Kathrin Schurmann, Monika Anton, Igor Ivanov, Constanze Richter#, Hartmut Kuhn+, Matthias ...

20 Pages·2011·2.26 MB·English

JBC Papers in Press. Published on May 10, 2011 as Manuscript M110.211821
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M110.211821
MOLECULAR BASIS FOR THE REDUCED CATALYTIC ACTIVITY OF THE NATURALLY OCCURING T560M
MUTANT OF HUMAN 12/15-LIPOXYGENASE THAT HAS BEEN IMPLICATED IN CORONARY ARTERY DISEASE

Kathrin Schurmann, Monika Anton, Igor Ivanov, Constanze Richter#, Hartmut Kuhn+, Matthias
Walther
*Institute of Biochemistry, University Medicine Berlin - Charité, Monbijoustr. 2, D-10117 Berlin, Germany;
#Institute of Food Chemistry and Toxicology, Technical University Berlin, Germany

Running head: mutant 12/15-LOX

+Address correspondence to: Dr. Hartmut Kuhn, Institute of Biochemistry, University Medicine Berlin -
Charité, Oudenarder Str. 16, 13347 Berlin, Germany; Tel. [phone], Fax [phone], e-
mail: [email protected]

Lipoxygenases (LOXs) have been enzymes and have been implicated in the
implicated in cardiovascular disease. A rare pathogenesis of inflammatory (3,4), degenerative
single nucleotide polymorphism causing T560M (5,6) and hyperproliferative (7,8) diseases. The
exchange has recently been described and this possible role of ALOX15 in cardiovascular diseases
mutation leads to a near null-variant of the has been a matter of discussion for many years
enzyme encoded for by the ALOX15 gene. When (9,10) and pro- and anti-atherogenic (11-13) D
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we inspected the 3D-structure of the rabbit activities have been reported in various animal n
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ortholog we localized T560 outside the active site atherosclerosis models. In humans, expression of ad
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and identified a hydrogen bridge between its side ALOX15 has been detected in atherosclerotic fro
chain and Q294. This interaction is part of a lesions (14) and structural analysis of deposited m
h
complex hydrogen bond network, which appears lipids suggested its catalytic activity in young ttp
to be conserved in other mammalian LOXs. human plaques (15). On the other hand, more recent ://w
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Q294 and N287 are key amino acids in this studies failed to detect significant amounts of w
network and we hypothesized that disturbance ALOX15 mRNA in advanced human lesions (16) .jbc
.o
of this hydrogen bond system causes the low but suggested significantly higher levels of ALOX5 rg
b/
activity of the T560M mutant. To test this transcripts. This finding and the pro-inflammatory y
g
hypothesis we first mutated T560 to amino acids character of ALOX5 is consistent with the ue
s
not capable of forming side chain hydrogen hypothesis that atherosclerosis is an inflammatory t o
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bridges (T560M, T560A) and obtained enzyme disease of the vessel wall (17,18). Ja
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variants with strongly reduced catalytic activity. In a population-based case-control study (19) ary
In contrast, enzymatic activity was retained after involving some 1800 subjects with clinically 24
T560S exchange. Enzyme variants with strongly significant coronary artery disease and some 1700 , 20
1
reduced activity were also obtained when we healthy controls a novel single nucleotide 9
mutated Q294 (binding partner of T560) and polymorphism (SNP) in the coding region of the
N287 (binding partner of Q294 and M418) to L. ALOX15 gene was reported. This non-synonymous
Basic kinetic characterization of the T560M SNP exchanges T560 to M (T560M) and in vitro
mutant indicated that the enzyme lacks a kinetic mutagenesis studies on the recombinant ALOX15
lag-phase but is rapidly inactivated. This data indicated a strong reduction of the catalytic activity
suggests that the low catalytic efficiency of the of the T560M mutant (19). Heterozygous allele
naturally occurring T560M mutant is caused by carriers had a significantly increased risk for
alterations of a hydrogen bond network coronary artery disease (adjusted odds ratio of 1.62;
interconnecting this residue with active site p=0.02). When this SNP was genotyped in the
constituents. Disturbance of this bonding patient cohort of the ARIC (Atherosclerosis Risk in
network increases the susceptibility of the Communities) study heterozygote carriers also
enzyme for suicidal inactivation. showed an increased risk for coronary artery disease
(19), which was borderline significant (adjusted
Lipoxygenases (LOXs) catalyze the hazards ratio 1.31; p=0.06). In both studies,
peroxidation of free and/or esterified homozygote carriers were too rare to draw
polyunsaturated fatty acids to corresponding conclusions. In an independent large-scale (some
hydroperoxy compounds (1,2). They constitute a 2600 participants) case-control study (20) a similar
heterogeneous family of lipid metabolizing trend towards an increased risk for myocardial
1
Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
infarction was observed for heterozygote allele each mutant, 5-10 clones were selected, screened
carriers of the T560M mutation (OR 1.7, p=0.06). for LOX expression and one clone was completely
The molecular basis for the strongly reduced sequenced to confirm mutagenesis.
catalytic activity of the T560M mutant has not been
explored in detail. Structural modeling on the basis Purification of recombinant ALOX15 – Wild-
of the X-ray coordinates of the rabbit ortholog type human ALOX15 and selected mutants were
(21,22) indicated that T560 is not an immediate affinity purified on a Ni-TED matrix open bed
constituent of the active site. Instead, it is localized column. For purification, LOX-active clones were
in a more flexible loop region that has no direct picked with a sterilized toothpick and 20 ml LB
contact to the catalytic center. This study was aimed medium containing ampicillin (0.1 mg/liter) were
at exploring the mechanistic basis for the low inoculated. After 8 h at 37°C, 15 ml were added to
catalytic efficiency of the naturally occurring 3 l LB medium containing ampicillin (0.1 mg/liter)
T560M mutant of ALOX15. Our data suggest that and bacteria were grown at 37°C overnight.
the loss in catalytic activity is caused by a Cultures were cooled to room temperature and after
disturbance of a hydrogen bond network that expression of recombinant LOX was initiated by
surrounds the bottom of the substrate binding addition of 1 mM IPTG further incubated for 4 h at
pocket and that these alterations induce an increased 25°C. Then cells were spun down, reconstituted in
susceptibility of the enzyme for catalytic PBS and lyzed with an EmulsiFlex-C5 high
inactivation. pressure homogenizer (Avestin, Ottawa, Canada).
Cell debris was spun down, the supernatant was D
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added to 0.5 ml Ni-TED (Machery-Nagel, Düren, n
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Materials and Methods Germany) and the suspension was incubated for 1h ade
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Chemicals - The chemicals used were at 4°C. The Ni-TED was transferred to an open bed fro
column, washed with washing buffer (50 mM m
obtained from the following sources: arachidonic h
aSceirdv a (5(HZe,8idZe,1lb1eZrg,1, 4GZ-eerimcoasnayt)e,t rHaePnLoCic staacnidd)a rdfsro omf pNraoHte2iPnOs 4w, e3re0 0e lumteMd wNiathC le, luptHio n 8b.0u)f fearn d( 50b omunMd ttp://w
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NaH PO , 300 mM NaCl, 200 mM imidazole, pH w
1H2EST-HE EfTroEm, 1C2a(y±m)-aHnE CTEhe, m1.5, Ss-oHdEiuTmE ,b oarnodh y1d5r(i±de)-, 8.0). 2The4 LOX activity of the elution fractions was .jbc.o
ampicillin from Life Technologies, Inc. tested employing a spectrophotometric assay. The brg/
(Eggenstein, Germany), isopropyl-ß- pooled LOX containing Ni-TED fractions were y g
thiogalactopyranoside (IPTG) from Carl Roth supplemented with 10% glycerol and shock frozen ues
GmbH (Karlsruhe, Germany). HPLC solvents were in liquid nitrogen. An electrophoretic purity of t on
purchased from Baker (Deventer, The Netherlands). about 90% usually was obtained. Jan
u
Restriction enzymes were obtained from Fermentas To determine the amount of LOX protein, ary
(St. Leon-Rot, Germany). Oligonucleotide synthesis Western Blot analyses of wild-type and mutant 24
was performed at BioTez (Berlin, Germany) and enzymes were quantified (supplement Fig. S1). , 20
1
Aliquots of the LOX containing Ni-TED fractions 9
nucleic acid sequencing was carried out at Eurofins
were separated by SDS gel electrophoresis, blotted
MWG Operon (Ebersberg, Germany). The E. coli
strain XL-1 blue was purchased from Stratagene on a nitrocellulose membrane and His-tagged
proteins were detected using an RGSHis antibody
(La Jolla, CA).
(Qiagen, Hilden, Germany) at 1:5000 dilution. As

Bacterial expression and site-directed secondary antibody an anti mouse IgG at a dilution
mutagenesis of ALOX15 – Wild-type human of 1:5000 was used (Sigma-Aldrich, Munich,
Germany).
ALOX15 and its mutants were expressed as N-
terminal his-tag fusion proteins in E. coli as In different batches of enzyme preparations
described before (23). For this purpose the cDNA we found that the specific activity of purified wild-
type human ALOX15 varied between 20 s-1 and 40
was cloned into the pQE-9 procaryotic expression
s-1 (under V condions) and this data is consistent
plasmid in such a way that the starting methionine max
of the LOX coding sequence was deleted. Because with corresponding values obtained under
comparable conditions for native and recombinant
of technical reasons the N-terminus was elongated
rabbit ALOX15 enzyme.
by additional amino acids including six consecutive
His. Site-directed mutagenesis was performed using
the QuikChangeTM Site-Directed Mutagenesis Kit Cloning, expression and activity assay of
mouse 5-LOX (alox5) – 500 µl mouse blood were
(Stratagene, Amsterdam, The Netherlands). For
treated with fMet-Phe-Leu for 30 min at 37°C and
2
total RNA was prepared using the QIAamp RNA expression was induced by the addition of IPTG (1
Blood Mini Kit from Qiagen (Hilden, Germany). mM final concentration). After 2 hours at 30°C
After reverse transcription (RevertAid H Minus bacteria were spun down, washed and reconstituted
reverse transcriptase, Fermentas, St. Leon-Rot, in 0,5 ml PBS. Arachidonic acid was added on ice
Germany) 100 ng of the resulting cDNA were used (100 µM final concentration) and cells were lyzed
as template to amplify mouse alox5 by PCR. The by sonication with a Labsonic U-tip sonifier (Braun,
PCR fragment was cloned into the pRSET A Melsungen, Germany). The mixture was incubated
expression vector (Invitrogen), which contains an for 10 minutes at 25°C, the hydroperoxy
N-terminal His-tag sequence, between unique Xho I compounds formed were reduced with sodium
and Hind III restriction sites. After sequencing, borohydride, and after acidification to pH 3 (acetic
recombinant alox5 was expressed in the E. coli acid) 0.5 ml methanol were added. The protein
strain BL21(DE3)pLysS (Invitrogen) as described precipitate was spun down, and aliquots of the clear
for ALOX15. The recombinant protein was purified supernatant were injected directly for quantification
on a Co-sepharose open bed column (BD Talon, BD of the LOX products to RP-HPLC. For most
Biosciences, Heidelberg, Germany) and elution of activity assays instead of the 5 ml overnight
the protein was achieved by 200 mM imidazole. cultures purified LOX fractions were used applying
The fractions containing 5-LOX activity were the same protocol.
pooled and used for activity assays. For time dependence measurements, the
As for ALOX15 catalytic activity of alox5 activity assays were incubated for the scheduled
was assayed by HPLC quantification of arachidonic time periods at 25°C and then the reaction was D
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acid oxygenation products. For this purpose stopped by addition of 500 µl ice-cold methanol. n
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aliquots of the purified enzyme preparation were Hydroperoxides were reduced with sodium ad
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incubated with arachidonic acid (100 µM) for 10 borohydride, samples were acidified with acetic fro
min in PBS containing 0.4 mM Ca2+, 0.1 mM ATP acid and protein precipitation spun down. Aliquots m
h
and 40 µg/ml phosphatidyl choline (final of the supernatant were used for RP-HPLC ttp
concentrations). Product preparation and HPLC quantification. ://w
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analysis were carried out as described for ALOX15. HPLC analysis was performed on a w
Shimadzu instrument equipped with a Hewlett- .jbc
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Expression and activity assay of human Packard diode array detector 1040 A by recording rg
b/
platelet 12-LOX (ALOX12) - Human platelet 12- the absorbance at 235 nm. Reverse phase-HPLC y
g
LOX (ALOX12) was expressed as his-tag fusion was carried out on a Nucleodur C18 Gravity ue
s
protein as described before (24). Enzyme column (Machery-Nagel, Düren, Germany; 250 x 4 t o
n
purification and activity assays of wild-type and mm, 5 µm particle size) coupled with a guard Ja
n
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mutant enzyme species were carried out as column (8 x 4 mm, 5-µm particle size). A solvent ary
described above for ALOX15. system of methanol/water/acetic acid (85/15/0.05, 24
by volume) was used at a flow rate of 1 ml/min. 15- , 20
1
Determination of iron content of wild-type HETE enantiomers were separated by chiral phase 9
ALOX15 and selected mutants – To determine the HPLC (CP-HPLC) as free fatty acids on a Chiralcel
iron content, LOX containing Ni-TED fractions OD column (Daicel Chem. Ind., Ltd.) using a
were further purified by FPLC using a Resource Q solvent system consisting of hexane/2-
column (GE Healthcare). The electrophoretically propanol/acetic acid (100/5/0.1, by vol.) and a flow
homogeneous FPLC fractions were concentrated, rate of 1 ml/min. 12-HETE enantiomers were
desalted by gel filtration and the iron content was separated as methyl esters after incubating with
measured by atom absorption spectroscopy on a diazomethane for 15 min at room temperature on
Perkin Elmer AA800 instrument equipped with the Chiralcel OD column using a solvent system of
AS800 autosampler. The iron content was related to hexane/2-propanol/acetic acid (100/2/0.1, by vol.)
LOX protein that was quantified and a flow rate of 1 ml/min.
spectrophotometrically (1 mg/ml pure ALOX15 has
an absorbance of 1.78 at 280 nm). Structural modeling and amino acid
sequence alignments – Human and rabbit ALOX15
HPLC-based activity assays - For routine proteins share 81% sequence identity. For the rabbit
activity assays, one sequenced clone was replated, enzyme, the crystal structure was solved and the
five well separated colonies were picked, and the pdb entry 2P0M was used to model the structure of
bacteria were cultured overnight at 37°C in 5 ml LB the human ortholog. The recently solved crystal
medium containing 0.1 mg/ml ampicillin. LOX structure of human 5-LOX (25) was employed to
3
construct a model of mouse 5-LOX (alox5). The detailed structural investigations indicated that the
VMD software package (26) was employed for T560-Q294 hydrogen bridge is part of a more
structural analysis and figure preparation. Hydrogen complex hydrogen bond network (Fig. 1D). Here,
bonds were identified with the Swiss PdbViewer Q294 and N287 appear to play major roles since
v4.0.1. each of these residues interacts with four different
Multiple sequence alignments were binding partners. Interestingly, this hydrogen bond
performed applying clustalW2 program provided by network interconnects T560 with M418, which has
the European Bioinformatics Institute (EBI). previously been shown to directly interact with
substrate fatty acids (24,27).
Statistics – The experimental raw data were
statistically evaluated employing the Microsoft Mutation of T560 to residues not capable of
Excel® software package (version 12.0). Means ± forming side chain hydrogen bridges impairs
standard deviations were calculated. For catalytic activity – The naturally occurring T560M
significance calculations the two-sided (type 2) mutant in the human enzyme lacks the hydrogen
Students’ t-test was employed. donating side-chain OH-group and the purified
recombinant protein exhibits a strongly impaired
catalytic activity (Fig. 2A, Table 1). This
Results corresponds well with the previously reported 5%
residual activity for this mutant when expressed in
T560 is not an active site residue but forms a
E. coli (19). Similar results were obtained when D
hydrogen bridge with Q294 – Human ALOX15 ow
wild-type human ALOX15 and its T560M mutant n
shares a high degree (81%) of sequence identity lo
with the ortholog rabbit enzyme and thus, the X-ray were expressed in human embryonic kidney cells ade
d
coordinates of the rabbit enzyme were employed to (19) and we confirmed this data in the current study fro
(not shown). The T560A mutant, which also lacks m
draw structural conclusions on the human enzyme. h
For clarity reasons, we use amino acid numbering trhesei duhayld croatgaelny ticd aocntaivtiintyg ofO oHn-lgyr 1o7u p%, (eTxahbibleit e1d). Ina ttp://w
of human ALOX15 throughout this paper. w
contrast, when T560 was mutated to S, an amino w
When we inspected the structure of the rabbit acid that carries a hydrogen donating OH-group, the .jbc
ALOX15 we found that T560 is not an active site .o
catalytic activity was largely retained. In fact, we rg
residue. Although it is localized in proximity to b/
measured a residual activity of more than 70% y
I417 (Fig. 1A), which contributes to form the g
bottom of the substrate-binding pocket (27), the (Table 1). This data confirms the catalytic ues
importance of the T560-Q294 hydrogen bridge. t o
closest distance between the two residues is 4.12 Å. n
Moreover, we found that these mutants exhibited Ja
This is beyond the binding distance of non-covalent n
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interactions. T560 is localized in a loop region that similar reaction specificities as the wild-type ary
lgarcokusp s(thaybdler osgeecno nddoanroyr )s tbrruicdtguersa lw eiltehm tehnet ss.i dIets c OhaHin- eanltzeyramtieo ns( Tianb lee nz1y)m, es-suugbgsetsrtaitneg inthtearta ctnioon mwaejorer 24, 201
induced. 9
oxygen of the amide group (hydrogen acceptor) of

Q294 (Fig. 1B), which is located in a neighboring
Q294L exchange also leads to a loss in
loop. This hydrogen bridge might stabilize this
catalytic activity – As suggested in Fig. 1B the
structural microenvironment.
hydroxy group of T560 hydrogen bridges with the
The rabbit ALOX15 undergoes structural
side chain of Q294. If this hydrogen bridge is
rearrangement when a ligand is bound at the active
important for the catalytic activity, mutation of
site and two major conformers (ligand-free, ligand
Q294 to a residue lacking a hydrogen acceptor
bound) have been described (22). The
should also lead to an inactive enzyme. Indeed,
conformational changes include dislocation of
when we tested the purified Q294L mutant in our
surface helix 2 and, to make room for ligand
HPLC-based activity assay we observed a strongly
binding, retreat of helix 18 from the active site. To
reduced catalytic activity (Fig. 2B). Further activity
explore whether the 3D-structure of the T560 region
assays using higher (5-times) enzyme concentration
is altered upon ligand binding we compared the two
of the Q294L mutant indicated a product pattern
conformers but did not find major structural
that was very similar to that of the wild-type
differences (Fig. 1C). In fact, the bonding distance
enzyme (84% 15-HETE, 16% 12-HETE, see
of the T560-Q294 hydrogen bridge was nearly
supplement Fig. S2). Taken together, the strongly
unchanged (2.62 Å for the ligand-free conformer vs.
impaired catalytic activity of the T560M mutant and
2.73Å for the ligand-bound conformer). More
the even lower activity of the Q294L variant
4
suggest the functional importance of the hydrogen (supplement Table S1). Except for the epidermal
bridge between T560 and Q294. LOXs the third key residue in this hydrogen bond
network (N287) is also conserved. Taken together,
N287 is a key residue in the hydrogen this data suggests that similar hydrogen bridges
bonding network and its mutation also inactivated may also exist in other LOX isoforms.
the ALOX15 - A second key element in this Recently, the crystal structure of human 5-
hydrogen bond network is N287 (Fig. 1D). Its side LOX has been solved (25) and we inspected this
chain amide forms hydrogen bridges with the ALOX5 structure for the existence of a similar
backbone of V288, with D415 and M418. When we hydrogen bond. Indeed, a hydrogen bridge was
mutated this residue to L (N287L), an amino acid present between T570 (corresponds to T560 in
that cannot form any side chain hydrogen bridges, ALOX15) and Q303 (corresponds to Q294 in
we obtained a mutant enzyme that exhibited less ALOX15) with a binding distance of 2.21 Å. On the
than 1 % residual catalytic activity (Table 2). We other hand, T560, Q294, and N287 of ALOX15 are
next replaced N287 with D. The side chain of not conserved in plant or prokaryotic LOXs. For
aspartate might act as hydrogen acceptor and thus these LOX-isoforms the hydrogen bond network
the hydrogen bridge with the backbone amide of may not be of functional importance.
V288 should have been retained. However, since
there is no amide nitrogen in the side chain, the Functional importance of T560 and Q294 in
hydrogen bridges to D415 and M418 (Fig. 1D) other mammalian LOX isoforms – If the hydrogen
should have been lost. Although the N287D mutant bond network identified for human ALOX15 is D
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exhibits a strongly reduced catalytic activity when conserved as suggested by the alignment data one n
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compared to the wild-type enzyme (Table 2) its would expect impaired catalytic activities when our ad
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reaction rate was significantly (p<0.016) higher mutagenesis scheme is applied to other LOX fro
than that of the N287L mutant. Finally, we created isoforms. To test this hypothesis we created m
h
the N287Q mutant. Asparagin (N) and glutamine (Q) corresponding mutants for human ALOX12 ttp
carry identical functional side chain residues but (platelet type 12-LOX) and mouse alox5 (5-LOX). ://w
w
differ with respect to their hydrocarbon chain length. Sequence alignments identified the target amino w
In principle, the side chain hydrogen bridges acids (T560 and Q294 for ALOX12 and T570 and .jbc
.o
formed by N287 might be retained in the N287Q Q303 for alox5, respectively) and we introduced an rg
b/
mutant despite the additional CH -group in the side M and an L at these positions. The results y
2 g
chain. When we compared the catalytic activity of summarized in Table 3 indicate that the mutant ue
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the purified N287Q mutant with that of the wild- enzyme species exhibited strongly impaired t o
n
type enzyme we found that the mutant enzyme catalytic activities and that the drop in activity was Ja
n
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exhibited only about 4 % residual activity (Table 2). more pronounced for the Q294L/Q303L exchanges ary
However, when compared with the N287D and as compared to T560M/T570M mutation. This data 24
N287L mutants the catalytic activity of the N287Q confirms the findings made for human ALOX15 for , 20
1
variant was significantly higher (Fig. 3). The two additional mammalian LOXs and suggest the 9
product pattern of the N287Q mutant was similar to functional importance of the identified hydrogen
that of the wild-type enzyme. bond network for these enzyme species.

The T560-Q294 hydrogen bridge may be The T560M mutant lacks a kinetic lag-phase
conserved in a large number of animal LOXs. In and is more susceptible for suicidal inactivation –
order to explore whether the T560-Q294 hydrogen To further explore the molecular basis for the low
bridge is peculiar for human ALOX15 we searched catalytic efficiency of the T560M mutant we
the publically available sequence databases and compared the progress curves of arachidonic acid
found that T560 of the human enzyme is strongly oxygenation (Fig. 4). We found that the wild-type
conserved in a large number of animal LOX- enzyme shows a pronounced kinetic lag-phase but
isoforms (supplement Table S1). In some cases T then the reaction rate increases continuously prior to
is exchanged for an S, which also acts as hydrogen enzyme inactivation. In contrast, no kinetic lag-
donor. In fact, our mutagenesis studies indicated phase was observed for the T560M mutant. Instead,
only a minor loss in catalytic activity for a T-to-S a continuous decline of the oxygenation rate was
exchange (Table 1). The binding partner of T560 in observed from the very beginning of the reaction
human ALOX15 (Q294) is conserved in almost and after 2 min the reaction has virtually ceased.
every LOX-isoform that contains a T or an S at the Addition of fresh substrate did not restart the
position aligning with T560 of ALOX15 reaction (Fig. 5) as indicated by HPLC analysis of
5
the reaction products and this data excludes network, which apparently stabilizes the structure of
shortage of fatty acid substrate as molecular basis this region of the protein, which lacks stable
for the impaired catalytic activity. In contrast, secondary structural elements such as helices or ß-
addition of fresh enzyme induced a further increase barrels. Moreover, via this hydrogen bond network
in product formation indicating suicidal inactivation T560 is interconnected with M418, which according
of the enzyme during fatty acid oxygenation as to the triade concept interacts with the methyl end
molecular basis for the loss in catalytic activity. of the substrate fatty acid (24,27). Thus, our data
This data suggested that the T560M mutant does suggest that exchange of a peripheral amino acid,
not exhibit a kinetic lag-phase but appears to be might induce via the hydrogen bond network
more susceptible for suicidal inactivation. conformational alterations at the catalytic center,
which are mirrored by a strongly impaired catalytic
Lack of iron incorporation is not responsible activity. More generally spoken, structural
for the reduced catalytic activity – LOXs contain alterations outside the active site might impact the
equimolar amounts of non-heme iron and iron enzyme activity if these alterations are translated to
incorporation into recombinant proteins might be a the catalytic center.
critical step when iron-containing enzymes are The question why the T560M exchange does
expressed in E. coli. To exclude that a difference in not alter the reaction specificity of the mutant
iron incorporation between wild-type and mutant enzyme was not addressed experimentally.
enzyme species is responsible for the reduced However, for alterations in the reaction specificity
catalytic activity of the enzyme species we the spatial properties of the amino acid side-chain D
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compared the iron content of wild-type ALOX15, are important since introduction of less space-filling n
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its T560M and N287D mutant. We quantified an residues at this position provide more space for ad
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iron load of 80.9±0.04%, 76.8±0.09% and deeper penetration of the substrate fatty acid into fro
80.4±0.03% for wild-type ALOX15, T560M and the substrate binding pocket (24, 27). In contrast, in m
h
N287D, respectively. This data indicates an 80% the wild-type enzyme the hydrogen bond network ttp
iron load of the recombinant enzyme and strongly interconnects T560 with the peptide backbone of ://w
w
suggest that the loss in catalytic activity may not be M418, which does not alter the spatial properties of w
related to impaired iron incorporation. this amino acid. Thus, the structural alterations .jbc
.o
induced by T560M and M418V(A) exchange are rg
b/
different and thus alterations in the positional y
g
Discussion specificity cannot necessarily be expected as ue
s
The role of ALOX15 in atherogenesis has functional consequence of T560M exchange. We t o
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been a matter of discussion for many years and pro- hypothesize that T560M exchange might impact the Ja
n
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and anti-atherogenic effects have been reported (9- alignment of fatty acid substrate at the active site in ary
16). Recent population-based case-control studies such a way that the rate of hydrogen abstraction 24
yielded evidence for an anti-atherogenic effect of from C13 is impaired but that the stereochemistry , 20
1
the enzyme (19). The molecular basis for this effect of oxygen insertion is hardly altered. It may be of 9
remains unclear but involvement of ALOX15 in interest in this context that the M418A mutant of
vasodilatation was discussed as a possible reason in the rabbit 12/15-LOX only exhibits 30% residual
one of these studies (20). However, since the catalytic activity (28).
T560M SNP is very rare in Caucasians (less than Detailed sequence comparison (supplement
1% heterozygous allele carriers) both studies were Table S1) suggests that this hydrogen bond
underpowered to draw definite conclusions on the network might be conserved in animal LOXs and
patho-physiological relevance of this SNP in our findings indicate that disruption of this network
myocardial infarction and/or coronary artery (mutations at T560 or Q294) induces a loss in
disease. enzymatic activity of ALOX15, ALOX12 and
Nevertheless, the T560M exchange occurs in alox5. Thus, our data suggest that this structural
vivo and its lacking catalytic activity was quite element is of functional importance for these and
surprising since the T-to-M exchange should not probably for other animal LOX isoforms.
directly affect the active site. Inspecting the 3D- It is interesting to note that T560M exchange
structure of the rabbit ALOX15, which shares 81% induced less severe functional consequences (partial
identity with the human enzyme, we found that the inactivation) than mutations at Q294 and this was
side chain hydroxy group of T560 hydrogen bridges the case for all three LOX isoforms tested. These
with the side chain oxygen of Q294. This hydrogen differences may become plausible if one considers
bridge is part of a more complex hydrogen bond the fact that T560 only forms a single hydrogen
6
bond within the hydrogen bridge network. In (35). However, when this methionine was
contrast, Q294 is involved in multiple hydrogen exchanged by site-directed mutagenesis to a residue
bonds (see figure 1 D) and thus, the structural that cannot be oxidized the enzyme still underwent
alterations induced by mutations of Q294 are suicidal inactivation. This data indicates that
expected to be more severe. methionine oxidation may not be a crucial process
When we characterized the catalytic activity in suicidal inactivation (36). Although the
of the T560M mutant in more detail we noticed that molecular basis of suicidal inactivation still remains
the difference in the reaction rate between wild-type unclear the T560M mutant of the human ALOX15
and mutant enzyme strongly depended on the might constitute a suitable model for further
duration of the incubation period (Fig. 4), investigations into this mechanism.
suggesting that the two enzyme species exhibited
different reaction kinetics. In general, kinetics of the
LOX reaction are characterized by two
peculiarities: i) Kinetic lag-phase (29,30) and ii)
suicidal enzyme inactivation (31). When we
recorded the kinetic progress curves for human
wild-type ALOX15 and its T560M mutant (Fig. 4)
we found that the wild-type enzyme follows the
usual reaction kinetics but that the T560M mutant
lacks the kinetic lag-phase. In fact, the reaction D
o
w
starts with maximal rate but the enzyme quickly n
lo
undergoes inactivation. These kinetic peculiarities ad
e
d
appear to be related to the hydrogen bond network fro
involving T560, Q294, and N287, which thus might m
h
play a role in the activation/inactivation processes. ttp
The detailed molecular basis, however, remains to ://w
w
be explored. w
The kinetic lag-phase has previously been .jbc
.o
related to oxidation of the non-heme iron from its rg
b/
silent ferrous state to its catalytically active ferric y
g
form (29,30). However, although oxidation of the ue
s
non-heme iron is involved in the activation process t o
n
more recent kinetic studies on the rabbit ALOX15 Ja
n
u
suggested an oxygen dependence of the activation ary
process (32). Although the detailed mechanism of 24
enzyme activation still remains elusive oxygen , 20
1
dependence has recently been confirmed 9
experimentally for another mammalian LOX
(33,34).
Some LOX-isoforms undergo suicidal
inactivation during fatty acid oxygenation
(31,35,36) and we confirmed this kinetic peculiarity
for both, recombinant wild-type human ALOX15
and its T560M mutant. However, the inactivation
rate of the mutant enzyme appears to be much faster
(Fig. 4) since the enzyme was completely
inactivated after 1-2 min. More detailed studies on
the molecular basis for this difference are difficult
since the principle mechanism of suicidal
inactivation of LOXs or other enzymes of the
arachidonic acid cascade such as cyclooxygenase
isoforms has not been clarified. Originally, it has
been suggested for LOXs that peroxide induced
oxidation of a single methionine residue at the
active site is the critical step in suicidal inactivation
7
Acknowledgement
Financial support for this study was provided by Deutsche Forschungsgemeinschaft (GRK1673).

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7069-7079

9
LEGENDS to SCHEMES and FIGURES

Figure 1 Localization of T560 in the 3D-structure of ALOX15 and its involvement in a network of
hydrogen bonds. A) T560 is not an immediate active site residue. Although it is located in proximity to
I417 there is no direct interaction to any of the triade constituents (F352, I417, M418, I592, numbering
according to human ALOX15). The shortest distance (4.12 Å) was measured between the backbone
carbonyl of I417 and the backbone nitrogen of T560, which is beyond the binding distance of non-
covalent interactions. B) The side chain OH-group of T560 hydrogen bridges with side chain oxygen of
Q294. This hydrogen bridge might contribute to stabilize the less well structured loop regions indicated in
green and light red. C) The loop regions surrounding T560 and Q294 do not undergo major structural
rearrangement upon ligand binding at the active site. In fact, the bonding distance of the T560-Q294
hydrogen bridge, which is 2.62 Å in the ligand-free structure, only increases to 2.72 Å in the ligand-
bound form. As indicated before, there is a pronounced relocation of helix 2 during ligand binding (grey –
ligand-free conformer, light blue – ligand-bound conformer, orange – arachidonic acid). D) Q294 is a key
residue in a hydrogen bond network, which connects the side chain of T560 with M418, a member of the
amino acid triade, which determines the positional specificity of mammalian 12/15-LOXs. Images were
prepared with VMD software package (26). Hydrogen bridges were determined with Swiss-PdbViewer,
v4.0.1.

D
o
w
Figure 2 T560M and Q294L exchanges inactivate human ALOX15. Wild-type and mutant enzymes n
lo
were expressed and purified as described in materials and methods. LOX concentrations of each a
d
e
pasresapyasra. tRioenp rweseernet aqtuivaen teifxiaemd pblye icmhmroumnaot-obglroatmtinsg a raen dsh eoqwuna.l Tamheo uyn-atsx ios fw eansz yscmaele wd eforer tuhsee dp riond tuhcet aacmtiovuitnyt d from
h
formed by the wild-type enzyme. In panel A the moblie phase of the HPLC system consisted of 80% ttp
methanol, 20% water, and 0.05% acetic acid resulting in longer retention times. Fort panel B the solvent ://w
w
composition was 85% methanol, 15% water, and 0.05% acetic acid as described in Materials and w
Methods. .jb
c
.o
rg
by/
g
Figure 3 Catalytic activity of various ALOX15 Q287 mutants. Wild-type and mutant enzymes were u
e
s
expressed and purified as described in materials and methods. LOX concentrations of each preparation t o
n
were quantified by immuno-blotting and equal amounts of enzyme were used in the activity assays. The Ja
n
Y-axis was normalized to the product amount formed by the N287Q mutant. Note that in comparison to ua
ry
table 2 the amount of enzyme used was increased threefold. 2
4
, 2
0
1
9
Figure 4 Time dependence of arachidonic acid oxygenation by purified wild-type human ALOX15
and its T560M mutant. Wild-type and T560M mutant were expressed and purified as described in
materials and methods. LOX concentrations of each preparation were quantified by immuno-blotting and
in in this experiment, the amount of T560M used was fivefold higher than the wild-type. Activity assays
were carried out as described and the reaction was stopped by the addition of 500 µl ice cold methanol.
After reduction of hydroperoxides and acidification samples were centrifuged and aliquots of the
supernatant used for HPLC analysis.

Figure 5 Suicidal inactivation of the T560M mutant of ALOX15 during arachidonic acid
oxygenation. The T560M mutant was expressed and purified as described in Materials and Methods.
Activity assays were carried out as described and the reaction was stopped after 4 min of incubation by
the addition of 500 µl ice cold methanol. Additional samples were further incubated for 4 min with no
additives, with fresh arachidonic acid or with fresh enzyme. After reduction of the hydroperoxides and
10