazoa (Lee et al., 2008; Toporkova et al.,
2017b). Detection of non-plant members expanded the CYP74 diversity
from family to clan (Nelson et al., 2013).
The most common CYP74 enzymes are AOSs and HPLs (Grechkin,
2002). The genes encoding these enzymes were found in the genomes of
Abbreviations: DES, divinyl ether synthase; HPL, hydroperoxide lyase; EAS, epoxyalcohol synthase; AOS, allene oxide synthase; 9-H(P)OD, (9S,10E,12Z)-9-hydro
(pero)xy-10,12-octadecadienoic acid; 9-H(P)OT, (9S,10E,12Z,15Z)-9-hydro(pero)xy-10,12,15-octadecatrienoic acid; 13-H(P)OD, (9Z,11E,13S)-13-hydro(pero)xy-
9,11-octadecadienoic acid; 13-H(P)OT, (9Z,11E,13S,15Z)-13-hydro(pero)xy-9,11,15-octadecatrienoic acid; IMAC, immobilised metal affinity chromatography; Me,
methyl; TMS, trimethylsilyl.
* Corresponding author.
** Corresponding author.
E-mail addresses: toporkova@kibb.knc.ru (Y.Y. Toporkova), grechkin@kibb.knc.ru (A.N. Grechkin).
https://doi.org/10.1016/j.phytochem.2020.112512
Received 25 March 2020; Received in revised form 2 September 2020; Accepted 4 September 2020
Available online 11 September 2020
0031-9422/© 2020 Elsevier Ltd. All rights reserved.

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
all flowering plants studied to date. DESs and EASs are less common and
studied enzymes. These enzymes were found in a small number of plant
species from different taxa.
m/z 123 (27%), m/z 109 (29%), m/z 95 (59%), m/z 81 (97%), and m/z
67 (100%). Its mass spectral pattern was identical to that of 9-[(1′E,3′Z)-
nonadienyloxy]-(8E)-nonenoic (colneleic) acid (Galliard and Phillips,
1972; Fammartino et al., 2007). Upon the hydrogenation over PtO2 the
product 1 turned to the compound the mass spectrum of which matched
that of 10-oxanonadecanoic acid (Me) (Toporkova et al., 2013). Thus,
the obtained data enabled to identify product 1 as colneleic acid. The
second most product of this reaction was compound 2. The electron
impact mass spectrum of product 2 (Me/TMS) exhibited M+ at m/z 398
(0.4%), [M – Me]+ at m/z 383 (0.6%), [M – MeO – TMSOH]+ at m/z 277
(0.8%), [M – C1/C8]+ at m/z 241 (2%), [M – C1/C9]+ at m/z 212 (1%),
[M – C1/C10]+ at m/z 199 (100%), m/z 155 (11%), m/z 129 (57%), and
[TMS]+ at m/z 73 (93%). The intense fragment at m/z 199 indicated the
presence of oxiranyl carbinol function with oxirane at C9/C10 and
secondary alcohol (TMS) at C11. Catalytic hydrogenation of product 2
over PtO2 followed by methylation and trimethylsilylation afforded
product whose mass spectrum matched that of 9,10-epoxy-11-hydrox-
yoctadecanoic acid (Me/TMS) (Toporkova et al., 2017a). Thus, the
mass spectral data substantiated the structure of 9,10-epoxy-11-hydrox-
y-12-octadecenoic acid for compound 2. Additionally, GC-MS analyses
revealed the presence of minor product 3 in both reactions. The mass
spectrum of product 3 (Me/TMS) exactly matched the spectrum of
9-hydroxynonanoic acid (Me/TMS) (Mukhtarova et al., 2011), thus
suggesting the enzymatic formation of 9-oxononanoic acid, the 9-HPL
product. Furthermore, when the products (Me/TMS) of NtDES and
LeDES incubation with 9-HPOD were analysed by GC-MS without the
preliminary NaBH4 reduction, product 3 was absent. Instead, a more
volatile product has been detected. The electron impact mass spectrum
of this volatile product (Me) exhibited [M – H]+ at m/z 185 (1%), [M –
CO]+ at m/z 158 (9%), [M – OMe]+ at m/z 155 (15%), [M – C8/C9]+ at
m/z 143 (27%), 87 (71%), as well as the methyl ester McLafferty rear-
rangement ion at m/z 74 (100%). The spectrum matched that of 9-oxo-
nonanoic acid (Me) (Mukhtarova et al., 2011), thus, substantiating the
structure of 9-hydroxynonanoic acid (Me/TMS) for compound 3.
The GC-MS profiles of NaBH4-reduced products (Me/TMS) of NtDES
and LeDES incubations with 9(S)-HPOT looked similar to those of 9(S)-
HPOD products (Fig. 3B, Supplementary Fig. S1B). The major product of
both incubations was compound 4 the mass spectrum of which exhibited
M+ at m/z 306 (8%), [M – C8’/9’]+ at m/z 277 (1%), m/z 240 (4%), [M –
C1’/C9’ – O]+ at m/z 169 (8%), [169 – MeOH]+ at m/z 137 (21%), [M –
C1/C9 – O]+ at m/z 121 (37%), m/z 93 (69%), m/z 79 (100%) and
corresponded to that of 9-[1′E,3′Z,6′Z-nonatrienyloxy]-(8E)-nonenoic
(colnelenic) acid (Me) (Galliard et al., 1973). The second most abundant
product of 9(S)-HPOT conversion by the LeDES was compound 5 eluted
after the 9-HOT (Me/TMS). Its mass spectrum possessed the following
fragments: M+ at m/z 396 (0.2%), [M – Me]+ at m/z 381 (1%), [M –
MeO]+ at m/z 365 (0.1%), [M – TMSOH]+ at m/z 306 (1%), m/z 257
(2%), m/z 211 (2%), [M – C1/C10]+ at m/z 197 (21.12%), m/z 155
(6%), m/z 131 (31%), m/z 107 (61%), and [TMS]+ at m/z 73 (100%).
The spectral patterns, particularly the characteristic fragment at m/z
197, indicated the oxiranyl carbinol structure with TMS-oxy function at
C11 and oxirane at C9,C10, i.e. the 9,10-epoxy-11-hydroxy-12,15-octa-
decadienoic acid (Me/TMS). At the same time, compound 5 was only a
minority upon the reaction of 9(S)-HPOT with NtDES (Fig. 3B). Finally,
the minor product of both reactions was the same above mentioned
9-HPL product 3, 9-hydroxynonanoic acid (Me/TMS).
Recently, it was found that some CYP74 enzymes are multifunctional
and catalyze the formation of products characteristic for different types
of enzymes. So, several CYP74C subfamily enzymes, previously
described as HPLs, possess the EAS activity towards some substrates
(Toporkova et al., 2018b). Then, the rice HPLs (CYP74E1 and CYP74E2)
exhibited side AOS activities (Kuroda et al., 2005). Furthermore, three
CYP74B HPLs (Toporkova et al., 2020), as well as two AOSs (carrot
DcAOS (CYP74B33, Gorina et al., 2019) and Arabidopsis AOS
(CYP74A1, Hughes et al., 2008)), were described to exhibit additional
EAS activity. Two more AOS, namely LuAOS (CYP74A1) and LeAOS3
(CYP74C3) were reported to possess minor HPL activity (Song et al.,
1993; Toporkova et al., 2008). A similar dualism of catalysis was first
time described in present work for some DESs. Three divinyl ether
synthases, LeDES (CYP74D1) of tomato, NtDES (CYP74D3) of tobacco,
and LuDES (CYP74B16) of flax producing not only divinyl ethers (DES
products), but also aldoacids (HPL products) and oxiranyl carbinols
(EAS products), are described in this work. Besides, the present work
reports the catalytic alterations of LuDES upon the site-directed
mutagenesis.
2. Results
2.1. Expression and purification of recombinant proteins NtDES, LeDES,
WT LuDES, LuDES A287G, and LuDES F291V
Recombinant enzymes NtDES (NP_001312606, GI:107799697),
LeDES (NP_001234527, GI:543675), and LuDES (ADP03054,
GI:310687282) have been obtained in a heterologous expression system
in E. coli cells and purified by IMAC.
The mutant forms of LuDES gene were constructed by PCR using
primers listed in Table 1 and Pfu DNA polymerase (Promega, USA). The
mutations were confirmed by DNA sequencing using the 3130 Genetic
Analyzer (Applied Biosystems). The multiple alignments of CYP74
amino acid sequences (Fig. 1) have been performed to reveal the
distinctive conserved features of the AOSs, HPLs, DESs within the
catalytically essential HBD domain (Toporkova et al., 2008, 2018b). The
choice of mutation sites was based on these analyses.
Characterization of reaction products of purified enzymes was per-
formed by GC-MS. The structural formulae of the identified products of
target enzymes are presented at Fig. 2. The peaks of main products in
TIC (total ion current) chromatograms have been quantified. The per-
centage of different kinds of products are presented in Table 2.
2.2. Product specificity exhibited by NtDES and LeDES
Recombinant NtDES and LeDES efficiently utilized 9(S)-HPOD and 9
(S)-HPOT, while as 13(S)-HPOD was the poor substrate. At the same
time, these enzymes were quite inactive towards 13(S)-HPOT. The GC-
MS analyses of NaBH4-reduced products (Me/TMS) of 9(S)-HPOD in-
cubation with NtDES and LeDES revealed products 1–3 (Fig. 3A, Sup-
plementary Fig. S1A). The main product of 9(S)-HPOD conversion by
both enzymes was compound 1 whose mass spectrum exhibited M+ at
m/z 308 (20%), [M – MeO]+ at m/z 277 (1%), [M – C6’/9’]+ at m/z 251
(5%), [M – C5’/9’]+ at m/z 237 (2%), [M – C1/C7]+ at m/z 165 (11%),
The 13(S)-HPOD was less efficient substrate for both enzymes.
Conversion 13(S)-HPOD resulted in the formation of a single product 6
(Fig. 3C, Supplementary Fig. S1C). Mass spectrum of product 6
possessed M+ at m/z 398 (0.86%), [M – Me]+ at m/z 383 (1%), [M – n-
pentyl]+ at m/z 327 (2%), [M – C12/C18]+ at m/z 285 (55%), and
[TMS]+ at m/z 73 (100%). The fragmentation patterns confirmed the
structure of 11-hydroxy-12,13-epoxy-9-octadecenoic acid (Me/TMS) for
compound 6. Catalytic hydrogenation of product 6 over PtO2 followed
by methylation and trimethylsilylation yielded product mass spectrum
of which corresponded to the structure of 11-hydroxy-12,13-
Table 1
Oligonucleotide primers used for site-directed mutagenesis of LuDES gene.
Primer
5′–3′ sequence
LuDES F291V F
LuDES F291V R
LuDES A287G F
LuDES A287G R
CTGGCATTCAACTCGgTCGAAGGATTTACCC
GGGTAAATCCTTCGAcCGAGTTGAATGCCAG
CAATTTATTATTTGTTCTGGgATTCAACTCGTTC
GAACGAGTTGAATcCCAGAACAAATAATAAATTG
2

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
Fig. 1. Multiple alignments of I-helix se-
quences of following CYP74s: As, Allium
sativum; AsDES, CAI30435; Ca, Capsicum
annuum; CaDES, ABH03632; CaHPL,
AAK27266; Cs, Citrus sinensis; CsAOS,
NP_001275835; Gm, Glycine max; GmAOS,
NP_001236445; Le, Solanum lycopersicum;
LeHPL, CAB43022; LeDES, AAG42261; Lu,
Linum usitatissimum; LuDES, ADP03054; Na,
Nicotiana attenuata; NaAOS, CAC82911; Nt,
Nicotiana tabacum; NtDES, AAL40900;
NtHPL, AAZ39884; Ra, Ranunculus acris;
RaDES, CYP74Q1, AJU57209; Sm, Selagi-
nella moellendorffii; SmDES1, CYP74M1,
EFJ19674; SmDES2, CYP74M3, EFJ34345;
St, Solanum tuberosum; StDES, CAC28152.
Hydroperoxide-binding domain is circled.
Sites with substitutions are marked with
arrows.
Fig. 2. Structural formulae of reaction products of target WT enzymes and their mutant forms. 1, colneleic acid; 2, 9,10-epoxy-11-hydroxy-12-octadecenoic acid; 3,
9-hydroxynonanoic acid; 4, colnelenic acid; 4a, (3′E)-colnelenic acid; 5, 9,10-epoxy-11-hydroxy-12,15-octadecadienoic acid; 6, 11-hydroxy-12,13-epoxy-9-octade-
cenoic acid; 7, (
ω5Z)-etherolenic acid; 8, (9Z)-12-hydroxy-9-dodecenoic acid; 9, (10E)-12-hydroxy-10-dodecenoic acid; 10, 11-hydroxy-12,13-epoxy-9,15-octadeca-
dienoic acid; 11, (
ω
5Z)-etheroleic acid; 12, 9-hydroxy-12,13-epoxy-10-octadecenoic acid; 13, 9,10-epoxy-13-hydroxy-11,15-octadecadienoic acid; 14, 9,10-epoxy-
13-hydroxy-11-octadecenoic acid. (3′E)-Colnelenic acid is a product of thermal isomerization of the ordinary (8E,1′E,3′Z,6′Z)-colnelenic acid occurring during the
GC analyses.
3

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
Table 2
Quantification of the DES, HPL and EAS products formed by recombinant NtDES, LeDES, WT LuDES and two mutant forms LuDES A287G and LuDES F291V upon
incubations with fatty acid hydroperoxides. The NaBH4–reduced products (Me/TMS) were analysed by GC-MS. The peaks of epoxyalcohols (EAS products), 9-hydrox-
ynonanoic acid (NaBH4-reduced 9-HPL product), (9Z)-12-hydroxy-9-dodecenoic and (10E)-12-hydroxy-10-dodecenoic acids (NaBH4-reduced 13-HPL products), as
well as divinyl ethers (DES products) in TIC chromatograms were quantified. Their percentage compositions are presented.
Substrate
9(S)-HPOD
9(S)-HPOT
13(S)-HPOD
13(S)-HPOT
DES
HPL
EAS
DES
HPL
EAS
tr.a
DES
HPL
EAS
DES
HPL
EAS
NtDES
64.6
55.2
0
8.9
tr.a
0
29.5
44.8
100
92.5
66.9
0
7.5
0
0
100
100
24.4
42.0
47.6
n.a.b
n.a.b
58.0
0
LeDES
9.9
23.2
95.2
87.5
72.3
0
0
WT LuDES
LuDES F291V
LuDES A287G
4.8
19.7
0
55.9
58.0
52.4
22.7
46.2
44.0
19.3
53.8
20.5
0
1.9
1.8
98.1
98.2
0
12.5
27.7
0
0
0
35.5
a
tr., trace quantity (less than 1% of total products).
No activity.
b
epoxyoctadecanoic acid (Me/TMS) (Toporkova et al., 2017a), thus
confirming the identification of compound 6. Thus, NtDES and LeDES
behaved mainly as the DES with minor EAS and HPL activities towards
9-hydroperoxides and as the EAS towards 13(S)-HPOD (Table 2). At the
same time, NtDES and LeDES were absolutely inactive towards 13
(S)-HPOT (Fig. 3D, Supplementary Fig. S1D).
2.3. Product specificity exhibited by LuDES
Recombinant LuDES effectively metabolized 13(S)-HPOD and 13(S)-
HPOT (Fig. 4). 9(S)-HPOD and 9(S)-HPOT were poorer substrates for
LuDES (Fig. 4A and B). The GC–MS analyses (Fig. 4D) of 13(S)-HPOT
conversion by the LuDES revealed the presence of a predominant
product 7, identified earlier as (ω5Z)-etherolenic acid (Me ester)
(Gogolev et al., 2012), mass spectrum of which possessed M+ at m/z 306
(14%), [M – C2H5]+ at m/z 277 (2%), [M ꢀ OCH3]+ at m/z 275 (2%),
[M ꢀ (CH2)6 – COOCH3]+ at m/z 163 (9%), 149 (28%), 131 (38%), 107
(36%), 81 (100%), and 55 (84%). Additionally, there were three minor
products 8–10 (Me/TMS derivatives of NaBH4-reduced products), see
Fig. 4D. The most distinctive fragments of the mass spectrum of product
8 (Me/TMS) were [M – H]+ at m/z 299 (0.4%), [M – Me]+ at m/z 285
(1%), and [M – Me – MeOH]+ at m/z 253 (5%). Overall, the spectrum
matched that of (9Z)-12-hydroxy-9-dodecenoic acid (Me/TMS), a hy-
droxy acid formed through the NaBH4 reduction of the aldoacid
(9Z)-12-oxo-9-dodecenoic acid, a primary product of 13-HPL (Mukh-
tarova et al., 2011). Catalytic hydrogenation of product 8 followed by
methylation and trimethylsilylation afforded compound identified as
12-hydroxydodecanoic acid (Me/TMS) by its mass spectrum with
prominent [M – Me]+ at m/z 287 and [M – Me – MeOH]+ at m/z 255
(Mukhtarova et al., 2011). Product 9 (eluted shortly after peak 8)
possessed M+ at m/z 300 (1%), [M – Me]+ at m/z 285 (1%), and [M – Me
– MeOH]+ at m/z 253 (5%). The spectrum corresponded to that of
(10E)-12-hydroxy-10-dodecenoic acid (Me/TMS) formed through the
NaBH4 reduction of traumatin, (10E)-12-oxo-10-dodecenoic acid, a
product of allylic isomerization of the (9Z)-12-oxo-9-dodecenoic acid.
Thus, compound 9 was also derived from a 13-HPL product. Catalytic
hydrogenation of product 9 followed by methylation and trimethylsi-
lylation resulted in the formation of the 12-hydroxydodecanoic acid
(Me/TMS) mentioned above.
Fig. 3. GC-MS analyses of products (Me/TMS) of recombinant NtDES in-
cubations with 9(S)-HPOD (A), 9(S)-HPOT (B), 13(S)-HPOD (C), and 13(S)-
HPOT (D). 1, colneleic acid (Me); 2, 9,10-epoxy-11-hydroxy-12-octadecenoic
acid (Me/TMS); 3, 9-hydroxynonanoic acid (Me/TMS); 4, colnelenic acid
(Me); 4a, (3′E)-colnelenic acid (Me); 6, 11-hydroxy-12,13-epoxy-9-octadece-
noic acid (Me/TMS). The structural formulae of products are presented in
Fig. 2. 9-HOD, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid; 9-HOT,
(9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid; 13-HOD, (9Z,11E,
13S)-13-hydroxy-9,11-octadecadienoic acid; 13-HOT, (9Z,11E,13S,15Z)-13-
hydroxy-9,11,15-octadecatrienoic acid.
Besides compounds 8 and 9, the additional minor peak of less vol-
atile product 10 (Me/TMS) was detected upon the 13-HPOT conversion.
Its spectrum possessed [M – Me]+ at m/z 381 (0.2%), [M – Ethyl]+ at m/
z 367 (0.2%), m/z 325 (1%), [M – C12/C18]+ at m/z 285 (41%), [M –
C1/C8]+ at m/z 239 (4%), m/z 181 (7%), m/z 163 (14%), m/z 155
(17%), m/z 135 (25%), m/z 121 (11%), and [TMS]+ at m/z 73 (100%). A
prominent fragment at m/z 285 resulted from a cleavage between
oxirane and C11 in the 11-hydroxy-12,13-epoxy moiety. [M ꢀ C12/
C18]+ при m/z 285 (60%); Hydrogenation of compound 10 gave its
saturated analogue mass spectrum of which matched that of 11-
4

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
abundant product of 13(S)-HPOD conversion by the LuDES was com-
pound 12. The mass spectrum of product 12 exhibited M+ at m/z 398
(0.7%), [M – Me]+ at m/z 383 (1%), [M – C14/C18]+ at m/z 327 (1%),
[M – C1/C8]+ at m/z 241 (6%), [TMS–O+=CH2] at m/z 103 (3%), m/z
99 (cleavage of oxirane at C12/C13, 18%), and [TMS]+ at m/z 73
(100%). The characteristic fragment at m/z 241 suggested the epox-
yalcohol structure with a secondary alcohol (TMS) function at C9.
Moreover, the data indicated the presence of oxirane at C12,C13. Cat-
alytic hydrogenation converted product 12 to a saturated derivative
whose mass spectrum approved the structures of 9-hydroxy-12,13-epox-
yoctadecanoic acid (Me/TMS) and 9-hydroxy-12,13-epoxy-10-octadece-
noic acid (Me/TMS) for hydrogenated product and compound 12,
respectively (Toporkova et al., 2018b).
The 9(S)-HPOT was the poorest substrate for LuDES. Conversion of 9
(S)-HPOT resulted in the formation of compounds 3 and 5 described
above, as well as the minor product 13 (Fig. 4B), whose mass spectrum
exhibited M+ at m/z 396 (0.4%), [M – Me]+ at m/z 381 (0.6%), [M –
MeO]+ at m/z 365 (0.4%), [M – MeCH CH CHCH ]+ at m/z 327 (7%),
–
–
2
2
m/z 311 (12%), [327 – TMSOH]+ at m/z 237 (21%), [M – C10/C18]+ at
+
–
–
m/z 185 (23%), [CH O –TMS] at m/z 103 (30%), m/z 75 (28%), and
2
[TMS]+ at m/z 73 (100%). The fragments at m/z 185 and 173 signifying
the presence of oxirane at C9, C10 and a secondary alcohol function
(TMS) at C13. Thus, the spectrum possessing also [M – Me]+ at m/z 381
approved the structure of oxiranyl vinyl carbinol 9,10-epoxy-13-hy-
droxy-11,15-octadecadienoic acid (Me/TMS) for compound 13 (Top-
orkova et al., 2018b).
The GC-MS analyses of 9(S)-HPOD conversion by the LuDES revealed
products 2 (described above) and 14 (Fig. 4A). The main fragmentation
patterns of compound 14 looked similar to those of compound 13 except
[M – Me]+ at m/z 383, thus approving the structure of oxiranyl vinyl
carbinol 9,10-epoxy-13-hydroxy-11-octadecenoic acid (Me/TMS) for
compound 14 (Toporkova et al., 2018b).
As a whole, data obtained indicated that LuDES possessed mainly
DES activity towards 13(S)-HPOT and mainly HPL activity towards 13
(S)-HPOD. At the same time, LuDES possessed the EAS activity towards
fatty acid 9-hydroperoxides with minor HPL activity towards 9(S)-HPOT
(Table 2). To analyze primary determinants governing the LuDES
catalysis we suggested performing site-directed mutagenesis.
Fig. 4. GC-MS analyses of products (Me/TMS) of recombinant WT LuDES in-
cubations with 9(S)-HPOD (A), 9(S)-HPOT (B), 13(S)-HPOD (C), and 13(S)-
HPOT (D). 2, 9,10-epoxy-11-hydroxy-12-octadecenoic acid (Me/TMS); 3, 9-
hydroxynonanoic acid (Me/TMS); 5, 9,10-epoxy-11-hydroxy-12,15-octadeca-
dienoic acid (Me/TMS); 6, 11-hydroxy-12,13-epoxy-9-octadecenoic acid (Me/
2.4. Product specificity exhibited by LuDES F291V and LuDES A287G
mutant forms
TMS); 7, (ω5Z)-etherolenic acid (Me); 8, (9Z)-12-hydroxy-9-dodecenoic acid
(Me/TMS); 9, (10E)-12-hydroxy-10-dodecenoic acid (Me/TMS); 10, 11-hy-
droxy-12,13-epoxy-9,15-octadecadienoic acid (Me/TMS); 12, 9-hydroxy-
12,13-epoxy-10-octadecenoic acid (Me/TMS); 13, 9,10-epoxy-13-hydroxy-
11,15-octadecadienoic acid (Me/TMS); 14, 9,10-epoxy-13-hydroxy-11-octade-
cenoic acid (Me/TMS). The structural formulae of products are presented in
Fig. 2. 9-HOD/T and 13-HOD/T are decrypted in Fig. 3.
LuDES is the only DES member of the CYP74B subfamily. Other
members of this subfamily are 13-HPLs. The LuDES sequence has some
peculiarities compared with 13-HPLs. In addition to results of experi-
ments on site-directed mutagenesis of LuDES obtained earlier (Top-
orkova et al., 2013), we analysed substitutions in other sites of HBD (the
I-helix groove motif) in the LuDES sequence. Unlike 13-HPLs, the second
site of HBD in the LuDES sequence contains an alanine instead of a
glycine. The sixth position of most AOSs and HPLs HBD sequences, as
well as the LuDES and Solanaceae DESs, contains an aromatic residue, as
a rule, the phenylalanine. Data obtained earlier (Hughes et al., 2008)
and results of 3D modelling using EsyPred program suggested that this
residue (Phe-291 in LuDES) could contact the substrate. We substituted
Phe-291 to Val to alter the size and hydrophobicity of the substrate
pocket and to manipulate contacts with the substrate and its alignment
relative to the haem iron as it was done before (Hughes et al., 2008).
Thus, we constructed and analysed LuDES A287G and F291V mutant
forms.
hydroxy-12,13-epoxyoctadecanoic acid (Me/TMS) (Toporkova et al.,
2017a). Thus, the obtained data pointed to a structure of 11-hydroxy-12,
13-epoxy-9,15-octadecadienoic acid (Me/TMS) for compound 10.
The GC-MS chromatogram of 13(S)-HPOD conversion by the LuDES
differed from that of 13(S)-HPOT (Fig. 4C). This reaction resulted
mainly in the formation of compounds 8 and 9 – products of 13-HPL
activity. The minor products were compounds 6a-6b, 11 and 12. The
compound 6 was 11-hydroxy-12,13-epoxy-9-octadecenoic acid (Me/
TMS) mentioned above. In this reaction this compound was presented as
two isomers 6a and 6b, presumably having a distinct stereo configura-
tion at C11 or C12. The mass spectrum of compound 11 possessed M+ at
m/z 308 (13%), [M ꢀ OCH3]+ at m/z 277 (2%), [M ꢀ (CH2)3–CH3]+ at
m/z 251 (4%), [M ꢀ (CH2)6–COOCH3]+ at m/z 165 (16%), 159 (17%),
135 (32%), 81 (77%), 67 (86%), and 55 (100%). The spectral pattern
was identical to that of (9Z,11E,1′Z)-12-(1′-hexenyloxy)-9,11-dodeca-
Generally, both substitutions (A287G and F291V) led to similar re-
sults (Fig. 5, Supplementary Fig. S2). Both mutants kept significant
catalytic activity compared with the WT enzyme (Table 3) and possessed
main EAS and minor HPL activities towards fatty acid 9-hydroperoxides
(Fig. 5A and B, Supplementary Figs. S2A–B). Conversion of 13(S)-HPOD
by both mutants resulted in the formation of comparable amounts of
aldoacids (HPL products) and epoxyalcohols (EAS products) with a
dienoic [(ω5Z)-etheroleic] acid (Me) (Hamberg, 1998). Then, the least
5

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
slight prevalence of the first ones (Fig. 5C, Supplementary Fig. S2C).
Conversion of 13(S)-HPOT by LuDES F291V mutant form led to the
formation of the same products but with a slight prevalence of the
epoxyalcohols (EAS activity), see Fig. 5D. DES activity disappeared in all
cases, excluding conversion of 13(S)-HPOT by LuDES A287G mutant
form (Supplementary Fig. S2D). Total enzymatic activity of this mutant
towards 13(S)-HPOT distributed (Table 2) as follows: HPL > DES > EAS
(44 : 35.5: 20.5, %/%). The most surprising observation was increasing
enzymatic activity of LuDES F291V and A287G mutant forms towards
fatty acid 9-hydroperoxides compared with WT enzyme (Table 3, Fig. 5A
and B, Supplementary Figs. S2A–B).
3. Discussion
The above-described results have shown that all three studied DESs
converted their preferred substrates mainly to divinyl ethers with mi-
norities of HPL and EAS products. NtDES (CYP74D3) and LeDES
(CYP74D1) exhibited mainly the DES activities (with minor HPL and
EAS activities) towards their preferred substrates, the 9-hydroperoxides.
The LuDES (CYP74B16) also behaved predominantly as a DES towards
its preferred substrate (13(S)-HPOT) with only minor side HPL and EAS
activities. Surprisingly, the divinyl ethers were the only minorities
(below 20% of total products) upon the LuDES incubation with 13(S)-
HPOD (Fig. 4C, Table 2). Furthermore, non-preferred substrates (13-
HPOD with NtDES or LeDES, as well as the 9-hydroperoxides with
LuDES) afforded mainly the EAS products.
The catalytic specificity of DESs was further altered by site-directed
mutagenesis. Two mutations within the I-helix groove region of LuDES,
namely the F291V and A287G, resulted in the full loss of DES activity
and the acquirement of the HPL/EAS activity. Only the 13-HPOT con-
version by the A287G mutant form still produced some divinyl ether
(ω5Z)-etherolenic acid (7) along with the prevalent HPL and EAS
products. Alterations of catalysis of CYP74 enzymes by the site-directed
mutagenesis have been described before, namely the conversions of AOS
to HPL conversions (Lee et al., 2008; Toporkova et al., 2008), DES to
AOS (Toporkova et al., 2013) and dual function HPL/EAS (CYP74C) to
AOS (Toporkova et al., 2018b). The mutation points were localized
within the active site, at the I-helix groove motif (HBD), or near the “F/L
toggle”, and the ERR-triad. For instance, the E292G substitution within
the I-helix groove motif of LuDES led to its conversion to AOS (Top-
orkova et al., 2013). The present work reports the novel kinds of cata-
lytic transformations of CYP74 enzymes upon the site-directed
mutagenesis, namely the DES to HPL and DES to EAS conversions.
The results of present work demonstrated that some divinyl ether
synthases of CYP74 family produce the epoxyalcohols besides with the
divinyl ethers. The relative yield of epoxyalcohols was substrate-
dependent and was elevated by the site-directed mutagenesis. These
observations are consistent with the literature data on product speci-
ficity of CYP74 enzymes (Song et al., 1993; Hughes et al., 2008; Top-
orkova et al., 2018b, 2020; Gorina et al., 2019), as well as some
nonclassical P450 AOSs of fungi (Hoffmann and Oliw, 2013; Hoffmann
et al., 2013). Synthesis of epoxyalcohols is the mechanistically relevant
general property of CYP74s and some other nonclassical P450s. It is
tightly related to the formation of the epoxyallylic radical, which is the
conceptual intermediate of all CYP74s and related enzymes (Fig. 6). The
recombination of hydroxyl radical (from compound II) to the epox-
yallylic radical gives the oxiranyl carbinol. The AOS, HPL, or DES re-
actions proceed through the same intermediate (Fig. 6). These
prerequisites create the versatility CYP74 enzymes and enable their in-
terconversions by the site-directed mutagenesis. DES to EAS conversions
by site-directed mutagenesis are first time described in present work (see
Fig. 7).
Fig. 5. GC-MS analyses of products (Me/TMS) of LuDES F291V mutant form
incubations with 9(S)-HPOD (A), 9(S)-HPOT (B), 13(S)-HPOD (C), and 13(S)-
HPOT (D). 2, 9,10-epoxy-11-hydroxy-12-octadecenoic acid (Me/TMS); 3, 9-
hydroxynonanoic acid (Me/TMS); 5, 9,10-epoxy-11-hydroxy-12,15-octadeca-
dienoic acid (Me/TMS); 6, 11-hydroxy-12,13-epoxy-9-octadecenoic acid (Me/
TMS); 8, (9Z)-12-hydroxy-9-dodecenoic acid (Me/TMS); 9, (10E)-12-hydroxy-
10-dodecenoic acid (Me/TMS); 10, 11-hydroxy-12,13-epoxy-9,15-octadecadie-
noic acid (Me/TMS). The structural formulae of products are presented in
Fig. 2. 9-HOD/T and 13-HOD/T are decrypted in Fig. 3.
Table 3
Kinetic parameters of reactions catalyzed by the WT LuDES and its mutant
forms.
Substrate Km,
μM
kcat, sꢀ 1 kcat/Km, sꢀ 1
μ
Mꢀ 1 Specificity, %
WT LuDES
13-HPOT 31.2
13-HPOD 80.3
623.7
1222.1
277.4
364.5
353.1
335.2
76.8
20
100
76.1
10.5
14.5
81.7
100
39.6
61.9
40.8
100
1.5
15.22
2.1
9-HPOT
9-HPOD
132.1
125.7
2.9
LuDES F291V 13-HPOT 64.9
13-HPOD 50.3
5.44
6.66
2.64
4.12
6.63
16.24
0.25
7.91
9-HPOT
9-HPOD
29.1
19.5
80.3
LuDES A287G 13-HPOT 39.4
13-HPOD 14.4
261.4
233.9
13.9
9-HPOT
9-HPOD
56.6
6.9
54.6
48.7
6

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
Fig. 6. The mechanisms of fatty acid hydroperoxide conversions by target DESs. R = HOOC(CH2)7–, R’ = n-butyl.
4. Experimental
usitatissimum L. (Linaceae) DES (LuDES, CYP74B16) open reading
frames (ORFs) have earlier been cloned into the vector pET32-Ek/LIC
(Novagen, USA) to yield the target recombinant proteins with His-tags
at N- and C-termini (Gogolev et al., 2012; Toporkova et al., 2013). Re-
combinant plasmid pET-23a containing the ORF of the LeDES has
generously been gifted by Prof. G. Howe (Michigan University, USA).
The resulting recombinant plasmids were transferred into Escherichia
coli cells. The Rosetta-gami (DE3)pLysS B strain was used for the prep-
aration of the LuDES and NtDES enzymes, and the BL21 (DE3)pLysS
strain was used for the preparation of the LeDES enzyme. Recombinant
genes were expressed in host cells as follows. An overnight culture (10
ml) of bacteria was inoculated into 1 L of Luria-Bertani medium sup-
plemented with mineral medium M9 (1:1, by volume). Bacteria were
grown at 37 ◦C, 250 rpm to an OD600 of 0.6. Expression of the target
4.1. Materials
Linoleic and α-linolenic acids, as well as the soybean lipoxygenase
type V, were purchased from Sigma. NaBH4 and silylating reagents were
purchased from Fluka (Buchs, Switzerland). (9S,10E,12Z)-9-Hydro-
peroxy-10,12-octadecadienoic (9-HPOD) and (9S,10E,12Z,15Z)-9-
hydroperoxy-10,12,15-octadecatrienoic (9-HPOT) acids were prepared
by incubation of linoleic and α-linolenic acids, respectively, with the
recombinant maize 9-lipoxygenase (GeneBank: AAG61118.1) (Wilson
et al., 2001) at 0 ◦C, Na-phosphate buffer (100 mM, pH 6.0), under
continuous oxygen bubbling. (9Z,11E,13S)-13-Hydroperoxy-9,11-octa-
decadienoic (13-HPOD) and (9Z,11E,13S,15Z)-13-hydroperoxy-9,11,
15-octadecatrienoic (13-HPOT) acids were obtained by incubation of
genes
was
induced
by
addition
of
0.5
mM
iso-
linoleic and
α
-linolenic acids, respectively, with the soybean lip-
propyl-β-D-1-thiogalactopyranoside to the medium. Simultaneously, the
medium was supplied with 5-aminolevulinic acid to facilitate the haem
formation. Induction was controlled by the 12% SDS-PAGE analysis of
crude cellular extracts. Bacteria were harvested by centrifugation for 15
min at 4500 rpm at 4 ◦C and lysed with BugBuster Protein Extraction
Reagent (Novagen, USA). Purification of His-tagged recombinant pro-
tein was performed using Bio-Scale Mini Profinity IMAC cartridge in
BioLogic LP chromatographic system (Bio-Rad, USA). The recombinant
enzymes were eluted from the cartridge using 50 mM histidine. The
recombinant protein concentrations were determined using the Quan-
t-iT™ Protein HS Assay Kit (Invitrogen, USA). The haemoprotein con-
centrations were estimated using the pyridine haemochromogen assay
(Schenkman and Jansson, 2006). The relative purity of recombinant
proteins was estimated by SDS-PAGE and staining of the gel with
oxygenase type V at 23 ◦C, Tris-HCl buffer (50 mM, pH 9.0), under
continuous oxygen bubbling. The extracted hydroperoxides (as free
carboxylic acids) were purified by normal phase HPLC (NP-HPLC) on the
Kromasil Si columns (7
under the isocratic
μ
m; 4.0 × 250 mm; Elsico, Moscow, Russia)
elution with the solvent mixture
hexane-isopropanol-acetic acid 98.1:1.8:0.1 (v/v) at a flow rate of 0.4
ml/min. Hydroperoxides were chromatographically pure and at least
98% optically pure, as judged by chiral phase HPLC (Chechetkin et al.,
2009).
4.2. Expression and purification of LuDES, NtDES and LeDES enzymes
Nicotiana tabacum L. (Solanaceae) DES (NtDES, CYP74D3) and Linum
7

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
Fig. 7. The unrooted phylogenetic tree of the CYP74 family. Classified CYP74 subfamilies are marked with their letter designations (A, B, C, etc.). Subfamilies
consisting of more than one member are outlined with unclosed curves (semi-ellipses). The following CYP74s were used for analysis: As, A. sativum; AsDES
(CYP74H1), CAI30435.1; At, Arabidopsis thaliana; AtAOS (CYP74A1), NP199079.1; AtHPL (CYP74B2), C74B2ARATH; Ca, C. annuum; CaHPL (CYP74B1),
NP001311810.1; CaDES (CYP74D4), NP001311513.1; Cas, Camellia sinensis; CasHPL (CYP74B24), BAU24783.1; Cs, Cucumis sativus; CsHPL/EAS/AOS (CYP74C31),
XP004137005.1; CsHPL/EAS (CYP74C1_Cs), NP001274399.1; Cm, Cucumis melo; CmHPL/EAS (CYP74C2), NP001284390.1; Dc, Daucus carota; DcAOS (CYP74B33),
XP_017248700.1; Gm, G. max; GmAOS (CYP74A1), NP001236432.1; GmHPL/EAS (CYP74C13_Gm), KRH29541.1; Hv, Hordeum vulgare, HvAOS2 (CYP74A3),
CAB86384.1; Le, L. esculentum; LeAOS1 (CYP74A1), CAB88032.1; LeAOS2 (CYP74A2), AAF67141.1; LeAOS3 (CYP74C3), NP001265949.1; LeHPL (CYP74B3),
AAF67142.1; LeDES (CYP74D1), NP001234527.1; Lu, L. usitatissimum; LuAOS (CYP74A1), sp|P48417.1; LuDES (CYP74B16), ADP03054.2; Mp, M. polymorpha,
MpAOS1, BAS32647.1; MpAOS2, BAS32648.1; Mt, Medicago truncatula; MtHPL/EAS (CYP74C13_Mt), XP003606860.1; MtHPL3 (CYP74B4), AAY30368.1; Nt,
N. tabacum; NtDES (CYP74D3), NP001312606.1; Os, Oryza sativa; OsAOS1 (CYP74A4), XP015631686.1; OsHPL2 (CYP74E1), EAY85033.1; Pa, Parthenium argen-
tatum; PaAOS (CYP74A1), sp|Q40778.2; Pd, Prunus dulcis; PdHPL (CYP74C5), CAE18065.1; Pg, Psidium guajava; PgHPL (CYP74B5), AAK15070.1; Pi, Petunia inflata;
PiCYP74C9, ABC75838.1; Pp, P. patens; PpAOS1 (CYP74A1), XP024380613.1; PpAOS2 (CYP74A8), XP024372097.1; PpHPL (CYP74G1), CAC86920.2; Ra, R. acris;
RaDES (CYP74Q1), AJU57209.1; Rj, R. japonicus; RjEAS (CYP74A88), QCR70269.1; Sm, S. moellendorffii; SmDES1 (CYP74M1), XP002979266.1; SmDES2
(CYP74M3), XP002964012.2; SmEAS (CYP74M2), EFJ26024.1; St, S. tuberosum; StAOS2 (CYP74A6), ABD15175.1; StAOS3 (CYP74C10), CAI30876.1; StHPL/EAS
(CYP74C4), XP006365486.1; StDES (CYP74D2), NP001305517.1; Zm, Zea mays; ZmAOS1 (CYP74A19), AAR33048.1; ZmHPL (CYP74F2), NP_001105255.2. The
multiple alignments of selected CYP74 amino acid sequences and phylogenetic tree building were made with MEGA7 software. Multiple alignment was performed
using the ClustalW method, phylogenetic tree was build using the maximum likelihood method based on the Poisson correction model (Zuckerkandl and Author-
Anonymous, 1965); the bootstrap consensus tree was inferred from 1000 replicates (Felsenstein, 1985). The analysis involved 45 amino acid sequences.
Coomassie brilliant blue R-250. Catalytic activity of the purified re-
combinant enzymes was determined by monitoring the decrease of the
signal at 234 nm in a РВ 2201 В spectrophotometer (SOLAR, Belarus)
acidified to pH 6.0, and the products were extracted with hexane/ethyl
acetate (1:1, v/v) mixture, then methylated with ethereal diazomethane
and trimethylsilylated with pyridine/hexamethyldisilazane/trimethyl-
chlorosilane (1:1:1, v/v) mixture at 23 ◦C for 30 min. The silylation
reagents were evaporated in vacuo. The dry residue was dissolved in
with substrate concentration 40 μM. The pH optima were determined in
previous studies: pH 7.0 is optimal for LeDES (Itoh and Howe, 2001) and
pH 7.5 is optimal for NtDES and LuDES (Fammartino et al., 2007;
Gogolev et al., 2012). The analyses were performed at 25 ◦C in 0.6 ml of
0.1 M Na phosphate buffer with corresponding pH.
100 μl of hexane and subjected to GC-MS analyses. When specified, the
products were reduced with NaBH4, then methylated and trimethylsi-
lylated. Alternatively, the products of NaBH4 reduction were hydroge-
nated over PtO2, then methylated and trimethylsilylated. Products
(without or with the preliminary NaBH4 reduction) were analysed as Me
esters/TMS derivatives (Me/TMS) by GC-MS as described before
(Grechkin et al., 2006).
4.3. Incubations of recombinant enzymes with fatty acid hydroperoxides
The recombinant WT enzymes and their mutant forms (10 μg) were
incubated with 100 μg of 9(S)-HPOD, 9(S)-HPOT, 13(S)-HPOD, or 13(S)-
HPOT in Na-phosphate buffer (100 mM, 10 ml), pH 7.0 (LeDES) or 7.5
(NtDES and LuDES), 23 ◦C, for 15 min. The reaction mixtures were
8

Y.Y. Toporkova et al.
Phytochemistry 179 (2020) 112512
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Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
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The authors are thankful for the financial support from the govern-
ment assignment for FRC Kazan Scientific Center of RAS, Russia
(obtaining and studies of catalytic properties of mutant forms LuDES
A287G and LuDES F291V). T.I. thanks the Russian Foundation of Basic
Research (RFBR, Russia, project no. 18-04-00508) for financial support
(study of the LeDES). E.S. thanks RFBR, Russia (project no. 18-34-
01012) for financial support (study of the wild-type LuDES). L.M.
thanks RFBR, Russia (project no. 20-34-70126) for financial support
(study of the wild-type NtDES).
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