Supplemental data

A dual positional specific lipoxygenase functions in the generation of flavour compounds during climacteric ripening of apple

Schiller D, Contreras C, Vogt J, Dunemann F, Defilippi B, Beaudry R, and Schwab W

Figures S1 – S9

Figure S1 Sequence similarity of apple LOX genes

Figure S2 Comparison of the deduced amino acid sequences of five apple LOX genes.

Figure S3 RT-PCR expression of LOX transcripts

Figure S4 SDS-PAGE of purified recombinant apple LOX proteins.

Figure S5 Effects of temperature and pH on the enzymatic activity of apple LOX enzymes.

Figure S6 LC-MS analysis of hydroperoxy fatty acids (HpODE) formed from linoleic acid catalyzed by LOX1:Md:1a (A) and LOX1:Md:1c (B).

Figure S7 Effect of substrate concentration on enzymatic activity of wild-type and mutant apple LOX.

Figure S8 Transient expression of select LOX genes

Figure S9 Factors that determine regio- and stereospecificity of LOX1:Md:1a

Figure S10 Characterization of ripening apple fruit.

Tables S1 – S6

Table S1 Identity of Malus x domestica lipoxygenases, and oligonucleotides used for gene amplification.

Table S2 Locus, description, forward and reverse primer sequence (5→3'), annealing temperature, expected PCR fragment size, and

optimum RT-PCR cycle number for putative LOX gene expression in skin tissue from ripening 'Jonagold' fruit.

Table S3 Locus, description, and forward and reverse primer sequences (5→3') for qPCR amplification of selected LOX genes and a GAPDH control gene in skin tissue from ripening 'Jonagold' fruit.

Table S4 Distribution of hydro(pero)xy products gained from wild-type and mutant apple LOX enzyme reaction with α-linolenic acid and arachidonic acid.

Table S5 Oligonucleotides used to amplify the MdLOX1a, MdLOX1c, MdLOX2a and MdLOX2b genes for subcloning into pRSET B and pYES2 expression vectors.

Table S6 Oligonucleotides used for site-directed mutagenesis of the MdLOX1a gene.

Fig

ure

 S1

Se

qu

en

ce

sim

ila

rity

of

ap

ple

LO

X g

en

es

. N

ucl

eo

tide

se

qu

en

ces

of

the

Ge

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an

k a

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an

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ing

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en

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f P

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nd

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.

Fig

ure

 S2

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ed

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of

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tein

se

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en

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the

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X1

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c, M

dL

OX

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, M

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om

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ich

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Figure S3 RT-PCR expression screening of LOX transcripts. RT-PCR analysis of LOX gene expression for ‘Jonagold’ apple fruit during ripening. Eight time points were selected based on distinct physiological stages (see Fig. 2). Total RNA was isolated from fruit at each time point and GAPDH (lower right-hand panel) was used as a control.

Figure S4 SDS-PAGE of purified recombinant apple LOX proteins. Lanes correspond to crude extracts obtained from yeast cells carrying expression constructs for LOX1:Md:1a (1), LOX1:Md:1c (3), LOX2:Md:2a (5), LOX2:Md:2b (7) and empty pYES2 vector (9). A 10% acrylamide gel was loaded with 30 µg crude extract and 5 µg IMAC-treated protein each and stained with Coomassie. Partially purified LOX protein is marked with a red frame (Lanes 2, 4, 6 and 8). In contrast, IMAC-treated protein from extracts with empty pYES2 vector contained no distinct protein band of approximately 100 kDa size (10).

LOX2:Md:2aLOX1:Md:1cLOX1:Md:1a LOX2:Md:2b pYES2

130 kDa →

70 kDa →

55 kDa →

35 kDa →

25 kDa →

250 kDa →

100 kDa →

1 2 3 4 5 6 7 8 9 10

0 10 20 30 40 50 600

20

40

60

80

100

A

0 10 20 30 40 50 600

20

40

60

80

100

B

0 10 20 30 40 50 600

20

40

60

80

100

C

0 10 20 30 40 50 600

20

40

60

80

100

D

2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

E

2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

F

2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

G

2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

H

temperature, °C pH

rela

tive

act

ivity

, %

rela

tive

act

ivity

, %

rela

tive

act

ivity

, %

rela

tive

act

ivity

, %

LO

X1:

Md

:1a

LO

X1:

Md

:1c

LO

X2:

Md

:2a

LO

X2:

Md

:2b

Figure S5 Effects of temperature and pH on the enzymatic activity of apple LOX enzymes. LOX activity rates of recombinant LOX1:Md:1a (A, E), LOX1:Md:1c (B, F), LOX2:Md:2a (C, G) and LOX2:Md:2b (D, H) with linoleic acid were assayed at variable temperatures and pH values. The optimal reaction temperature for each enzyme was determined at 5 to 55°C and a pH of 7 (A-D). For determination of pH optimum, the reaction was run across a pH range of 3 to 10 at 25°C in the following buffers: sodium citrate for pH 3 to 6 (white squares), sodium phosphate for pH 6 to 8 (grey squares), and Tris-HCl for pH 8 to 10 (black squares), respectively (E-H). Values are the means ±SD for at least three independent measurements.

Figure S6 LC-MS analysis of hydroperoxy fatty acids (HpODE) formed from linoleic acid catalyzed by LOX1:Md:1a (A) and LOX1:Md:1c (B). Residual linoleic acid was monitored at m/z 279 [C18H32O2-H]-. The formation of HpODE was monitored at m/z 293 [C18H32O4-H2O-H]- (-MS top and bottom). Presence of 13-HpODE is confirmed by the fragment ions m/z 195 and 113 (-MS2(293) top) while fragment ions m/z 185 and 125 are indicative of 9-HpODE (-MS2(293) bottom).

linoleic acid m/z 279 -All MS

HpODE m/z 293 -All MS

0.00.51.01.52.0

0.00.51.01.52.0

0 5 10 15 20 25 30 Time [min]

Inte

nsity

(x1

07 )

311

293-MS, 20.9min

113

123 141 155 179 195

249

275

294-MS2(293)0

20406080

100

020406080

100

50 100 150 200 250 300 m/z

Inte

nsity

[%]

A LOX1:Md:1a

311

293-MS, 21.0min

125

185

197 212 249 277

294-MS2(293)

020406080

100

020406080

100

50 100 150 200 250 300 m/z

Inte

nsity

[%]

0.0

1.0

2.0

3.0

0.0

1.0

2.0

3.0

linoleic acid m/z 279 -All MS

HpODEm/z 293 -All MS

0 5 10 15 20 25 30 Time [min]

Inte

nsity

(x1

07 )

B LOX1:Md:1c

Figure S7 Effect of substrate concentration on enzymatic activity of wild-type and mutant apple LOX. LOX activity rates of recombinant LOX1:Md:1a (A) and LOX1:Md:1c (B) as well as mutant Gly567Ala protein (C) were monitored across a range of concentrations (2.5 to 100 µM) of linoleic acid (LA) and α-linolenic acid (LnA). The data were then fitted using Michaelis-Menten equation to calculate kinetic characteristics Km

and Vmax.

LA

0 20 40 60 80 1000

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

[S], µM

Ra

te,

µm

ol m

in-1

mg

-1

A

Wil

d-t

ype

LO

X1:

Md

:1a

LA

0 20 40 60 80 1000

2

4

6

8

10

12

14

16

[S], µM

Ra

te,

µm

ol m

in-1 m

g-1

B

LA

0 20 40 60 80 1000

2

4

6

8

10

12

14

[S], µM

Ra

te,

µm

ol m

in-1 m

g-1

C

Wil

d-t

ype

LO

X1:

Md

:1c

Mu

tan

t G

ly56

7A

la p

rote

in

Figure S8 Transient expression of selected LOX genes. Transient expression of MdLOX7a, MdLOX7c, and MdLOX1a (A, annotated as 9-LOX, 40µm ruler) and MdLOX2a, MdLOX4a, and MdLOX6b (B, annotated as 13-LOX, 30, 20, and 5 µm ruler, respectively) in tobacco leaves after three days by confocal microscopy as described by Brandizzi et al. (2002). The left column shows chloroplast autofluorescence, the middle column shows the protein fused with YFP, and the right column shows the overlay image.

Factors that determine regio- and stereospecificity of LOX1:Md:1a

Until now there is no comprehensive theory which illustrates all mechanisms underlying positional specificity of plant LOXs (Feussner and Wasternack, 2002). In

principal, it is often explained by a combination of factors, such as depth of substrate binding pocket (Gillmor et al., 1997) as well as substrate orientation

(Gardner, 1989). Fatty acid substrates presumably enter the hydrophobic environment of the enzyme active site with their methyl end first. The site of substrate

oxygenation is then determined by the depth of the substrate-binding pocket. In 13-LOXs this space is usually limited due to bulky amino acids (histidine or

phenylalanine) in the so-called Sloane position (Sloane et al., 1995). In contrast, 9-LOX harbour smaller amino acids (e.g., valine), which allows the substrate to

enter deeper into the binding pocket. For plant LOXs, a second theory has been proposed, where the site of dioxygenation is determined by the accessibility of

a conserved arginine residue at the bottom of the pocket (Hornung et al., 1999). In the case of 13-LOX, the arginine residue is shielded by space-filling amino

acids in the Sloane position. However, in 9-LOX the positively charged residue is accessible and allows the substrate to enter in an inverse head-to-tail

orientation favouring a penetration with its carboxylic group first.

In the case of LOX1:Md:1a, fatty acid substrates are most likely penetrating the active site with their carboxyl end first, as it was proposed by Boeglin et al.

(2008). Given that the enzyme harbours a valine residue (Val582) in the Sloane position, the carboxyl group of the substrate is supposed to form a salt bridge

with the conserved Arg732 residue at the bottom of the pocket (Figure 5). This might explain why mutation of Arg268 near the entry site had no effect on

enantiomeric composition and regiochemistry of hydroperoxides gained from reaction with all tested fatty acids. However, attempts to alter LOX1:Md:1a

specificity in favour of a 13(S)-LOX activity failed. The Val582Phe mutant, which should result in a straight substrate orientation, still produced considerably high

amounts of 13(R)-HpODE from LA and small amounts of 9-HpODE consisting of a racemic mixture of enantiomers. In general, substitution of a small residue in

the Sloane position with a histidine or phenylalanine is able to convert linoleate 9-LOX or dual-positional specific LOX to pure 13-LOX and vice versa (Hornung

et al., 1999; Hornung et al., 2008). However, in some cases bulkiness of this residue is not the sole determinant of positional specificity (Hughes et al., 2001).

The unchanged stereochemistry of 13-hydroperoxides produced by Val582Phe indicated that fatty acid substrates were still able to enter the enzyme active site

in inverse orientation. The bulky phenylalanine could, however perturb the oxygenation at C9-position sterically leading to the unspecific formation of 9-

hydroperoxides.

The major influence of a single active site amino acid on LOX stereospecificity has only recently been described by Coffa and Brash (2004). The so-called Coffa

site is conserved as an alanine in (S)- and a glycine in (R)-LOX. Evidence for this theory has been provided by mutagenesis of the alanine residue in wild-type

(S)-LOX to glycine, leading to mutant (R)-LOX with dual positional specificity (Coffa et al., 2005; Boeglin et al., 2008). In LOX1:Md:1a, mutation of the wild-type

Gly567 residue to alanine changed the dual positional (R)-LOX to an 9(S)-LOX with a product formation similar to LOX1:Md:1c for all three tested substrates

(Table 2; supplementary data Table S4). Additionally, the alanine substitution yielded a 10-fold and 8.5-fold increase in catalytic efficiency of LA- and LnA-

hydroperoxidation, respectively (Table 4). This is in accordance to an earlier report on a 6-fold drop in LA turnover caused by the substitution of alanine by

glycine in the Coffa site of soybean LOX-1 (Coffa et al., 2005). The volume of the side chains in this position might not only have effects on the oxygen access to

the activated pentadienyl radical, but also change the way the substrate interacts with the catalytic iron center (Coffa et al., 2005). In general, stereochemical

control of LOX activity is thought to involve a switch in the position of oxygenation on the substrate (Coffa and Brash, 2004). A glycine promotes oxygenation

near the catalytic iron atom resulting in (R)-stereochemistry. Whereas the larger alanine residue allows oxygenation only deep in the active site cavity and gives

(S)-hydroperoxides. An inversion of substrate orientation is therefore not essentially required to produce 9- and 13-hydroperoxides from LA with the same

enzyme.

In addition, a model for steric control of LOX activity was proposed in which oxygen is directed to a specific site of the substrate via a postulated side channel

(Knapp et al., 2001). In LOX1:Md:1a, three residues (Leu521, Leu572, Ile578) border this channel where it should intersect the substrate cavity. We found that

Ile578 aligns with a isoleucine residue discussed to determine O2 availability in soybean LOX-1 (Knapp et al., 2001; Ivanov et al., 2010). Mutation of the

corresponding Ile553 in the soybean LOX to a bulky phenylalanine residue seemed to constrict the putative oxygen binding channel leading to a drastic

decrease in catalytic efficiency (Knapp and Klinman, 2003). Although, mutation of Ile578 to leucine had nearly no effects on LOX1:Md:1a activity, recombinant

Ile578Leu protein lost some of its regiospecificity producing more 9-hydroperoxides from LA and LnA (Table 2, supplementary data Table S4). In contrast,

mutation of Leu572 to isoleucine seemed to strongly impede oxygen access to the substrate binding cavity leading to an almost complete loss of enzymatic

activity. Considering the idea that Ile578 might be involved in sterically directing oxygen to the substrate in favour of the C13 position of LA and LnA, the

mutation to leucine might enable oxygen a better access to the C9 position farther down the pocket. Furthermore, we found that Ile578Leu promoted

oxygenation at the C9- and C5-position of AA (supplementary data Table S4). Based on a model predicting inverse orientation of AA substrate in the active site,

this is also consistent with our earlier conclusion.

Product specificity of LOX1:Md:1a can therefore be explained as a combination of the following factors: i) A valine residue (Val582) in the Sloane position allows

fatty acid substrates to penetrate the active site in inverse head-to-tail orientation. ii) A glycine residue (Gly567) in the Coffa site sterically enables oxygenation at

both C9 and C13 position in the carbon chain of LA. iii) The shape of the substrate binding pocket, including a proposed oxygen channel, favours oxygenation at

C13 over C9, thereby determining product proportions.

The amino acid composition of the entrance site has an important influence on substrate affinity and catalytic efficiency of LOX as well (Knapp and Klinman,

2003; Coffa et al., 2005; Palmieri-Thiers et al., 2011). It was demonstrated that penetration of fatty acid substrates into the active site of olive LOX1 requires the

movement of the side chains of Phe277 and Tyr280 (Palmieri-Thiers et al., 2011). Both sequence positions are highly conserved among plant LOX

(LOX1:Md:1a: Phe277, Tyr280). Thus, it can be assumed that mutagenesis of the nearby Ile566 (Figure 5) to a more space-filling amino acid would also affect

enzyme activity. Indeed, LOX activity of the Ile566Phe mutant was nearly completely abolished. The Ile566 residue is bordering the entry to the substrate

binding pocket and is located in helix 11 together with conserved residues, which have been shown to influence kinetic efficiency and regiospecificity of LOX

(Coffa et al., 2005). It appears, that replacement of this residue with a phenylalanine entailed a constriction of the substrate channel leading to perturbed

accessibility of the binding pocket.

Figure S9 Factors that determine regio- and stereospecificity of LOX1:Md:1a.

Figure S10 Characterization of ripening apple fruit.

Characterization of ripening apple fruitBriefly, the first harvest took place on 4 Sept. in 2009. After the initial harvest dates, ‘normally-ripening’ fruit were harvested twice per week, every three to four days until ripening was imminent as judged by the average internal ethylene being greater than 0.1 µL L-1. At that time additional fruit were harvested and thereafter allowed to ripen in a high humidity, controlled environment chamber at 15 °C. Fruit were examined every three to four days until the conclusion of the study on 27 Oct. (day 53) in 2009. On each evaluation date, the 5 fruit having an internal ethylene content nearest the median of a 20-fruit sample were selected for analysis of CO2 production and volatile emissions. As previously described by Contreras and Beaudry (2013), respiration was determined for whole fruit in a flow-through system at ambient temperature (22 ±1 °C). Fruit were sealed in 1-L Teflon containers (Savillex Corporation, Minnetonka, MN) flushed at approximately 40 mL min-1 with air. Volatile analysis was performed on the headspace of the 1-L containers following a 20 min incubation period during which the flow of air through the container was stopped. Volatiles were collected using a solid phase micro extraction (SPME) fiber (65 µm thickness PDMS-DVB, Supelco Inc., Bellefonte, PA) and separated by gas chromatography (HP-6890, Hewlett Packard Co., Wilmington, DE). Volatile detection was by time-of-flight mass spectrometry (TOFMS) using electron impact ionization (Pegasus II, LECO Corp., St. Joseph, MI). Identification of all quantified compounds was achieved by comparison of the mass spectrum with authenticated reference standards and with spectra in the National Institute for Standard and Technology (NIST) mass spectral library (Search version 1.5). Volatile compounds were quantified by calibrating with a known amount of an authenticated, high-purity standard mixture of 28 volatilized alcohols, aldehydes, and esters as previously described (Contreras and Beaudry, 2013). Following volatile analysis, fruit tissue was sampled by removing the epidermis and 2-3 mm of cortex with a manual rotary peeler (Model 8, Goodell, Antrim, NH), and immediately frozen in liquid nitrogen and stored at -80 °C. Tissue from five of the ten selected fruit was pooled into each of two biological replicates. Data for ethylene, respiration and volatile production was used to identify critical stages of development for LOX expression analysis (Figure 2). Eight stages were identified: immature apple (stage 1), mature with low levels of ethylene (stage 2), mature, low levels of ethylene just prior to the detection of hexyl esters (stage 3), mature/ripening with low but increasing levels of ethylene and low levels of hexyl esters (stage 4), ripening, autocatalytic ethylene synthesis engaged and rapidly increasing ester emissions (stage 5), ripening, at the peak of the respiratory climacteric (stage 6), ripe, at the peak of ester emissions and the onset of the decline in respiration (stage 7), and overripe/senescent with declining ester synthesis and respiratory activity (stage 8).

References:Boeglin, W.E., Itoh, A., Zheng, Y., Coffa, G., Howe, G.A., and Brash, A.R. (2008). Investigation of substrate binding and product

stereochemistry issues in two linoleate 9-lipoxygenases. Lipids 43, 979-987.Coffa, G., and Brash, A.R. (2004). A single active site residue directs oxygenation stereospecificity in lipoxygenases, stereocontrol is

linked to the position of oxygenation. Proc. Natl. Acad Sci. USA 101, 15579-15584.Coffa, G., Imber, A.N., Maguire, B.C., Laxmikanthan, G., Schneider, C., Gaffney, B.J., and Brash, A.R. (2005). On the relationships of

substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant. J. Biol. Chem. 280, 38756-38766.

Contreras, C., and Beaudry, R. (2013). Lipoxygenase-associated apple volatiles and their relationship with aroma perception during ripening. Postharv. Biol. Technol. 82, 28-38.

Feussner, I., and Wasternack, C. (2002). The lipoxygenase pathway. Annu. Rev. Plant Biol. 53, 275-297.Gardner, H.W. (1989). Soybean lipoxygenase-1 enzymically forms both (9S)-and (13S)-hydroperoxides from linoleic acid by a pH-

dependent mechanism. Biochim. Biophys. Acta 1001, 274-281.Gillmor, S.A., Villaseñor, A., Fletterick, R., Sigal, E., and Browner, M.F. (1997). The structure of mammalian 15-lipoxygenase reveals

similarity to the lipases and the determinants of substrate specificity. Nat. Struct. Mol. Biol. 4, 1003-1009.Hornung, E., Kunze, S., Liavonchanka, A., Zimmermann, G., Kühn, D., Fritsche, K., Renz, A., Kühn, H., and Feussner, I. (2008).

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia. Phytochemistry 69, 2774-2780.

Hornung, E., Walther, M., Kühn, H., and Feussner, I. (1999). Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis. Proc. Natl. Acad Sci. USA 96, 4192-4197.

Hughes, R., West, S., Hornostaj, A., Lawson, D., Fairhurst, S., Sanchez, R., Hough, P., Robinson, B., and Casey, R. (2001). Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers. Biochem. J. 353, 345-355.

Ivanov, I., Heydeck, D., Hofheinz, K., Roffeis, J., O’Donnell, V.B., Kuhn, H., and Walther, M. (2010). Molecular enzymology of lipoxygenases. Arch. Biochem. Biophys. 503, 161-174.

Knapp, M.J., and Klinman, J.P. (2003). Kinetic studies of oxygen reactivity in soybean lipoxygenase-1. Biochemistry 42, 11466-11475.Knapp, M.J., Seebeck, F.P., and Klinman, J.P. (2001). Steric control of oxygenation regiochemistry in soybean lipoxygenase-1. J. Am.

Chem. Soc. 123, 2931-2932.Palmieri-Thiers, C., Alberti, J.-C., Canaan, S., Brunini, V., Gambotti, C., Tomi, F., Oliw, E.H., Berti, L., and Maury, J. (2011).

Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies. Arch. Biochem. Biophys. 509, 82-89.

Sloane, D.L., Leung, R., Barnett, J., Craik, C.S., and Sigal, E. (1995). Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase, the side-chain geometry of amino acids 417 and 418 determine positional specificity. Protein Eng. 8, 275-282.

Tab

le S

1 Id

en

tity

of

Ma

lus

x d

om

es

tic

a li

po

xy

ge

na

se

s,

an

d o

lig

on

uc

leo

tid

es

us

ed

fo

r g

en

e a

mp

lifi

ca

tio

n

Locu

s G

enB

ank

acc.

no.

D

escr

iptio

n U

pper

prim

er

Low

er p

rimer

A

nnea

ling

tem

pera

ture

A

mpl

icon

le

ngth

E

ncod

ed

prot

ein

leng

th

5‘

pos

ition

a

sequ

ence

b

5‘ p

ositi

ona

sequ

ence

c

(°C

) (b

p)

(am

ino

acid

s)

MD

P00

0045

0991

KC

7064

80

MdL

OX

1a

-48

TTA

TT

CA

CA

AC

AT

TC

TT

TG

C

+26

71

AC

GC

TT

GT

TT

GA

TC

CC

ATA

C

61.4

27

19

863

MD

P00

0031

2397

M

dLO

X1b

-2

4 G

AA

AC

TG

GA

GG

TC

CG

AC

+

2853

G

GT

CA

TAC

TT

CTA

GC

ATA

TC

AC

58

.4

2877

92

0

MD

P00

0042

3544

KC

7064

81

MdL

OX

1c

-37

AT

TC

GT

GTA

AA

GC

AA

AG

CA

G

+26

80

GG

TC

ATA

CT

TC

TAG

CA

TAT

CA

C

57.0

27

17

862

MD

P00

0014

6677

K

C70

6482

M

dLO

X1d

-9

A

GA

TC

AA

AG

AT

GC

TG

CA

TT

G

+28

81

CA

AA

CA

AA

GA

AT

CA

CA

GA

AG

C

58.1

28

90

952

MD

P00

0087

4800

K

C70

6483

M

dLO

X2a

-5

9 G

GA

TT

CA

AA

CT

TT

CT

CG

AA

C

+27

75

CC

AC

CA

CC

AC

CT

CA

AA

ATA

A

65,7

28

34

906

MD

P00

0075

5511

KC

7064

85

MdL

OX

2b

-10

GA

AG

AA

GA

AG

AT

GG

CA

CT

GA

CTA

AA

C

+27

35

CTA

AA

TG

TT

GT

TG

AG

AG

TC

ATA

TC

G

61.9

27

45

905

a

from

the

tra

nsla

tion

star

t co

don

b

unde

rline

d ba

ses

indi

cate

the

tra

nsla

tion

star

t co

don

c

unde

rline

d ba

ses

indi

cate

the

tra

nsla

tion

stop

cod

on

Tab

le S

4

Dis

trib

uti

on

of

hy

dro

(pe

ro)x

y p

rod

uc

ts g

ain

ed

fro

m w

ild

-ty

pe

an

d m

uta

nt

ap

ple

LO

X e

nzy

me

re

ac

tio

n w

ith

α-

lin

ole

nic

ac

id a

nd

ara

ch

ido

nic

ac

id.

Pro

po

rtio

ns

of

H(p

)OT

E a

nd

H(p

)ET

E p

rod

uct

s a

re g

ive

n a

s p

er

cen

t o

f to

tal p

rod

uct

am

ou

nt

ob

tain

ed

fro

m s

pe

cific

LO

X a

ctiv

ity w

ith L

nA

an

d A

A,

resp

ect

ive

ly.

H

(p)O

TE

pro

duct

s (%

) H

(p)E

TE

pro

duct

s (%

)

enzy

me

13

9

15

12

11

9 8

5

LOX

1:M

d:1a

89

.8

10.2

41

.5

16.8

11

.6

15.9

6.

1

8.1

LOX

1:M

d:1c

7.

2

92.8

11

.8

4.4

53

.2

4.0

8.

4

18.2

LOX

2:M

d:2a

90

.2

9.8

44.7

16

.3

9.6

12.3

8.

2

8.9

LOX

2:M

d:2b

97

.0

3.0

97.4

0.

7

1.1

0.2

0.

3

0.3

Arg

268A

la

91.9

8.

9 34

.9

11.1

17

.0

19.5

10

.9

6.6

Gly

567A

la

2.7

97

.3

7.4

8.5

44

.1

7.2

9.

1

23.7

Ile57

8Leu

80

.5

19.5

17

.1

14.9

9.

9 28

.9

7.8

21

.4

Val

582P

he

90.9

9.

1 29

.8

11.8

23

.7

13.7

6.

4

14.6

Tab

le S

5

Oli

go

nu

cle

oti

de

s u

se

d t

o a

mp

lify

th

e M

dL

OX

1a

, Md

LO

X1

c, M

dL

OX

2a

an

d M

dL

OX

2b

ge

ne

s

for

su

bc

lon

ing

in

to p

RS

ET

B

an

d p

YE

S2

ex

pre

ss

ion

ve

cto

rs.

Prim

er n

ame

S

eque

nce

pRS

ET

-L1a

_Xho

I_fo

r A

TAA

TAC

TC

GA

GC

AT

GT

TG

CA

CA

AC

CT

GC

T

pRS

ET

-L1a

_Nco

I_re

v A

AT

GT

CC

CA

TG

GT

TAG

ATA

GA

GA

CG

CT

GT

TG

pRS

ET

-L1c

_Xho

I_fo

r A

GA

AC

TC

GA

GA

AT

GC

TG

CA

GA

ATA

TC

GT

TG

pRS

ET

-L1c

_Kpn

I_re

v A

GC

TG

GTA

CC

TTA

AA

TT

GA

GA

CA

CT

GT

TG

G

pRS

ET

-L2a

_Xho

I_fo

r A

TAA

CT

CG

AG

AA

TG

GC

AC

TG

GC

TAA

AC

pRS

ET

-L2a

_Kpn

I_re

v C

CC

CT

GG

TAC

CT

CA

TAT

CG

AA

ATA

CTA

TT

C

pRS

ET

-L2b

_Xho

I_fo

r A

CTA

CT

CG

AG

AA

TG

GC

AC

TG

AC

TAA

AC

pRS

ET

-L2b

_Kpn

I_re

v T

GC

TG

GTA

CC

TC

ATA

TC

GA

AA

TAC

TAT

TAG

G

pYE

S_L

OX

his_

Kpn

I_fo

r C

AA

GG

TAC

CA

AC

AC

AA

TG

TC

TG

GT

TC

TC

AT

CA

T

pYE

S_L

1a_N

otI_

rev

G

TT

GC

GG

CC

GC

TTA

GA

TAG

AG

AC

G

pYE

S_L

1c_X

baI_

rev

G

GC

TC

TAG

AT

TAA

AT

TG

AG

AC

AC

TG

TT

GG

pYE

S_L

2a/b

_Not

I_re

v

AC

TG

CG

GC

CG

CT

CA

TAT

CG

AA

ATA

CTA

TT

Tab

le S

6

Oli

go

nu

cle

oti

de

s u

se

d f

or

sit

e-d

ire

cte

d m

uta

ge

ne

sis

of

the

Md

LO

X1

a g

en

e.

Mut

ant

Met

hode

F

orw

ard

prim

erb

R

ever

se p

rimer

b

Arg

268A

la

oeP

CR

a

L1a_

R26

8A_f

or

CA

AG

AG

AT

GA

AG

CA

TT

TG

GT

CA

CT

TG

L1

a_R

268A

_rev

C

AA

GT

GA

CC

AA

AT

GC

TT

CA

TC

TC

TT

G

Ile56

6Phe

oe

PC

Ra

L1a_

I566

F_f

or

GTA

CA

TC

AA

CG

CA

TT

TG

GT

AG

AG

G

L1a_

I566

F_r

ev

CC

TC

TA

CC

AA

AT

GC

GT

TG

AT

GTA

C

Gly

567A

la

Qui

kCha

nge

L1

a_G

567A

_for

C

CA

TG

TAC

AT

CA

AT

GC

AA

TT

GC

TA

GG

GG

AA

TC

CT

CC

TTA

AT

GC

L1

a_G

567A

_rev

G

CA

TTA

AG

GA

GG

AT

TC

CC

CT

AG

CA

AT

TG

CA

TT

GA

TG

TAC

AT

GG

Leu5

72Ile

Q

uikC

hang

e

L1a_

L572

I_fo

r T

TG

GC

AG

GG

GA

AT

CC

TC

AT

TAA

TG

CT

CG

CG

GA

GT

TATA

GA

G

L1a_

L572

I_re

v

CT

CTA

TAA

CT

CC

GC

GA

GC

AT

TAA

TG

AG

GA

TT

CC

CC

TG

CC

AA

Ile57

8Leu

Q

uikC

hang

e

L1a_

I578

L_fo

r C

CT

TAA

TG

CT

CG

CG

GA

GT

TT

TAG

AG

TC

GA

CA

GT

TT

TT

CC

AG

C

L1a_

I578

L_re

v

GC

TG

GA

AA

AA

CT

GT

CG

AC

TC

TAA

AA

CT

CC

GC

GA

GC

AT

TAA

GG

Val

582P

he

oeP

CR

a

L1a_

V58

2F_f

or

GT

TATA

GA

GT

CT

AC

AT

TC

TT

TC

CA

GC

TAG

L1

a_V

582F

_rev

C

TAG

CT

GG

AA

AG

AA

TG

TA

GA

CT

CTA

TAA

C

Thr

775L

eu

Qui

kCha

nge

L1

a_T

775L

_for

G

AC

TG

TAC

TT

GG

TAT

TG

CC

TT

GA

TT

GA

GA

TT

TT

GT

CA

AG

GC

L1

a_T

775L

_rev

G

CC

TT

GA

CA

AA

AT

CT

CA

AT

CA

AG

GC

AA

TAC

CA

AG

TAC

AG

TC

a

ampl

ifica

tion

of g

ene

frag

men

ts w

as a

chie

ved

in c

ombi

natio

n w

ith p

YE

S_L

OX

his_

Kpn

I_f

and

pYE

S_L

1a_N

otI_

r (r

efer

to

tabl

e S

5)

b

unde

rline

d ba

ses

indi

cate

bas

e ex

chan

ges

for

dire

cted

mut

agen

esis ;

bas

es in

ital

ic in

dica

te c

onse

rvat

ive

base

exc

hang

es t

o m

inim

ize

the

risk

of h

airp

in a

nd s

elf-

dim

er f

orm

atio

n