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L. Sooman et al. / Biochimica et Biophysica Acta 1861 (2016) 108–118
differences between these 9R- and 9S-DOX domains in regard to the
supra- and antarafacial hydrogen abstraction and oxygen insertion?
To answer the first question we decided to recombinantly express
the tentative 9R-DOX-AOS of Aspergillus niger (EHA25900). Regarding
the second question, the only characterized enzyme for comparison is
9S-DOX-AOS of F. oxysporum [8]. We therefore decided to express a
second enzyme of the putative 9S-DOX-AOS subfamily and we chose
Colletotrichum graminicola (teleomorph Glomerella graminicola). This
would allow us to compare two 9S-DOX-AOS and their homologues
with the putative 9R-DOX-AOS subfamily, consisting of EHA25900
(A. niger), AGH14485 (A. terreus) and homologues of three other
Aspergilli. The sequence similarities within and between these two
subfamilies are illustrated by a phylogenetic tree (Fig. 1B).
The overall objective of this report was to study the structural basis
of the 9R- and 9S-DOX-AOS activities. The first goal was to determine
the catalytic properties of the tentative 9R-DOX-AOS (EHA25900) of
A. niger with homology to (DOX)-9R-AOS of A. terreus. The second
goal was to characterize the tentative 9S-DOX-AOS (EFQ27323) of
C. graminicola with homology to 9S-DOX-AOS of F. oxysporum. The
third goal was to use the sequence information for homologous
enzymes to determine whether site-directed mutagenesis of the DOX
domains of 9R-DOX-AOS could alter the chirality of the hydroperoxide
and allene oxide from R to S at C-9.
supernatants were used immediately or frozen at −80 °C until needed.
9S- and 9R-DOX-AOS of A. niger and C. graminicola were expressed in at
least three independent expression experiments.
2.3. Site-directed mutagenesis of recombinant proteins
Site-directed mutagenesis was performed according to the
QuickChange protocol (Stratagene) with 10 ng of the pUC57 constructs
as templates, oligonucleotides (44-nt), and Pfu DNA polymerase (16
cycles). PCR products were incubated with DpnI (37 °C, 2 h) to digest
methylated DNA. Gel electrophoresis confirmed amplification of one
distinct PCR product, which was then used for transformation of E. coli
(NEB5α) cells by heat shock. All mutations, except for the heme thiolate
replacements, were confirmed by sequencing before sub cloning
to pET101D-TOPO vectors described above. The Cys1075Ser and
Cys1052Ser replacements of EHA25900 and EFQ27323, respectively,
were confirmed by expression of recombinant enzyme with abolished
9-AOS but retained 9-DOX activities.
2.4. Enzyme assays
Recombinant proteins of the crude cell lysate (in 0.05 M Tris–HCl
(pH 7.6)/5 mM EDTA/10% glycerol) were incubated with 100 μM
18:2n-6, other fatty acids, or HPODE for 30–40 min on ice.
[11S-2H]18:2n-6 was only incubated for 10 min and immediately
treated with NaBH4 to convert the α-ketol to 9,10-dihydroxy-12(Z)-
octadecamonoenoic acid (9,10-DiHOME). The reactions (0.3–0.5 ml)
were terminated with methanol (1–2 vols.) and proteins were removed
by centrifugation. The metabolites were extracted on octadecyl silica
(SepPak/C18), eluted with ethyl acetate, evaporated to dryness, diluted
in ethanol (50 μl), and 10 μl were subject to LC-MS/MS analysis.
Triphenylphosphine or NaBH4 were used to reduce hydroperoxides to
alcohols for steric analysis.
2. Materials and methods
2.1. Materials
Fatty acids were dissolved in ethanol and stored in stock solutions
(50–100 mM) at −20 °C. 16:3n-3 (99%), 18:2n-6 (99%), 18:3n-6
(99%), and 18:3n-3 (99%) were from VWR. 18:1n-9, 18:1n-6, 20:2n-6,
13
and [
C ]18:2n-6 (98%) were from Larodan (Malmö, Sweden).
18
[11S-2H]18:2n-6 (N99% 2H) was prepared as described [14]. S and R
stereoisomers of 9- and 13-HPODE were prepared enzymatically or by
photooxidation as described [8,15]. Rac 9-HPODE was prepared by
autoxidation of linoleic acid followed by purification by reverse phase
(RP) and normal phase (NP) HPLC [16]. 9,14-DiHODE was prepared by
13
autoxidation of 18:2n-6 as described [17]. [
C ]13S-HPODE was pre-
18
2.5. LC-MS analysis
pared with soybean LOX (Lipoxidase, Sigma). Phusion DNA polymerase
and chemically competent Escherichia coli (NEB5α) were from New
England BioLabs. Restriction enzymes were from New England BioLabs
and Fermentas. Champion pET101D Directional TOPO Kit was from
Invitrogen. Gel extraction kit and Pfu DNA polymerase were from
Fermentas. RNaseA, lysozyme, deoxyribonuclease I, and ampicillin
were from Sigma. Sequencing was performed at Uppsala Genome Cen-
ter (BMC, Uppsala University). The open reading frames of EHA25900
(A. niger) and EFQ27323 (C. graminicola) in pUC57 vectors were ordered
from GenScript (Piscatawy, NJ 08854). PCR primers were ordered from
TIB Molbiol (Berlin, Germany). SepPak columns (silicic acid and C18
silica) were from Waters.
RP-HPLC with MS/MS analysis was performed with a Surveyor MS
pump (ThermoFisher) and an octadecyl silica column (5 μm;
2.0 × 150 mm; Phenomenex), which was eluted at 0.3–0.4 ml/min
with methanol/water/acetic acid, 750/250/0.05. The effluent was
subject to electrospray ionization in a linear ion trap mass spectrometer
(LTQ, ThermoFisher). The heated transfer capillary was set at 315 °C,
the ion isolation width at 1.5 amu (4 amu for analysis of 2 H-labeled
metabolites), the collision energy at 35 (arbitrary scale), and the tube
lens varied between 90 and 120 V. PGF1α was infused for tuning. Sam-
ples were injected manually (Rheodyne 7510) or by an auto sampler
(Surveyor Autosampler Plus, ThermoFisher).
NP-HPLC with MS/MS analysis was performed with a silicic acid
column (5 μm; Kromasil 100SI, 250 × 2 mm, Dalco Chromtech) using
3% isopropyl alcohol in hexane for separation of oxidized fatty acids
(0.3–0.5 ml/min; Constametric 3200 pump, LDC/MiltonRoy). The efflu-
ent was combined with isopropyl alcohol/water (3/2; 0.2–0.3 ml/min)
from a second pump (Surveyor MS pump; [18]). The combined effluents
were introduced by electrospray ionization into the ion trap mass
spectrometer above. Chiral phase (CP)-HPLC was performed in the
same way. Isomers of 9-HODE were separated by chromatography on
Chiralcel OB-H or on Reprosil Chiral-AM. The former was eluted at
0.5 ml/min with hexane/isopropyl alcohol/acetic acid, 95/5/0.01 [19],
and mixed post-column with isopropyl alcohol/water (0.25 ml/min).
Stereoisomers of α-ketols and 9-HODE were resolved on Reprosil
Chiral-AM (5 μm, 250 × 2 mm; Dr. Maisch), eluted at 0.2 ml/min with
hexane/ethanol/acetic acid, 95/5/0.025, and mixed with isopropyl
alcohol/water (0.15 ml/min).
2.2. Expression of recombinant proteins
The open reading frames of EHA25900 and EFQ27323 in pUC57
were transferred to pET101D-TOPO vectors by PCR technology accord-
ing to Invitrogen's instructions. Competent E. coli (BL21) Star cells
were transformed with the expression constructs by heat shock. Cells
were grown until they obtain an A600 of 0.6–0.8 in 2xYT medium
(Tryptone 16, yeast extract 10 g, NaCl 5 g) at 37 °C (220 rpm) prior to
addition of 0.1 mM isopropyl-β-D-galactopyranoside to induce protein
expression. After 5 h of moderate shaking (~100 rpm) at room temper-
ature (20 °C), the cells were harvested by centrifugation (13,000 rpm,
4 °C; 25 min), suspended in 50 mM Tris–HCl (pH 7.6)/5 mM EDTA/10%
glycerol with lysozyme and deoxyribonuclease I. The suspension was
frozen and thawed twice and then sonicated (Bioruptor Next Gen,
10 × 30 s, 4 °C). Cell debris was removed by centrifugation and the