Supporting Information
Material and methods
5
Fatty acid substrates
Fatty acid standards and substrates, including palmitoleic acid (C16:1∆9Z), elaidic acid
(C18:1∆9E), oleic acid (C18:1∆9Z), 11-cis-octadecanoic acid(C18:1∆11Z),11-trans-octadecanoic
acid(C18:1∆11E),12-trans-octadecanoic acid(C18:1∆12E), linoleic acid (C18:2∆9Z,12Z), conjugated
linoleic acids (C18:2∆9Z,11E, C18:2∆9E,11E, and C18:2∆10E,12Z), α-linolenic acid (C18:3∆9Z,12Z,15Z), γ-
10
linolenic acid (C18:3∆6Z,9Z,12Z), pinolenic acid (C18:3∆5Z,9Z,12Z), stearidonic acid (C18:4∆6Z,9Z,12Z,15Z)
were purchased from Nu-Check Prep (Elysian, MN) and Sigma Aldrich (St. Louis, MO).
Strains and plasmids
Strains and plasmids used in this study are listed in Table S1. For cloning, expression, and
15
biotransformation experiments, E. coli ER2566 was transformed with two constructed plasmids
following standard procedures (Sambrook and Russell 2001).
Table S1: The strains and plasmids used in this study
Name
Strains
L. acidophilus
E. coli ER2566
ER2566_ lht13
ER2566_lht10
Plasmids
pET-15b
pET-15b/LHT-10
pET-15b/LHT-13
Description
Reference or
source
LMG 11470 (Synonym of ATCC 4796)
F– λ– fhuA2 [lon] ompT lacZ::T7 gal endA1 [dcm]
ER2566 harboring lht-13 in pET-15b
ER2566 harboring lht-10 in pET-15b
LMG
NEB
In this study
In this study
T7 promoter, T7 terminator, bacterial pBR322 origin (ori)
and Ampr
pET-15b carries cloned lht1 from L. acidophilus
pET-15b carries cloned lht2 from L. acidophilus
Novagen
In this study
In this study
20
1
Primers
Primers for amplification of LHTs in this study were listed in Table S2. Amplification of LHT10 and LHT-13 using each forward and reverse primer set, L. acidophilus genomic DNA, and a
phusion high-fidelity DNA polymerase (New England Biolabs, Herfordshire, UK) kit following
5
recommendation of suppliers and standard procedures .
Table S2: Primers used for amplification of LHTs in this study.
Target DNA name
Sequence (5’ to 3’)*
lht-1(HMPREF0492_0906)
LHT-13 F
5′-TTCATATGTATTATTCCAATGGTAATTACGA-3′
LHT-13 R
5′-GCCTCGAGTTAGACTAAATTTGCTTCTTTAAGTA-3′
lht-2(HMPREF0492_0997)
LHT-10 F
5′-ATCATATGCATTATAGTAGTGGTAATTATG-3′
LHT-10 R
5′-CGCTCGAGCTAAACCAACTTATACTTCTTAAGC-3′
*
Restriction enzyme (NdeI and XhoI) site were underlined
10
Molecular mass determination
The subunit molecular mass of the putative fatty acid hydratases, LHT-13 and LHT-10, from L.
acidophilus were examined by SDS-PAGE. The molecular mass of the native enzyme was
determined by gel filtration chromatography using a Sephacryl S-300 HR 16/60 preparative15
grade column (Amersham Biosciences, Uppsala, Sweden). The purified enzyme solution was
applied to the column and eluted with 50 mM citrate-phosphate buffer (pH 5.0) containing 150
mM NaCl at a flow rate of 1 ml/min. The column was calibrated with ferritin (400 kDa),
catalase (206 kDa), albumin (75 kDa), and carbonic anhydrase (29 kDa) as reference proteins
(Amersham Biosciences).
20
Effects of reaction conditions on enzyme activity of 10- and 13-HOD conversion
Unless otherwise stated, the reaction was performed in 50 mM citrate-phosphate buffer (pH 5.0)
2
containing 5 mM linoleic acid, 0.1 mg/ml (84 U/ml) linoleate 13-hydratase (LHT-13) or 0.5
mg/ml (39 U/ml) linoleate 10-hydratase (LHT-10), and 4% (v/v) ethanol at 35°C in a 2 ml-tube
for 10 min. To examine the effects of pH and temperature on the activity of LHT-13 from L.
acidophilus, the pH was varied from 4.0 to 6.0 using 50 mM citrate-phosphate buffer and the
5
temperature was varied from 25 to 45°C. The thermal stability of the enzyme was determined by
incubating the enzyme for 90 min at temperatures ranging from 25 to 60°C. After incubation,
the remaining activity was determined under the standard reaction conditions. The effect of
solvent on the activity of the 13-LHT was investigated using ethanol, methanol, butanol, 2propanol, hexane, DMSO, and acetone. The effect of ethanol concentration on enzyme activity
10
was examined by varying its concentration from 0 to 8% (v/v). The determination of reaction
conditions for LHT-10 was also performed as same manner.
Preparation of the hydroxy fatty acid standards
The enzymatic reaction products, including 10-HOD, 10-HODE (cis-12,15), 10-HSA, 13-HOD,
15
13-HODE (cis-5,9, -6.9, and cis-12,15) and 10,13-diHOD, were isolated through twice time
extraction using two volumes of ethyl acetate. After the organic layers were separated from the
aqueous phase, the organic extract containing 10-HOD, 10-HSA, and 13-HOD were dried with
anhydrous Na2SO4 or MgSO4 and concentrated by evaporation in vacuo. The concentrate was
purified using a silicic acid column chromatography (Risser and Perkins 1966) with methanol
20
gradient (0−5%, v/v) in chloroform by step wise manner. 10,13-diHOD was also obtained with
the same method except for 1−10% methanol gradient in chloroform. 10-HODE (cis-12,15) and
13-HODE (cis-5,9, -6.9, and cis-12,15) were prepared by semi-preparative HPLC using HPLC
system (Agilent 1260, Palo Alto, CA, USA) coupled to a UV detector at the absorbance at 202
nm with a reverse phase Nucleosil C18 column (250 × 10 mm, 5 μm particle size; Phenomenex,
25
Torrance, CA, USA). The products were eluted with a gradient of solvent A
(acetonitrile/water/acetic acid; 50:50:0.1, v/v/v) and solvent B (acetonitrile/acetic acid; 100:0.1,
3
v/v) at 3.0 ml/min. The hydroxy fatty acids were obtained with high purity (>97%) and higher
purity (>99%) were used for structure determination and standards for quantification,
respectively.
5
Quantification methods details of GC-FID
Prior to quantify the products, 0.5 mM of C18:0 as an internal standard was added to monitor
the extraction recovery. The supernatant (200-500 μl) obtained from the centrifugation and
filtration of biotransformation reactant was extracted twice times with twice volumes of ethyl
acetate and dried with anhydrous MgSO4 or Na2SO4. Since the presence of water inhibited
10
sylilation, the supernatant was completely dried.
- Calibration: Calibration curves were established with 13-HOD, 13-HODE, 10-HOD, and 10HSA that were purified from enzymatic conversion products by repetitive semi-preparative
HPLC. The purity of each product was checked by GC-MS as over 99%. Each standard solution
was prepared in triplicate by serial dilution in MeOH with 20 mM stock solution. The range of
15
standard for 13-HOD and 10-HSA were six points from 0.3 to 20 mM and 10-HOD and 13HODE were five points from 0.3 to 10 mM. The linearity of peak area versus concentration for
each hydroxyl fatty acid product was determined within the concentration range, and the
aliquots were injected and run. The calibration curve constructed was evaluated by its
correlation coefficient (Supplementary Table 1). The calibration equation from minimum three
20
replicate experiments demonstrated the linearity of the method. The standard deviation of the
slope for the calibration curve was as relative standard deviation (RSD).
- Precision and accuracy: The precision of the analytic method was determined by repeatability.
Five or six different concentration samples (0.3−10 mM or 0.3−20 mM) were analyzed three
times. The RSD value for precision was below 3%.
4
- Recovery: To determine the recovery and to study the interference of formulation additives,
the recovery experiments were performed by adding the known amount of HFA with three
different concentrations. The percent analytical recovery values were calculated by comparing
the concentration obtained from the known samples with additional concentrations.
5
A
B
C
D
10
Figure S1. Calibration graphs of standard for quantification of the biotransformed hydroxy fatty
acids in this study. (A) 13-HOD (B) 13-HODE (cis-6,9) (C) 10-HOD (D) 10-HSA.
5
Table S3. Summary of calibration and linearity of standards.
*
Correlation
%RSD**
Hydroxy fatty acids
Range (points)
Regression equation
13-HOD
0.3 – 20 mM (6)
Y = 8.602x + 0.2414
0.9996
0.1254
13-HODE (cis-6,9)
0.3 – 10 mM (5)
Y = 8.552x + 0.2338
0.9988
0.0853
10-HOD
0.3 – 20 mM (6)
Y = 8.4617x + 0.245
0.9989
0.0813
10-HSA
0.3 – 10 mM (5)
Y = 8.611x + 0.2227
0.9997
0.1755
*
coefficient
Y=ax+b, ** relative standard deviation
O-acetyl-mandelic derivatization of HFAs for stereo analysis
5
A 10 mg sample of purified HFA was treated with 2 ml of a 12% borontrifluoride in
dichloromethane and the mixture was heated on a water bath (at 90°C) for 10 min. The reaction
mixture was evaporated to dryness under a N2 stream after extraction with 2 ml of n-hexane.
The 1uL of sample was subjected to gas chromatography to identify the methyl ester.
O-Acetyl-mandelic derivatization from HFA methyl ester was performed as followed by the
10
published methods (el-Sharkawy et al. 1992; Yang et al. 1993). Simply, the 10 mg (0.032 mmol)
of methyl-13-hydroxy-9-octadecenoate and methyl-10-hydroxystearate was dissolved in 10 mg
(0.47mmol) of dicyclohexylcarbodiimide in 2 ml of CH2C12 and then this mixture added to a 2
ml of CH2Cl2 solution of O-acetylmandelic acid (0.047 mmol) and 4-(N,N-dimethylamino)pyridine mixture. The mixture was stirred over an ice bath for 5 h and then filtered. The reactant
15
was dried under nitrogen to give a crude residue containing mainly (S)-(+)-O-acetylmandelate
ester of HFA. The 5mg of methyl-10-hydroxy-12-octadecenotate and methyl-13-hydroxy-6,9octadecadienoate was used for synthesis of mandeleate derivatives by same procedures. The
obtained products were dissolved in DMSO-d6 for proton-NMR analysis. Peak integration was
performed using ACD/NMR processor.
20
6
Results
Identification of two hyratases in L. acidophilus genome and sequence comparison
Supplementary Figure 2 shows putative two hydratase genes in L. acidophilus ATCC 4356
5
genome. The genes encoding LHT-13 and LHT-10 was comprised 590 amino acids encoded by
1,783 base pairs (HMPREF0492_0906 gene) and EEJ75841 protein was comprised 591 amino
acids encoded by 1,786 base pairs (HMPREF0492_0997 gene). EEJ76183 protein showed 58%
amino acid sequence identity with EEJ75841 protein. EEJ76183 protein showed 94−100% and
more than 90 % sequence identities with hydratases of L. acidophilus (Supplementary Table 3)
10
and its related strains including L. amylovorus, L. helveticus, and L. crispatus, respectively
(Supplementary Table 4). EEJ76183 protein was also homologous with MCRA proteins from
the genus Pediococcus (78% identity) and Streptococcus (70%). This protein showed 42−60%
amino acid sequence identity with the characterized linoleate 10-hydratases and oleate
hydratases (Table S5). EEJ75841 protein was higher identity with MCRA proteins from L.
15
acidophilus and related strains (more than 80% identity) but showed lower identity with two
hydratases of L. mucosae (77% and 58% identity), L. buchneri (59%
and 30%), Pediococcus
claussenii (56% and 34%), respectively (Supplementary Table 4). EEJ75841 protein exhibited
45−68% amino acid sequence identity with the characterized linoleate 10-hydratases and oleate
hydratases (Supplementary Table 5). Both proteins were homologous with 67 kDa Streptococcal
20
MCRA family proteins and oleate hydratases.
7
Figure S2. Genetic loci of the two hydratases in L. acidophilus ATCC 4356. Shade box and
percentage means relative identity between LHT-13 and LHT-10.
5
8
8
Table S4: Comparison of two hydratase protein sequences in L. acidophilus strains containing
9
LHT-13 and LHT-10.
Name
Protein accession
(amino acid residues)
Gene name
Identity
(%)
For LHT-13
L. acidophilus ATCC 4796
HMPREF0492_0906
EEJ76183 (590)
100
L. acidophilus CIP 76.31
LACIP7613_01498
CDF67336
100
L. acidophilus CIRM-BIA 442
LACIRM442_00103
CDF69017 (590)
100
L. acidophilus CIRM-BIA 445
LACIRM445_00351
CDF70787 (590)
100
L. acidophilus DSM 9126
LADSM9126_01212
CDF72610 (590)
100
L. acidophilus DSM 20242
LADSM20242_00691
CDF74590 (590)
100
L. acidophilus La-14
LA14_0588
YP_007937442 (590)
100
L. acidophilus NCFM
LBA0555
YP_193467 (590)
100
L. acidophilus 30SC
LAC30SC_02835
YP_004286876 (590)
94
L. acidophilus ATCC 4796
HMPREF0492_0997
EEJ75841 (591)
100
L. acidophilus CIP 76.31
LACIP7613_00033
CDF67433
99
L. acidophilus CIRM-BIA 442
LACIRM442_00386
CDF69112 (591)
99
L. acidophilus CIRM-BIA 445
LACIRM445_00448
CDF70883 (591)
99
L. acidophilus DSM 9126
LADSM9126_01305
CDF72702 (591)
99
L. acidophilus DSM 20242
LADSM20242_01398
CDF74685 (591)
99
L. acidophilus La-14
LA14_0679
YP_007937532 (591)
99
L. acidophilus NCFM
LBA0649
YP_193559 (591)
99
L. acidophilus 30SC
LAC30SC_03260
YP_004291763 (591)
99
(590)
For LHT-10
10
11
-9-
(591)
12
Table S5: Sequence identities of (A) LHT-13 and (B) LHT-10 from L. acidophilus ATCC 4356
13
with orthologous proteins in Lactobacillales.
Strains
Protein accession
Amino acids in
length
Identity (%)
For LHT-13
Lactobacillus acidophilus ATCC 4796
Lactobacillus amylovorus GRL 1112
Lactobacillus helveticus R0052
Lactobacillus crispatus ST1
Lactobacillus kefiranofaciens ZW3
Lactobacillus sp. ASF360
Lactobacillus mucosae LM1
Lactobacillus gasseri ATCC 33323
Pediococcus claussenii ATCC BAA-344
Streptococcus mutans UA159
Lactobacillus otakiensis JCM 15040
Lactobacillus buchneri NRRL B-30929
Pediococcus pentosaceus IE-3
For LHT-10
EEJ76183
YP_004031359
YP_006656360
YP_003601030
YP_004562912
EMZ19002
EHT17106
YP_814320
YP_005005459
NP_721921
GAD16444
YP_004397927
CCG90649
590
590
590
590
590
590
590
590
590
589
589
589
589
100
94
92
90
90
87
82
80
72
72
69
68
65
Lactobacillus acidophilus ATCC 4796
Lactobacillus ultunensis DSM 16047
Lactobacillus amylovorus GRL 1112
Lactobacillus crispatus ST1
Lactobacillus helveticus R0052
Lactobacillus kefiranofaciens ZW3
Lactobacillus sp. ASF360
Lactobacillus gasseri ATCC 33323
Lactobacillus mucosae LM1
EEJ75841
EEJ72450
YP_004031446
YP_003601030
YP_006656262
YP_004562795
EMZ18971
YP_815151
EHT17106
EHT15500
YP_005005459
YP_005004553
NP_721921
NP_720953
YP_004397927
YP_004397517
CCG90649
CCG90677
591
591
591
591
591
591
591
591
590
591
590
558
589
591
589
564
589
557
100
95
93
92
93
93
86
84
77
58
58
35
58
56
59
30
56
34
Pediococcus claussenii ATCC BAA-344
Streptococcus mutans UA159
Lactobacillus buchneri NRRL B-30929
Pediococcus pentosaceus IE-3
14
15
- 10 -
16
Table S6: Sequence identities of LHT-13 and LHT-10 from L. acidophilus ATCC 4356 with other characterized hydratases.
Protein
(Gene name)
Microorganism
Name of enzymes
Accession
number
Identity
(%)
Reference
L. acidophilus ATCC 4356
Macrococcus caseolyticus KCTC 3582
Lysinibacillus fusiformis KCTC 3454
L. acidophilus NCFM
Streptococcus pyogenes NZ131
Stenotrophomonas maltophilia KCTC 1773
Bifidobacterium breve NCIMB 702258
Elizabethkingia meningoseptica 3266
Linoleate 13-hydratase
Linoleate 10-hydratase
Oleate hydratase
Oleate hydratase
Linoleate 10-hydratase
Linoleate 10-hydratase
Oleate hydratase
Oleate hydratase
Oleate hydratase
EEJ76183
EEJ75841
B9E972
EFI68504
YP_193559
B5XK69.2
CAQ45596
ADY18551
ACT54545
100
58
60
60
58
56
52
50
42
This study
This study
(Joo et al. 2012a)
(Kim et al. 2012)
(Volkov et al. 2013)
(Volkov et al. 2010)
(Joo et al. 2012b)
(Rosberg-Cody et al. 2011)
(Bevers et al. 2009)
L. acidophilus ATCC 4356
Linoleate 10-hydratase
EEJ75841
100
This study
L. acidophilus NCFM
Streptococcus pyogenes NZ131
Lysinibacillus fusiformis KCTC 3454
Macrococcus caseolyticus KCTC 3582
Stenotrophomonas maltophilia KCTC 1773
Bifidobacterium breve NCIMB 702258
Elizabethkingia meningoseptica 3266
Linoleate 10-hydratase
Linoleate 10-hydratase
Oleate hydratase
Oleate hydratase
Oleate hydratase
Oleate hydratase
Oleate hydratase
YP_193559
B5XK69.2
EFI68504
B9E972
CAQ45596
ADY18551
ACT54545
100
68
63
62
54
51
45
(Volkov et al. 2013)
(Volkov et al. 2010)
(Kim et al. 2012)
(Joo et al. 2012a)
(Joo et al. 2012b)
(Rosberg-Cody et al. 2011)
(Bevers et al. 2009)
For LHT-13
EEJ76183
(HMPREF0492_0906)
For LHT-10
EEJ75841
(HMPREF0492_0997)
17
- 11 -
Expression, purification, and determination of molecular mass
The both expressed and purified protein showed a single band in SDS-PAGE with a molecular mass
of approximately 68 kDa, which is consistent with the calculated value of 68.1kDa based on the 590
and 591 amino acids and six histidine tag (Supplementary Fig. 3A and 3B). The molecular mass of the
5
native protein was estimated using gel filtration chromatography to be 68 kDa, indicating that it is a
monomer (Supplementary Fig. 3C). The molecular masses of oleate hydratases of Elizabethkingia
menungoseptica (Bevers et al. 2009), Bifidobacterium breve (Rosberg-Cody et al. 2011), and
Macrococcus caseolyticus (Joo et al. 2012a), and linoleate hydratase of S. pyogenes (Volkov et al.
2010) were 65−70 kDa as a monomer.
10
Substrate specificity of purified recombinant LHT-13 and identification reaction products of
LHT-13 and LHT-10.
Supplementary Table 7 shows relative activity (as for LA is 100%) and ssubstrate specificity of
purified recombinant LHT-13. The biotransformed 13-HODE(13-OH, 6Z,9Z) from GLA and 1315
HODE(13-OH, 9Z,15Z) from ALA were showed in Figure S4. No activity was detected for
palmitoleic acid (C16:1∆9Z),elaidic acid (C18:1∆9E), oleic acid (C18:1∆9Z), 11-cis-octadecanoic
acid(C18:1∆11Z),11-trans-octadecanoic acid(C18:1∆11E),12-trans-octadecanoic acid(C18:1∆12E),cis-9conjugated linoleic acid(C18:2∆9Z,11E), all trans-conjugated linoleic acid (C18:2∆9E,11E), eicosenoic
acids (C20:1∆7Z, C20:1∆9Z, C20:1∆11Z), arachidonic acid (C20:4∆5Z,8Z,11Z,14Z), and erucic acid (C22:1∆13Z).
20
Identification of detailed structure determination by 1H and
13
C-NMR spectroscopy was shown in
Figure S5 and S6. The biotransformed 10-HSA from OA and 10-HOD (10-OH, 12Z) from LA were
showed in Figure S7.
Optimized reaction conditions for the production of 13-HFAs by LHT-13.
25
To obtain maximal productivity of each 13-HFAs using LHT-13, optimum pH, temperature, and
solvent concentrations were investigated. Maximal enzyme activity was observed at pH 5.0
(Supplementary Fig. 11A) and at 35°C (Supplementary Fig. 11B). The production of 13-HOD from
12
linoleic acid was highest using ethanol among the solvents tested (Supplementary Fig. 12A), and its
optimal concentration was 4% (v/w) (Supplementary Fig. 12B).
13
A
B
5
C
14
Figure S3. SDS-PAGE analysis of the purified hydratase for recombinant (A) EEJ76183 and (B)
EEJ75841 from L. acidophilus in E. coli. Lane 1, molecular mass marker proteins (130, 100, 75, 55,
40, 35, and 25 kDa); lane 2, crude extract; lane 3, purified putative fatty acid hydratase. The protein
extract was prepared from culture of recombinant E. coli cells grown in the Riesenberg medium and 20 h
5
after induction of the gene expression with 0.1mM IPTG. The expressed (LHT-13 and LHT-10) band was
indicated by an arrow. (C) Determination of molecular mass of the purified native enzyme using gel
filtration chromatography. The reference proteins were ferritine (400 kDa), catalase (206 kDa),
conalbumin (75 kDa), and carbonic anhydrase (29 kDa). The putative fatty acid hydratase eluted at a
position corresponding to 68 kDa.
10
15
Table S7: Substrate specificity of the putative fatty acid hydratase of L. acidophilus. Asterisk means
hydroxylation position.
Substrate
LA(18:2 n-6, 9Z,12Z)
Product
Relative
activity (%)a
13S-HOD (13-OH, 9Z)
100
GLA (18:3 n-6, 6Z,9Z,12Z)
13S-HODE(13-OH, 6Z,9Z)
13
PLA (18:3 n-6, 5Z,9Z,12Z)
13-HODE (13-OH, 5Z, 9Z)
7
10-HOD(18:1, 10-OH, 12Z)
10,13-diHOD
6
ALA (18:3 n-3, 9Z,12Z,15Z)
13S-HODE(13-OH, 9Z,15Z)
Traceb
CLA (18:2, 10E, 12Z)
13-HOD(13-OH, 10E)
Trace
5
a
The relative activity of 100% in the enzyme reactions was 814 U/mg.
b
Trace: Each product clearly formed peak at GC compared with control reaction but their conversion yield was
no more than 3% or less.
16
A
B
5
Figure S4. GC-MS of the product formed from (A) α- and (B) γ-linolenic acid by recombinant
purified LHT-13 enzyme reaction.
10
17
H-NMR spectra (400 MHZ, CDCl3): δ ppm [integration, multiplicity, coupling constant J (HZ)],
5.35(1H, m, 10.8), 5.35(1H, m, 10.5), 3.60(1H, m), 2.32(2H, t, 7.4), 2.13(2H, m), 2.0(2H, q), 1.61(2H,
m), 1.52(2H, m), 1.48(2H, m), 1.43(2H, m), 1.26 (2H, m), 1.26 (2H, m), 1.26 (2H, m), 1.26 (2H, m),
1.26 (2H, m), 1.26(2H, m), 1.26(2H, m), 0.89(3H, t, 6.8).
1
5
Figure S5. 1H-NMR spectra of 13-hydroxy-9(Z)-octadecenoic acid
10
18
C-NMR spectrum (100 MHZ, CDCl3): δ ppm (multiplicity), 178.9(s), 130.7(d), 129.4(d), 72.1(d),
13
37.6(t), 37.4(t), 34.0(t), 31.1(t), 29.6(t), 29.1(t), 29.0(t), 29.0(t), 27.2(t), 25.5(t), 24.8(t), 23.8(t), 22.8(t),
5
14.2(q).
Figure S6. 13C-NMR spectra of 13-hydroxy-9(Z)-octadecenoic acid.
10
19
Proton-NMR spectral data of the minor products
- 13S-hydroxy-6Z,9Z-octadecadienoic acid (500 MHz, CD3Cl): δ ppm [integration, multiplicity,
coupling constant J (HZ)], 5.39 (m, 4H, 6Z, 10.7), 5.36 (m, 4H, H9, 10.6), 3.63 (m, 1H, H-13),
2.80 (dd, 2H), 2.35 (t, 2H), 2.22-2.06 (m, 6H), 1.69-1.25 (m, 12H), 0.89 (t, 3H, H-18).
5
- 13-hydroxy-5Z,9Z-octadecadienoic acid (500 MHz, CD3Cl): δ ppm [integration, multiplicity,
coupling constant J (HZ)], 5.29 (m, 4H, H-5, 10.6), 5.35 (m. 4H, H-9, 10.8), 3.65 (m, 1H, H-13),
2.80 (dd, 2H), 2.79-2.0 (m, 8H), 1.49-1.29 (m, 12H), 0.86 (t, 3H, H-18).
- 13S-hydroxy-9Z,15Z-otadecenoic acid (500 MHz, CD3Cl): δ ppm [integration, multiplicity, coupling
constant J (HZ)], 5.44 (m, 2H, H9, 10.7), 5.33 (m, 2H, H10), 5.30 (m, H16), 5.24 (m, H15, 10.6),
10
3.51 (m, H-13), 2.72 (m, 2H), 2.25 (t, 2H), 2.20 (m, 2H), 2.22 (dd, 2H), 1.96 – 1.21 (m, 12H),
0.89 (t, 3H, H-18).
- 13-hydroxy-10E-otadecenoic acid (500 MHz, CD3Cl) (partial): δ ppm [integration, multiplicity,
coupling constant J (HZ)], 5.60 (m, 2H, H-9, 15.0), 5.44 (m, 1H, H-10), 3.82 (m, 1H, H-13), 2.32
(m, 2H), 2.04 (m, 2H), 1.93 (m, 2H), 1.82 (m, 2H), 1.60 (m, 2H), 1.40 – 1.18 (m, 12H), 0.86 (t,
15
3H, 7.5, H-18).
- 10S-hydroxy-12Z-octadecenoic acid (500 MHz, CD3Cl) (partial): δ ppm [integration, multiplicity,
coupling constant J (HZ)], 5.48 (m, 2H, H-12, 10.6), 3.54 (m, 1H, H-13), 2.33 (m, 2H), 2.22 (dd,
2H), 2.0 (m, 2H), 1.80 (m, 2H), 1.32 – 1.24 (m, 14H), 0.89 (t, 3H, 7.5, H-18).
- 10-hydroxy-12Z,15Z-octadecadienoic acid (500 MHz, CD3Cl) (partial): δ ppm [integration,
20
multiplicity, coupling constant J (HZ)], 5.49 (m, 1H), 5.40 (m, 1H, 10.7, H-12), 5.36 (m, 1H),
5.30 (m, H15, 10.6), 3.64 (m, H-10), 2.79 (m, 2H), 2.35 (m, 2H), 2.26 (dd, 2H), 2.08 (m, 2H, H17), 1.60 (m, 2H, H-3), 1.45 (m, 2H, H-9) , 1.28 – 1.21 (m, 12H), 0 (t, 3H, 7.5, H-18).
20
Figure S7. 1H-NMR spectra of 13-hydroxy-6(Z),9(Z)-octadecenoic acid
5
Proton- (A) and
13
C- (B) NMR spectra of 13-hydroxyoctadec-6,9-dienoic acid. NMR data of the isolated
product: H NMR (500 MHz, CDCl3) δ 5.39 (m, 2H, 10.7), 5.36 (m, 2H, 10.6), 3.63 (m, 1H, H-13), 2.80 (dd,
1
2H), 2.35 (t, 2H), 2.22-2.06 (m, 6H), 1.69-1.25 (m, 12H), 0.89 (t, 3H, H-18). 13C NMR (125 MHz, CDCl3) δ
178.8 (C-1), 129.5 (C-6), 129.3 (C-7), 128.4 (C-9, C-10), 71.8 (C-13), 37.3, 37.0, 33.7, 31.8, 28.9, 26.7, 25.6,
25.2, 24.3, 23.5, 22.6, 14.0 (C-18).
10
21
A
B
5
Figure S8. GC-MS of the product formed from (A) oleic acid and (B) α-linolenic acid by LHT-10
10
enzyme reaction.
22
A
B
5
Figure S9. Proton-NMR spectroscopy of (S)-(+)-O-acetylmandelic ester of 13S-hydroxy-9(Z)octadecenoic acid methyl ester for determination of stereochemistry. (A) Proton NMR spectra
(600MHz, DMSO-d6), the peaks derived from mandelic acid was underlined: δ ppm [integration,
10
multiplicity, coupling constant J (HZ)], 7.45 (2H, dd), 7.36 - 7.39 (3H, m), 5.86 (1H, s), 5.7 - 5.3 (1H,
m, H-9, 10.6), 4.79 (1H, m, H-10), 3.67 (1H, s, H-13), 3.33 (m, 2H), 3.19 (3H, s, OCH3), 2.28 (3H, t,
CH3CO), 2.12 - 1.92 (4H, m), 1.72 - 1.23 (7H, m), 1.15 - 0.96 [18H, m], 0.88 (2H, m), 0.72 (t, 3H, H18). (B) Chiral (R/S) distribution of (S)-(+)-O-acetylmandelic ester of 13S-hydroxy-9(Z)-octadecenoic
acid. Examined S-form was 98% and R-form was 2% and thus obtained enantiomeric excess (% ee)
15
was 96%.
23
5
A
B
C
D
Figure S10. Chiral analysis of the minor product of 13-LHT and 10-LHT using 1H-NMR
10
spectroscopy. (A) (S)-(+)-O-acetylmandelic ester of 13-hydroxy-9(Z),15(Z)-octadecadienoic acid
methyl ester (B) (S)-(+)-O-acetylmandelic ester of 13-hydroxy-6(Z),9(Z)-octadecadienoic acid (C)
(S)-(+)-O-acetylmandelic ester of 10-hydroxy-9(Z)-octadecenoic acid (D) (S)-(+)-O-acetylmandelic
ester of 10-hydroxy-9(Z),15(Z)-octadecadienoic acid. Examined % ee was 98, 98, 97, and 82 %,
respectively.
15
24
A
B
5
Figure S11. Effects of pH and temperature on the activity of LHT-13 from L. acidophilus. (A) pH
effect. The reactions were performed in 50 mM citrate-phosphate buffer (pH 4.0−6.0) containing 5 m
M linoleic acid, 0.1 mg/ml enzyme, and 4% (v/v) ethanol at 35°C for 10 min. (B) Temperature
effect. The reactions were performed by varying the temperature from 25 to 60°C in 50 mM citrate10
phosphate buffer (pH 6.5) containing 5 mM linoleic acid, 0.1 mg/ml enzyme, and 4% (v/v) ethanol for
10 min.
25
A
B
5
Figure S12. Effect of solvent on the activity of LHT-13 from L. acidophilus. (A) Effect of solvent
type. The reactions were performed in 50 mM citrate-phosphate buffer (pH 5.0) containing 5 mM
linoleic acid, 0.1 mg/ml enzyme, and 2% (■) or 4% (□) (v/v) solvent at 35°C for 10 min. (B) Effect of
solvent concentration. The reactions were performed by varying the ethanol concentration from 0 to
10
8%in 50 mM citrate-phosphate buffer (pH 5.0) containing 5 mM linoleic acid and 0.1 mg/ml enzyme
at 35°C for 10 min. Data represent the means of three experiments and error bars represent the
standard deviation.
26
A
B
5
C
Figure S13. Characteristics of cofactor effect on the 13-LHT activity. (A) Multiple alignment of FADbinding regions (1-98 of PDB 4IA6) from 13-LHT (EEJ76183 from L. acidophilus) with the
10
characterized oleate hydratases (ADY18551 from B. breve, CAQ45596 from S. maltophilia,
ZP_07049769 from L. fusiformis, B9E972 from M. caseolyticus, ACT54545 from E. meningoseptica)
and linoleate hydratases (EEJ75841 from L. acidophilus ATCC 4796, YP_193559 from L. acidophilus
NCFM, B5XK69.2 from S. pyogenes) (see also Table S6). Conserved residues were indicated asterisk.
Critical residues of FAD-binding region for enzyme activity were shown as black box, whereas
15
variable residues were shown as gray box. (B) UV-visible spectrum of holoenzyme (blue), apoenzyme
(pink), and FAD (red). The concentrations of the holoenzyme and apoenzyme were approximately 10
mg/ml in 50 mM citrate-phosphate buffer (pH 5.0) and the concentration of FAD is 40 μM. (C) Effect
27
of FAD concentration on the activity of cis-12 linoleate hydratase from L. acidophilus. The effect on
FAD concentration was evaluated.The reactions were performed by varying the concentration from 0
to 4 mMin 50 mM citrate-phosphate buffer (pH 5.0) containing 5 mM linoleic acid, 0.1 mg/ml
enzyme, and 4% (v/v) ethanol at 35°C for 10 min.
5
28
H-NMR spectra (400 MHZ, CDCl3): δ ppm [integration, multiplicity, coupling constant J (HZ)], 3.87
1
(1H, m), 3.66 (1H, m), 2.34 (2H, t, 7.4), 2.21 (2H, m), 1.95 (2H, m), 1.63 (2H, m), 1.51 (2H, m), 1.48
5
(2H, m), 1.31 (2H, m), 1.31 (2H, m), 1.31 (2H, m), 1.31 (2H, m) 1.31 (2H, m) 1.31 (2H, m) 1.31 (2H,
m) 1.31 (2H, m), 0.89 (3H, t, 6.8).
Figure S14. 1H-NMR spectra of 10,13-diHOD
10
29
C-NMR spectrum (100 MHZ, CDCl3): δ ppm (multiplicity), 178.5(s), 73.3(d), 73.2(d), 42.8(t),
13
38.2(t), 33.9(t), 31.8(t), 29.4(t), 29.2(t), 29.1(t), 29.0(t), 28.9(t), 28.9(t), 25.2(t), 25.2(t), 24.6(t), 22.6(t),
5
14.0(q).
Figure S15. 13C-NMR spectra of 10,13-diHOD
10
30
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