Phospholipases a1 from armillaria ostoyae provide insight into the substrate recognition of a/b-hydrolase fold enzymes

Article abstract of DOI:10.1007/s11746-012-2050-x

Four enzymes with phospholipase A¹(PLA¹) activity were purified from the fruiting bodies of the basidiomycete Armillaria ostoyae. The enzymes (PLA1-1, -2, -3 and -4) showed similar isoelectric points (4.3, 3.9, 4.0 and 4.0) and apparent molecular masses in the range of 35-47 kDa. Mass spectrometric analyses of proteolytic fragments revealed sequences homologous to a/b-hydrolase fold enzymes. The enzymes share one conserved region with fungal phospholipases B and the active site sequence with bacterial esterases and PLA¹s. PLA¹-1 cleaves phospholipids and lysophospholipids with an optimum activity at pH 5.3. In contrast, PLA1-2, -3 and -4 are characterized by broad pH optima in the slightly acidic to neutral range and are additionally capable of hydrolyzing mono- and diglycerides as well as fatty acid methyl esters. All enzymes favor glycerol-based lipids with a single medium-sized fatty acid moiety in the sn-1 position but show reduced activity towards the corresponding 1, 2-diacyl derivatives with bulky long-chain or inflexible saturated fatty acid moieties in the sn-2 position. The enzymes prefer zwitterionic phospholipid substrates and are unable to hydrolyze triglycerides. From the selectivity of these broad-spectrum a/b-hydrolase fold enzymes towards the different classes of their substrates a regiospecific steric hindrance and a head group recognition are concluded.

Full text of DOI:10.1007/s11746-012-2050-x

J Am Oil Chem Soc (2012) 89:1435–1448
DOI 10.1007/s11746-012-2050-x
ORIGINAL PAPER
Phospholipases A from Armillaria ostoyae Provide Insight
1
into the Substrate Recognition of a/b-Hydrolase Fold Enzymes
Martin Dippe
Andrea Sinz
•
Mathias Q. M u¨ ller
Renate Ulbrich-Hofmann
•
•
Received: 16 August 2011 / Revised: 15 January 2012 / Accepted: 2 March 2012 / Published online: 18 March 2012
Ó AOCS 2012
Abstract Four enzymes with phospholipase A (PLA )
1
saturated fatty acid moieties in the sn-2 position. The
1
activity were purified from the fruiting bodies of the
enzymes prefer zwitterionic phospholipid substrates and
are unable to hydrolyze triglycerides. From the selectivity
of these broad-spectrum a/b-hydrolase fold enzymes
towards the different classes of their substrates a regio-
specific steric hindrance and a head group recognition are
concluded.
basidiomycete Armillaria ostoyae. The enzymes (PLA -1,
1
-
2, -3 and -4) showed similar isoelectric points (4.3, 3.9, 4.0
and 4.0) and apparent molecular masses in the range of
5–47 kDa. Mass spectrometric analyses of proteolytic
3
fragments revealed sequences homologous to a/b-hydro-
lase fold enzymes. The enzymes share one conserved
region with fungal phospholipases B and the active site
sequence with bacterial esterases and PLA s. PLA -1
Keywords Armillaria ostoyae ꢀ Fatty acid specificity ꢀ
a/b-Hydrolase fold enzyme ꢀ Phospholipase A ꢀ
1
1
1
cleaves phospholipids and lysophospholipids with an
Substrate selectivity
optimum activity at pH 5.3. In contrast, PLA -2, -3 and -4
1
are characterized by broad pH optima in the slightly acidic
to neutral range and are additionally capable of hydrolyz-
ing mono- and diglycerides as well as fatty acid methyl
esters. All enzymes favor glycerol-based lipids with a
single medium-sized fatty acid moiety in the sn-1 position
but show reduced activity towards the corresponding
Abbreviations
CID
Collision-induced dissociation
1,2-dipalmitoyl-sn-glycero-3-
phosphocholine
DPPC
DOPC
DSPC
EDTA
EGTA
HEPES
1,2-dioleoyl-sn-glycero-3-phosphocholine
1,2-distearoyl-sn-glycero-3-phosphocholine
Ethylenediaminetetraacetic acid
Ethylene glycol tetraacetic acid
1
,2-diacyl derivatives with bulky long-chain or inflexible
0
N-(2-hydroxyethyl)-piperazine-N -
(2-ethanesulfonic acid)
Data from mass-spectrometric peptide sequencing are available in the
PRIDE database (http://www.ebi.ac.uk/pride/) under the accession
numbers 17662–17665.
LPPC
LTQ
MES
PA
1-palmitoyl-sn-glycero-3-phosphocholine
Linear quadrupole ion trap
2-Morpholinoethanesulfonic acid
Phosphatidic acid
Electronic supplementary material The online version of this
article (doi:10.1007/s11746-012-2050-x) contains supplementary
material, which is available to authorized users.
PC
Phosphatidylcholine
M. Dippe (&) ꢀ R. Ulbrich-Hofmann
Institute of Biochemistry and Biotechnology,
Martin-Luther University Halle-Wittenberg,
Kurt-Mothes-Str. 3, 06120 Halle, Germany
e-mail: martindippe@gmx.de
PE
Phosphatidylethanolamine
Phosphatidylglycerol
PG
PI
Phosphatidylinositol
0
PIPES
PLA1
PLA2
PLB
PMSF
Piperazine-N,N -bis(ethanesulfonic acid)
Phospholipase A1
M. Q. M u¨ ller ꢀ A. Sinz
Phospholipase A2
Institute of Pharmacy, Martin-Luther University
Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4,
Phospholipase B
0
6120 Halle, Germany
Phenylmethanesulfonyl fluoride
1
23

1
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J Am Oil Chem Soc (2012) 89:1435–1448
in some enzymes the selectivity is determined by only a
few amino acid positions as shown for the esterase from
Bacillus subtilis. The enzyme can be converted to a mon-
oacylglycerol lipase by a single amino acid exchange [11].
PNGase F
POPC
Peptide-N-glycosidase F
1-Palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine
PS
Phosphatidylserine
A variant of the Staphylococcus hyicus PLA created by
1
SDS
Sodium dodecyl sulfate
introduction of defined amino acid residues of the closely
related triglyceride lipase from Staphylococcus aureus is
characterized by an increased activity towards triglycerides
and, vice versa, a strongly reduced affinity for phospho-
lipids [12]. However, additional regulatory elements such
as the so-called lid segment may also contribute to sub-
strate selectivity [13].
SDS-PAGE SDS-polyacrylamide gel electrophoresis
U
Unit
Introduction
Acyl ester hydrolases (EC 3.1.1) are ubiquitous enzymes
catalyzing the hydrolytic cleavage of carboxylic acid esters.
They are involved in many physiological processes from
digestion to signal transduction [1, 2]. Due to their impor-
tance in preparative chemistry and in industrial processes,
many acyl ester hydrolases have been isolated from micro-
bial sources such as bacteria and filamentous fungi [3]. Most
of these enzymes are characterized by a catalytic serine
nucleophile and a typical a/b-fold [4]. Despite these mech-
anistic and topological similarities, acyl ester hydrolases
differ widely in their substrate utilization. With respect to
their lipid substrates, esterases, mono-, di-, and triglyceride
lipases, phospholipases A (PLA) and lysophospholipases are
distinguished. The phospholipases A are further subdivided
into phospholipases A (PLA s, EC 3.1.1.32) and phospho-
Recently, PLA1 activity has been found in fruiting
bodies of the basidiomycete Armillaria ostoyae, which was
shown to be responsible for phospholipid degradation
during sporogenesis [14]. In this paper we report on the
purification and characterization of this enzymatic activity
yielding four new enzymes with PLA1 activity but
uncommon substrate selectivity. The enzymes show simi-
larities to both fungal PLB and microbial and plant PLA1s.
While one of these PLA s is specific for phospholipids, the
1
three other enzymes are also capable of converting neutral
lipids. From kinetic analyses, a strong dependence of the
activity on the chain length and saturation of fatty acids
and on the head group of phospholipids is concluded.
Based on the results, a model for the control of substrate
selectivity is proposed.
1
1
lipases A (PLA s, EC 3.1.1.4) according to their regio-
2
2
specificity in the cleavage of the sn-1 or sn-2 acyl moieties in
glycerophospholipids. Enzymes which act on both positions
are called phospholipases B (PLB, EC 3.1.1.5). The assign-
ment of phospholipases to PLB, however, must be consid-
ered with caution because they are difficult to differentiate
Experimental Procedures
Materials
from PLA s due to the acyl migration from the sn-2 to the
1
Phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylglycerol (PG) and phosphatidic acid (PA), all
from soybean, and 1,2-dioleoyl-sn-glycero-3-phosphocho-
line (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine
(DSPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-
choline (POPC) were products from Lipoid (Ludwigshafen,
Germany). Sphingomyelin from bovine brain was obtained
from Enzo (Farmingdale, USA). 2-Lysophospholipids and
1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine were
received from AvantiPolar (Alabaster, USA). 1-Palmitoyl-
2(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine was from
Invitrogen (Carlsbad, USA) and Triton X-100 from Appli-
chem (Darmstadt, Germany). Sequencing grade trypsin was
purchased from Roche Diagnostics (Mannheim, Germany).
All other phospholipids, fatty acids, fatty acid esters, enzyme
inhibitors and peptide-N-glycosidase F (PNGase F) from
Elizabethkingia meningoseptica were from Sigma (St. Louis,
USA). Soybean fatty acids were prepared from PC as
described elsewhere [14]. All other chemicals were of the
highest purity commercially available.
sn-1 position in 1-lysophospholipids.
However, a great number of enzymes cannot be clearly
assigned to one or other of the groups because they hydrolyze
a broad range of substrates. For example, the calcium-inde-
pendent cytosolic PLA s catalyze the hydrolysis of sn-1-
2
bound fatty acid moieties in phospholipids if no acyl moiety
at the sn-2-position is present and they are also active
towards lysophospholipids [5]. Endothelial lipases acting on
triglycerides also cleave phospholipids [6]. The lipolytic
enzyme from guinea pig pancreas shows PLA - [7] but also
1
mono-, di- and triglyceride lipase activity [8]. Additionally,
it converts galactolipids [9]. Moreover, many enzymes that
hydrolyze lysophospholipids function additionally as acyl-
transferases by transferring the fatty acid moiety of one
lysophospholipid molecule to another, an activity exhibited
by lysophospholipase from rabbit lung [10].
Extensive mutational studies on acyl ester hydrolases
have shed light on the molecular basis of the multifaceted
selectivity towards different classes of lipids. Interestingly,
1
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J Am Oil Chem Soc (2012) 89:1435–1448
Preparation of Crude Extracts
1437
against 20 mM sodium acetate buffer, pH 6.5, containing
1
mM EDTA.
Fruiting bodies from A. ostoyae were collected from
infected spruce trees (Picea abies) in the Harz mountains,
Germany. After grinding in liquid nitrogen, the resulting
powder was suspended in 20 mM sodium acetate buffer,
pH 6.5, containing 1 mM ascorbic acid, 1 mM ethylene-
For purification of PLA -1 under high salt conditions,
1
the procedure was modified by addition of 200 mM NaCl
to the buffers used for washing and elution.
Determination of PLA Activity During Purification
1
-
1
diaminetetraacetic acid (EDTA) (2 l kg of fruiting bod-
ies). The suspension was homogenized with a hand blender
and centrifuged for 20 min at 16,0009g. For the removal
of spores, the supernatant was subjected to one freeze–thaw
cycle with subsequent centrifugation (20 min, 27,0009g).
Reactions (20 ll) contained 12.5 mM PC from soybean,
5 mM sodium dodecyl sulfate (SDS) and 25 mM Triton
X-100 in 100 mM sodium acetate buffer, pH 5.6. Due to the
2?
absence of Ca -inducible phospholipase activity in extracts
of Armillaria ostoyae, this ion was not included in the assay.
Residual EDTA deriving from the buffers used in enzyme
purificationdidnotcauseanydisturbance. Afterincubationin
microplates for 0–90 min under shaking (400 rpm) at 30 °C,
fatty acid concentrations were determined by the NEFA C kit
according to Hoffmann et al. [15] using a standard curve of
soybean fatty acids. Enzyme activity was obtained from the
linear fit of the fatty acid concentration as a function of the
reaction time by the software SigmaPlot 11.0 (Systat Soft-
ware, USA). One unit (U) represents the amount of enzyme
releasing 1 lmol of fatty acid per minute.
Hydrophobic Interaction Chromatography
Ammonium sulfate (4 M) was added to 450 ml of crude extract
to yield a final concentration of 500 mM. The extract was
applied to 20 ml of Phenyl Sepharose 6 Fast Flow (high sub-
stitution, GE Healthcare, Little Chalfont, United Kingdom) on
¨
an Akta FPLC system (GE Healthcare), and proteins were
eluted with 20 mM sodium acetate buffer, pH 6.5, containing
1
mM EDTA and subsequently with a gradient of the same
buffer containing 0–20 % (v/v) acetonitrile. According to the
profile of eluted PLA activity, fractions were combined to three
1
main fractions (I–III), which were dialyzed against 20 mM
sodium acetate buffer, pH 6.5, containing 1 mM EDTA.
Protein Determination
For crude extracts, the method by Bradford [16] was used
because of the presence of ascorbic acid. In all other cases,
protein concentration was determined by the bicinchoninic
acid kit from Pierce (Rockford, USA), according to the
manufacturer’s instructions. Protein concentrations were
calculated using standard curves of bovine serum albumin.
Ion Exchange Chromatography
Anion exchange chromatography was performed on Source
1
5Q matrix (GE Healthcare). Samples obtained from
hydrophobic interaction chromatography (Supporting infor-
mation, Fig. S1) were loaded onto 3.6 ml of matrix material
andtheproteins were elutedbya gradient of 0–500 mMNaCl
in 20 mM sodium acetate, pH 6.5, containing 1 mM EDTA.
Samples obtained from hydroxyapatite chromatography
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Electrophoresis was performed in 12.5 % (w/v) poly-
acrylamide gels according to Laemmli [17]. The gels were
stained with silver [18] or with Coomassie Brilliant Blue
G250 [19]. Apparent molecular masses were estimated
from two independent experiments according to [20] using
molecular markers from Fermentas (Burlington, Canada).
(
Fig. S1) were applied to 1.5 ml of matrix material and the
proteins were eluted by a gradient of 0–300 mM NaCl in
0 mM sodium acetate buffer, pH 5.0, containing 1 mM
2
EDTA. In both cases, fractions containing PLA activity
1
were dialyzed against 20 mM sodium acetate buffer, pH
6
.5, containing 1 mM EDTA.
Isoelectric Point (pI) Determination
Hydroxyapatite Chromatography
Proteins were focused in a PhastGel IEF 3-9 applying
the Phast Electrophoresis System (GE Healthcare, Little
Chalfont, UK). pI values were estimated using molecular
markers (pI 3–10) from Serva (Heidelberg, Germany).
Preparations from ion exchange chromatography were sup-
0
plemented with 10 mM piperazine-N,N -bis(ethanesulfonic
acid) (PIPES)/NaOH, pH 6.5, and 3 mM MgCl2 and
applied to 3.5 ml of hydroxyapatite BioGel HTP (Bio-Rad,
Hercules, USA). The matrix was washed with 10 mM
PIPES/NaOH, pH 6.5, and proteins were eluted by a
gradient from 0 to 150 mM sodium phosphate buffer, pH
Deglycosylation
Enzymes were digested with PNGase F (80 U per mg of
6
.5. Fractions with PLA activity were pooled and dialyzed
1
PLA protein) according to the protocol of the supplier.
1
1
23

1
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J Am Oil Chem Soc (2012) 89:1435–1448
Samples of the digested proteins were analyzed by SDS-
PAGE as described above.
Kinetic Measurements
Reactions were performed in microplates by mixing 20 ll
of substrate solution (0.1–20 mM phospholipid, acylglyc-
erol or fatty acid methyl ester, and Triton X-100 in
the molar ratio of 1:2) with 20 ll of purified enzyme
(0.1 mU in 100 mM 2-morpholinoethanesulfonic acid
(MES)/NaOH, pH 5.6). After incubation for 0–90 min
under shaking (400 rpm) at 30 °C, 10 ll of 500 mM
Nano-HPLC/Nano-ESI-LTQ-Orbitrap Mass
Spectrometry
The purified enzymes (4 lg each) were applied to SDS-
PAGE as described above and stained by Coomassie
Brilliant Blue G250. PLA bands were excised from the gel
1
0
and treated with PNGase F according to the manufacturer’s
instruction. After reduction with 10 mM dithiothreitol and
carbamidomethylation with 55 mM iodoacetamide, the
proteins were in-gel digested with 0.2 lg of trypsin over-
night [21]. For mass spectrometric analyses according to
N-(2-hydroxyethyl)-piperazine-N -(2-ethanesulfonic acid)
(HEPES)/NaOH, pH 7.0, were added to adjust the reaction
mixture to neutral pH. The release of free fatty acids was
determined by the NEFA C kit as described in the previous
section using standard curves of the corresponding fatty
acids. From the initial rates as a function of the substrate
concentration, the maximum rate (Vmax), the substrate
concentration at half-maximum saturation (S0.5) and the
Hill coefficient h were calculated by the Hill equation using
the software SigmaPlot 11.0 (Systat Software, USA). All
experiments were performed in triplicate.
[
22], the proteolytic peptide mixtures were fractionized by
reversed-phase chromatography on an Ultimate nano-
HPLC system (Dionex, Idstein, Germany), including a
concentration and desalting step (C18 precolumn: Acclaim
˚
PepMap, 300 lm 9 5 mm, 5 lm, 100 A) before separa-
tion (C18 separation column: Acclaim, 75 lm 9 250 mm,
˚
3
lm, 300 A). The tryptic peptides were eluted using a
0-min linear gradient of 5–80 % (v/v) acetonitrile in 0.1%
9
pH Dependence
-
1
formic acid at a flow rate of 300 nl min , followed by
washing with 80 % acetonitrile (15 min). ESI–MS/MS
analysis was performed in positive ionization mode using a
LTQ-Orbitrap XL mass spectrometer (ThermoFisher Sci-
entific, Bremen, Germany), which was directly coupled to
the nano-HPLC system via a nano-ESI source (Proxeon,
Odense, Denmark). Each high-resolution mass spectrum
PLA activity was determined towards mixed micelles
1
consisting of 5 mM 1,2-dipalmitoyl-sn-glycero-3-phos-
phocholine (DPPC) and 10 mM Triton X-100 as described
in the previous section using the following buffers: 50 mM
sodium formate buffer (pH 3.0), sodium acetate buffer (pH
3.5–5.3), MES/NaOH (pH 5.6–6.75), HEPES/NaOH (pH
7.0–7.5) or Tris/HCl (pH 8.0–9.0). The ionic strength was
adjusted with NaCl to 50 mM. The experiments were
performed in triplicate.
(
m/z 300–2,000, R = 60,000) was followed by collision-
induced dissociation tandem mass spectrometry (CID–MS/
MS) of the five most intense signals in the linear ion trap
(
(
LTQ). Fragment ions were either analyzed in the orbitrap
R = 7,500) or in the LTQ. For data acquisition, the soft-
Inhibitor Studies
ware XCalibur 2.0.7 (ThermoFisher) was combined with
DCMS link 2.0 (Dionex).
-
1
Purified enzyme (10 mU ml in 20 mM HEPES/NaOH, pH
7.0) was incubated with 1 mM of phenylmethanesulfonyl
fluoride (PMSF), iodoacetamide, 4-bromophenacyl bromide
or 5 mM ethylene glycol tetraacetic acid (EGTA) at 25 °C.
After 12 h the remaining enzyme activity was assayed
towards 5 mM DPPC/10 mM Triton X-100 at pH 5.6 as
described above. Samples without inhibitor treatment were
used as references. The experiments were performed in
triplicate.
Amino acid sequences were identified by Mascot MS/MS
ion search [23]. Sequences showing significant similarity to
GXSXG-lipasepatterns, whicharetypicalfortheactivesiteof
serine hydrolases [24], were identified using the BLAST tool
[25]. Furthermore, highly conserved PLB regions were used
as templates for the alignment. These regions were identified
by comparing amino acid sequences of the six fungal PLB
from Cryptococcus neoformans var. neoformans (GenBank
ID: AAF61964.1), Candida albicans (GenBank ID:
XP_713822.1), Neurospora crassa(GenBankID: O42790.2),
Penicillium chrysogenum (GenBank ID: CAA42906.1),
Saccharomyces cerevisiae (GenBank ID: CAA88523.1) and
Aspergillus fumigatus (GenBank ID: Q9P8P4.2), corre-
sponding to the motifs Y40-S49, P96-T112, S140-A151,
A257-W283, F323-L337, L390-L406, W428-R440, F463-
N478, P483-Y502, W531-S543 and C555-G566 in the PLB
from Cryptococcus neoformans var. neoformans.
Esterase Assay with 4-Nitrophenyl Acetate
Hydrolysis of 4-nitrophenyl acetate (2 mM in 50 mM MES/
NaOH, pH 5.6, 10 mM Triton X-100) by the enzymes
-1
(7.5 mU ml ) was analyzed in glass cuvettes at 30 °C by
continuous determination of the absorbance at 360 nm. The
concentration of the product 4-nitrophenol was calculated
on the basis of an experimentally determined extinction
1
23

J Am Oil Chem Soc (2012) 89:1435–1448
1439
3
-1
-1
coefficient (e = 2.43 9 10 M cm ). The experiments
shown recently to contain high PLA activity [14], there-
1
0
were performed in duplicate.
fore this species was chosen for protein purification and
characterization.
Fluorimetric Determination of Regiospecificity
During the purification of the protein(s) showing PLA1
activity, surprisingly, four enzymes could be distinguished.
The purification scheme comprised hydrophobic interac-
tion chromatography on Phenyl Sepharose, anion exchange
chromatography at varying pH on Source 15Q, and
hydroxyapatite chromatography (Supporting information,
Fig. S1). In the initial step of purification on Phenyl
Sepharose, the activity could be partitioned into three
distinct peaks representing enzymes with different hydro-
phobicity (Fig. 1a). The three active fractions (corre-
sponding to the elution volumes 18–34, 40–58, and
Hydrolysis of 1-palmitoyl-2(1-pyrenedecanoyl)-sn-glycero-
3
-phosphocholine was assayed according to the method of
Radvanyi et al. [26].
Results
Purification of PLA Enzymes from A. ostoyae Fruiting
1
Bodies
80–120 ml in Fig. 1a) were separately subjected to three
In contrast to many enzymes produced by filamentous
fungi [27], there is little information about acyl ester
hydrolases from basidiomycetes. Of several basidiomyce-
tes tested, the fruiting bodies of the species A. ostoyae were
further chromatographic steps (anion exchange at pH 6.5,
hydroxyapatite, anion exchange at pH 5.0), yielding four
enzymes called PLA -1, PLA -2, PLA -3 and PLA -4.
1
1
1
1
Interestingly, PLA -3 and PLA -4 co-eluted during the first
1
1
Fig. 1 Elution profiles of
PLA s from A. ostoyae. a Three
1
active fractions in hydrophobic
interaction chromatography.
The crude extract was applied to
Phenyl Sepharose as described
in ‘‘Experimental Procedures’’.
The active peaks represent
PLA
PLA
1
-1 (I), PLA
-3 ? PLA -4 (III).
-3 and
-4 by anion exchange
1
-2 (II), and
1
1
b Separation of PLA
PLA
1
1
chromatography on Source 15Q
at pH 5.0. Peak III of the initial
hydrophobic interaction
chromatography (Fig. 1a),
which was further purified on
Source 15 Q at pH 6.5 and
hydroxyapatite as described in
the ‘‘Experimental Procedures’’
section, was applied to Source
1
PLA activity towards soybean
5 Q at pH 5.0. Filled circles
1
PC/Triton X-100/SDS; solid
lines absorbance at 280 nm;
dashed lines acetonitrile or
NaCl concentration of the eluent
1
23

1
440
J Am Oil Chem Soc (2012) 89:1435–1448
three steps of purification, but could be separated by anion
exchange at pH 5.0 (Fig. 1b). Therefore, these two proteins
seem to be very similar in their molecular properties. In the
case of PLA -2, -3 and -4, the proteins were homogeneous
1
in SDS-PAGE after the anion exchange step at pH 5.0,
whereas for PLA -1 an additional hydroxyapatite chro-
1
matography step at high salt conditions was necessary to
obtain pure protein (Fig. 2a–c). The data of the purifica-
tions are summarized in Table 1. From 1 kg of fruiting
bodies, 11 (PLA -1), 25 (PLA -2), 171 (PLA -3) and 139
1
1
1
(
PLA -4) lg of pure protein were obtained. The purifica-
1
tion factors were 5,650- (PLA -1), 2,610- (PLA -2), 1,250-
1
1
(
PLA -3) and 2,400 (PLA -4).
1
1
Molecular Masses, Isoelectric Points and Glycosylation
of the PLA1s
The molecular masses of PLA -1, PLA -2, PLA -3 and
1
1
1
PLA -4 derived from SDS-PAGE (Fig. 2) were
1
4
6.7 ± 0.5, 32.9 ± 0.9, 39.2 ± 0.6 and 35.4 ± 0.4 kDa.
Upon treatment with PNGase F (Supporting information,
Fig. S2), which cleaves the bond between asparagine and
glucosamine residues in glycoproteins [28], the electro-
phoretic mobilities of PLA -2, PLA -3 and PLA -4
1
1
1
revealed lower apparent molecular masses of 30.1 ± 0.4,
5.2 ± 0.5 and 33.0 ± 0.8 kDa. Therefore, these three
3
proteins seem to be glycosylated. In contrast, no significant
difference indicating an asparagine-linked carbohydrate
moiety was detected for PNGase F-treated PLA -1
1
(
46.9 ± 0.6 kDa). The pI values of the proteins were 4.3,
3
.9, 4.0, and 4.0 for PLA -1, PLA -2, PLA -3, and PLA -4,
1 1 1 1
respectively.
Mass Spectrometry-Based Amino Acid Sequence
Alignment
To obtain initial information on the similarity of the new
PLA s to characterized acyl ester hydrolases, the PLA s
Fig. 2 SDS-PAGE of PLA
Silver-stained SDS-PAGE gels of enzyme fractions of the various
purification steps yielding PLA -1 (a), PLA -2 (b), PLA -3 and
PLA -4 (c) were produced as described in ‘‘Experimental Proce-
dures’’. The lanes represent the molecular markers with molecular
masses as indicated in the figure (lane 1), the crude extract (lane 2),
the fractions I (a), II (b), and III (c) after hydrophobic interaction
chromatography corresponding to Fig. 1a (lane 3), the corresponding
active enzyme fractions after anion exchange at pH 6.5 (lane 4),
hydroxyapatite chromatography (lane 5), anion exchange at pH 5.0
1
s from A. ostoyae during purification.
1
1
were subjected to proteolytic digestion by trypsin. The
resulting peptides were analyzed by MS and MS/MS and
compared to amino acid sequences of known proteins. For
1
1
1
1
each PLA , 10 peptides were identified on average. The
1
PLA peptides identified showed high similarity to amino
1
acid sequences of serine hydrolases. For example, the
fragment GRAPGETTVPDR, which is present in all four
(
(
lane 6), and hydroxyapatite purification under high-salt condition
lane 7). PLA -3 and PLA -4, which co-eluted in the first three steps
PLA s is partially conserved in fungal PLBs (Fig. 3) but
1
1
1
cannot be found in other PLA s, bacterial esterases or
1
of purification and were separated only by anion exchange at pH 5.0
(Fig. 1b), are shown in lanes 6a and 6b in c
fungal triglyceride lipases. On the other hand, PLA -1,
1
PLA -3 and PLA -4 strongly differ from common fungal
1
1
PLB, which possess an A/SGLSGGXW consensus core. In
contrast, the PLA enzymes are characterized by amino
1
(Fig. 3). According to its AXSXG motif, PLA
displays similarity to yeast lipase 2 and PLA from maize
(Fig. 3) and to the lipase from Bacillus subtilis [29].
1
-1 also
acid sequences, which are highly similar to the catalytic
motifs of bacterial esterases and lysophospholipases
1
1
23

J Am Oil Chem Soc (2012) 89:1435–1448
1441
1
Table 1 Purification of PLA enzymes from A. ostoyae fruiting bodies
-
1
-1
Purification step
Specific activity (lmol min mg )/activity yield (%)
PLA -1 PLA -2
1
1
PLA
1
-3
1
PLA -4
Phenyl Sepharose
0.01/14.0
0.12/10.4
1.04/9.4
50.5/2.5
56.5/2.1
0.07/9.0
0.24/5.2
3.64/4.8
26.1/2.2
0.27/75.0
1.17/70.6
11.8/45.2
12.5/7.1
Source 15Q, pH 6.5
Hydroxyapatite
Source 15Q, pH 5.0
Hydroxyapatite (high salt)
24.0/11.0
Specific activities towards PC/Triton X-100/SDS were determined according to ‘‘Experimental Procedures’’. The activity yield was related to the
-1
-1
-1
l ). The specific activity of the crude extract was 0.01 lmol min mg
-1
activity of the crude extract (10.6 lmol min
Fig. 3 Alignment of PLA
Sequences of peptides obtained by digestion of A. ostoyae PLA
trypsin were identified by tandem mass spectrometry according to
‘Experimental Procedures’’. The deviation of the experimental
Mrexp) from the theoretical masses (Mrtheo) is given in ppm.
Underlined methionine residues were oxidized. PLA fragments
1
fragments with motifs of a/b-hydrolases.
GenBank ID: AAA79183.1; Baccl, Bacillus clausii lysophospholi-
pase, GenBank ID: YP_176371.1; Strsv, Streptomyces sviceus
lysophospholipase, GenBank ID: ZP_06921311.1; Bacpu, Bacillus
pumilus esterase/lipase, GenBank ID: YP_001488906.1) and other
serine hydrolases (Sacce2, Saccharomyces cerevisiae triglyceride
1
s with
‘
(
1
1
lipase2, GenBank ID: CAY78566.1; Zeama, Zea mays PLA ,
shown are homologous to conserved regions of fungal PLBs (Aspfu2,
Aspergillus fumigatus PLB2; Canal1, Candida albicans PLB1; Neucr,
Neurospora crassa PLB; Pench, Penicillium chrysogenum PLB;
Sacce1, Saccharomyces cerevisiae PLB1; Cryne1, Cryptococcus
neoformans var. neoformans PLB1) or catalytic motifs of bacterial
esterases (Bacli, Bacillus licheniformis esterase/acyl transferase,
GenBank ID: ACG31809.1). Dark grey: amino acid positions
characteristic for serine hydrolase consensus cores; light gray:
positions identical in the found sequences and in sequences from
peptide libraries; framed: positions conserved in fungal PLBs
(identical in C80 % of the selected sequences)
Influence of pH and Effectors on Enzyme Activities
conditions used, the maximum activities of PLA -2, -3 and
1
-4 were 51, 16 and 28 % compared to PLA -1, which had
1
The pH dependence of the activity towards DPPC in mixed
micelles showed strong differences for the four PLA1s
the highest activity of all the enzymes.
The enzymes did not show any dependence on metal
(
Fig. 4). PLA -1 is active in the range from pH 4.0 to 6.0
1
ions, which was tested at pH 5.6 for MgCl (0–40 mM),
2
with a distinct optimum at pH 5.3, whereas the other
enzymes were most active under less acidic or neutral
CaCl (0–40 mM) and KCl (0–120 mM) and at pH 7.0 for
2
CaCl (0–40 mM). The enzymes were not inhibited by
2
conditions. In contrast to PLA -1, they exhibited optimum
1
EGTA, demonstrating the absence of tightly bound metal
activity over a broad range of pH (PLA -2: pH 6.0–7.25;
1
ions. The PLA s also proved to be relatively insensitive to
1
PLA -3: pH 5.3–6.0; PLA1-4: pH 5.0–6.0). Under the
1
serine- or cysteine-modifying reagents. Thus, treatment
1
23

1
442
J Am Oil Chem Soc (2012) 89:1435–1448
(
1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) and DPPC),
sphingolipid (sphingomyelin), mono-, di- and triacylgly-
cerols (1-monoolein, 1,2-diolein, triolein) and fatty acid
alkyl ester (methyl oleate) were applied in the presence of
Triton X-100. The glycerophospholipids LPPC and DPPC
were hydrolyzed by all four enzymes. Surprisingly, how-
ever, a remarkable hydrolytic degradation of 1-monoolein,
1,2-diolein and methyl oleate was also observed for
PLA -2, -3 and -4. However, no significant reaction rate
1
-
1
-1
(
B0.3 lmol min mg ) was detectable with triolein or
the fatty acid amide in sphingomyelin. The missing con-
version of the triolein by all of the enzymes is in accor-
dance with our findings that the crude enzyme extracts
from A. ostoyae do not show any lipase activity toward
triglycerides in the absence of detergents as well as in the
presence of Triton X-100 or taurocholate [14].
Fig. 4 pH-activity profiles of A. ostoyae PLA
mM DPPC in micelles containing 10 mM Triton X-100 by PLA
open circles), PLA -2 (filled circles), PLA -3 (filled inverted
-4 (open triangles) was assayed according to
1
s. The hydrolysis of
5
(
1
-1
For a detailed comparison, kinetic studies with the
cleavable lipids were performed (Fig. 5). With only few
exceptions, the reaction rates as a function of the substrate
concentration were characterized by sigmoid kinetics,
which could be fitted by the Hill function. The resulting
1
1
triangles), and PLA
1
‘‘Experimental Procedures’’
with PMSF or iodoacetamide for 12 h resulted in only a
kinetic parameters are shown in Table 2. PLA -1 is
1
small loss of activity (C80 % residual activity). In contrast,
restricted to the conversion of glycerophospholipids and
exhibited only very low activity towards the acylglycerols
and methyl oleate even at high substrate concentrations.
Under conditions of substrate saturation, the hydrolysis rate
of LPPC exceeded that of DPPC by a factor of 3.8 while
the S0.5 value for LPPC was nearly tenfold higher. For the
other enzymes a similar preference for the monoacyl
phospholipid LPPC at substrate saturation compared to the
diacyl compound DPPC was observed; however, the S0.5
values were in the same range. Based on the Hill coeffi-
cients, the hydrolysis reaction showed positive coopera-
tivity in the case of DPPC (h = 1.7–2.1) but not in the case
of LPPC (h B 1.0).
4
-bromophenacyl bromide significantly inhibited PLA -1
1
(
(
8 % residual activity) and, to a certain extent, also PLA -2
1
55 % residual activity) but did not affect PLA -3 or PLA -4.
1
1
Regiospecificity in the Cleavage of Phospholipids
With regard to the regiospecificity of the purified A. ostoyae
phospholipases, the hydrolytic rates for the diacylphospho-
lipid POPC were compared with those for the structurally
related 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocho-
line. In the presence of 5 mM phospholipid and 10 mM
Triton X-100, POPC was readily hydrolyzed (122.6 ± 1.1,
-
1
-1
61.5 ± 0.6, 18.9 ± 0.3, 23.1 ± 0.3 lmol min mg by
PLA -1, PLA -2, PLA -3, PLA -4, respectively), whereas
The enzymes PLA -2, PLA -3, and PLA -4 showed a
1
1
1
1
1
1
1
similar trend in the conversion of neutral lipids. Compared
to 1,2-diolein, the activity towards 1-monoolein was
strongly favored. Moreover, the kinetics also showed a
higher degree of positive cooperativity for the diacyl
compound (h = 2.2–3.2) than for the monoglyceride
the lack of a cleavable sn-1 ester linkage in 1-O-hexadecyl-2-
oleoyl-sn-glycero-3-phosphocholine [6, 30] prevented the
hydrolytic degradation of this lipid. The rate of hydrolysis
-
1
-1
was B0.3 lmol min mg for all four enzymes. More-
over, the enzymes were shown to be unable to cleave the
fluorogenic 1-palmitoyl-2(1-pyrenedecanoyl)-sn-glycero-3-
(
h = 1.4–2.0). The S0.5 values for 1,2-diolein were in the
millimolar range (C2.2 mM) and higher by a factor of 5–8
than for 1-monoolein (Table 2).
phosphocholine which is another typical PLA -specific
2
substrate [26] (data not shown), indicating therefore that they
Interestingly, the simple fatty acid ester methyl oleate
was also degraded by PLA -2, PLA -3, and PLA -4. The
act specifically as PLA1s.
1
1
1
maximal hydrolytic rates, however, were at least threefold
lower compared to both monoacyl glycerolipids (LPPC
and 1-monoolein) (Fig. 5). Moreover, higher S0.5 values
Substrate Selectivity Towards Different Classes
of Lipids
([1.2 mM) and a decrease in cooperativity (h = 1.0–1.2)
In order to probe the substrate spectrum of the PLA1s,
compounds representing different classes of natural lipids
were selected. Mono- and diacylglycerophospholipids
were observed (Table 2). 1-Monoolein and DPPC showed
a substrate inhibition at high concentrations ([5 mM)
(Fig. 5).
1
23

J Am Oil Chem Soc (2012) 89:1435–1448
1443
Fig. 5 Rates of hydrolysis of different lipids catalyzed by PLA -1 (a),
1
PLA -2 (b), PLA -3 (c) or PLA -4 (d) as a function of the substrate
1 1 1
concentration. The hydrolysis rates of LPPC (open circles), DPPC (filled
circles), 1-monoolein (filled inverted triangles), 1,2-diolein (open
inverted triangles) or methyl oleate (filled diamonds) were determined
as described in ‘‘Experimental Procedures’’. Curves were fitted accord-
ing to the Hill model not considering data points in the range of substrate
inhibition
Substrate Selectivity in Phospholipid Hydrolysis
The influence of the degree of fatty acid unsaturation in
long-chained phosphatidylcholines is demonstrated in
Fig. 6b. Compared to the hydrolysis of the rigid DSPC, the
hydrolysis of DOPC was accelerated by a factor of C4.9.
Interestingly, even slightly higher reaction rates were
obtained for POPC having one oleoyl moiety in the sn-2
position only. Otherwise, phospholipids carrying two
polyunsaturated fatty acids such as the natural soya-PC,
which mainly contains linoleic and linolenic acid [31],
were degraded with the same rate as POPC.
The hydrolytic rates of the fungal PLA s were compared
1
for a series of diacylphospholipids differing in molecular
properties such as in the length and saturation of the
hydrophobic acyl moieties, as well as in the type of the
polar head group. Kinetic analyses of the conversion of
short-chain (1,2-dioctanoyl-sn-glycero-3-phosphocholine)
and unsaturated (DOPC) phosphatidylcholines confirmed
that the reactions were performed at substrate saturation
(
data not shown). Figure 6a demonstrates the strong
As shown in the previous section, the presence of a
phosphoalcoholic head group in the substrate was crucial
dependence of the PLA reaction rates on the chain length
1
of the fatty acid moieties in the 1,2-diacyl-sn-glycero-3-
phosphocholines. Hydrolysis proceeded at least two orders
of magnitudes faster for all four enzymes with the short-
chain 1,2-dioctanoyl-sn-glycero-3-phosphocholine than with
the corresponding diarachidoyl compound.
for PLA -1 but not for the other enzymes. Nevertheless, the
1
phosphocholine moiety strongly influenced the kinetic
constants and cooperativity in PLA -2, -3 and -4 as con-
1
cluded from a comparison of DOPC and 1,2-diolein
(Table 2). Accordingly, the kind of head group also proved
1
23

1
444
J Am Oil Chem Soc (2012) 89:1435–1448
Table 2 Kinetic parameters for
lipid hydrolysis catalyzed by
-1
-1
)
Substrate
LPPC
Enzyme
S
0.5 (mM)
V
max (lmol min mg
h
1
the PLA enzymes
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
PLA
1
-1
1
-2
1
-3
1
-4
1
-1
1
-2
1
-3
1
-4
1
-1
1
-2
1
-3
1
-4
1
-1
1
-2
1
-3
1
-4
1
-1
1
-2
1
-3
1
-4
1
-1
5.11 ± 1.09
0.24 ± 0.02
0.30 ± 0.03
0.22 ± 0.02
0.52 ± 0.03
0.32 ± 0.02
0.26 ± 0.02
0.36 ± 0.02
b.d.
194.2 ± 14.6
169.1 ± 4.8
80.0 ± 1.8
92.4 ± 2.7
51.4 ± 1.4
43.0 ± 1.5
17.0 ± 0.5
19.8 ± 0.5
b.d.
0.7 ± 0.1
0.8 ± 0.1
1.0 ± 0.1
0.8 ± 0.1
1.9 ± 0.2
1.9 ± 0.2
2.1 ± 0.4
1.7 ± 0.2
b.d.
DPPC
1
1
-Monoolein
,2-Diolein
0.34 ± 0.01
0.48 ± 0.03
0.43 ± 0.02
b.d.
226.2 ± 3.6
74.8 ± 2.2
103.0 ± 2.5
b.d.
2.0 ± 0.1
1.4 ± 0.1
1.5 ± 0.1
b.d.
2.63 ± 0.12
2.47 ± 0.20
2.17 ± 0.09
n.d.
14.8 ± 0.5
13.7 ± 0.5
22.4 ± 0.6
3.2 ± 0.3
38.0 ± 1.8
17.3 ± 0.6
18.9 ± 0.8
102.5 ± 2.5
51.1 ± 1.3
15.8 ± 0.3
21.1 ± 0.5
3.2 ± 0.4
2.2 ± 0.2
2.3 ± 0.2
n.d
Methyl oleate
1.57 ± 0.20
1.21 ± 0.13
1.16 ± 0.10
0.73 ± 0.05
0.52 ± 0.04
0.47 ± 0.02
0.57 ± 0.04
1.0 ± 0.1
1.1 ± 0.1
1.2 ± 0.1
1.4 ± 0.1
1.4 ± 0.1
1.6 ± 0.2
1.3 ± 0.1
DOPC
Parameters were obtained as
described in ‘‘Experimental
Procedures’’
PLA -2
1
PLA
1
-3
-4
b.d. below detection limit,
n.d. not determined
PLA
1
to be important for phospholipid turnover. All phospholipid
compounds analyzed were degraded by all four enzymes
exhibited a less distinct selectivity for the chain length of
the fatty acids. In accordance with the results obtained for
lysophosphatidylcholines, methyl oleate as an unsaturated
ester was not preferred to the corresponding saturated
compound methyl stearate. However, activities of all
(
Fig. 6c). Throughout, the zwitterionic phospholipids PC
and PE were preferred to the anionic species, with PC as
the more favored substrate. For the anionic lipids, the order
in the rates of hydrolysis was PG [ phosphatidylinositol
-
1
-1
enzymes were marginal (0.001–0.196 lmol min mg
towards the very short-chain 4-nitrophenyl acetate, which
)
(
PI) [ phosphatidylserine (PS) [ PA for PLA -1, PG [
1
PA [ PI [ PS for PLA -2, PA [ PG = PI [ PS for
is a typical esterase substrate.
1
PLA -3 and PG [ PI [ PA [ PS for PLA -4.
1
1
Cleavage of Monoacyl Substrates
Discussion
In the hydrolysis of lysophosphatidylcholines, the depen-
PLA Enzymes from A. ostoyae are New
1
dence of PLA activity on the chain length as well as the
1
Representatives of the a/b-Hydrolase Fold Enzymes
saturation of the acyl moiety (Fig. 7a) significantly differed
from that of the corresponding diacyl phospholipids
To the best of our knowledge this study presents the first
(
Fig. 6a, b). The activity was optimal for lysophosphati-
report on the isolation and characterization of PLA s from
1
dylcholine with 10 (PLA -2, PLA -3, PLA -4) or 12
1
fungal fruiting bodies. Chromatographic methods, such as
hydrophobic interaction, anion exchange and hydroxyapa-
tite chromatography, which are commonly used in the
isolation of fungal or mammalian PLB-type enzymes [32–
34], were also successfully employed in this study (Fig. 1,
Table 1) and yielded four different enzymes as homoge-
neous proteins (Fig. 2). Due to their activity toward POPC
1
1
(
PLA -1) carbon atoms. The influence of unsaturation,
1
which was probed for 1-stearoyl-sn-glycero-3-phospho-
choline and its unsaturated oleoyl analogue, was minimal.
Comparable results were obtained for fatty acid methyl
esters, which were significantly cleaved by PLA -2,
1
PLA -3, and PLA -4 only (Fig. 7b). However, the PLA s
1
1
1
1
23

J Am Oil Chem Soc (2012) 89:1435–1448
1445
Fig. 7 Selectivity in the hydrolysis of monoacyl substrates by the
A. ostoyae PLA s. The hydrolysis rates of 2-lysophospholipids (a) and
1
fatty acid methyl esters (b) of varying chain length and different
saturation were determined at conditions of substrate saturation
(
‘
5 mM monoacyl substrate/10 mM Triton X-100) as described in
‘Experimental Procedures’’
Hitherto, the most abundant type of phospholipid-
hydrolyzing enzymes described in fungi is PLB, which
belongs to the protein family of a/b-hydrolase fold
enzymes [27]. For example, the production of multiple
PLB enzymes was reported for Candida albicans [35] and
Saccharomyces cerevisiae [36]. Apart from the dominant
group of PLBs, there are only a few reported examples of
phospholipid-specific enzymes from fungi, such as the
Fig. 6 Selectivity in phospholipid hydrolysis by the A. ostoyae
1
PLA s. The hydrolysis rates of phosphatidylcholines with fatty acid
lipase-related PLA s from Mucor javanicus [37] and
1
moieties of varying chain length (a), long-chain phosphatidylcholines
with different saturation (b) and phospholipids from soybean with
different head groups (c) were determined at conditions of substrate
saturation (5 mM phospholipid/10 mM Triton X-100) as described in
Aspergillus oryzae [38], which also belong to the structural
family of a/b-hydrolases. The enzymes presented in this
paper (PLA -1, PLA -2, PLA -3 and PLA -4) share several
1
1
1
1
‘‘Experimental Procedures’’
features with both types of fungal a/b-hydrolase fold
enzymes, such as their low pI values [38–40] and the
N-linked glycosylation [40–42] found for PLA -2, -3 and
1
but inactivity towards 1-O-hexadecyl-2-oleoyl-sn-glycero-
-phosphocholine and 1-palmitoyl-2-(1-pyrenedecanoyl)-
-4. In particular, PLA -1 resembles the PLBs from Sac-
1
3
charomyces cerevisiae [43] or cryptococci [44] due to its
acidic pH optimum (Fig. 4) and independence of metal
ions. In contrast, the other PLA s (especially PLA -2)
sn-glycero-3-phosphocholine, the enzymes were attributed
to the PLA family of phospholipases.
1
1
1
1
23

1
446
J Am Oil Chem Soc (2012) 89:1435–1448
exhibit a broad-range activity from slightly acidic to neu-
tral pH (Fig. 4) and seem, therefore, to be more similar to
acyl residues in diacyl compounds (phosphatidylcholines
as well as diglycerides) strongly reduces the enzyme
activity. In phosphatidylcholines, this effect is alleviated by
a decrease in chain-length (Fig. 6a) or an increased flexi-
bility due to an introduction of double bounds in the sn-2
positioned acyl moiety (Fig. 6b). Therefore, the sn-2
pocket seems to play a key role in the regulation of sub-
strate acceptance. Like the A. ostoyae enzymes, other a/b-
hydrolase fold enzymes favor lysophosphatidylcholines
over their diacyl derivatives [39, 53] or phospholipids with
unsaturated fatty acids over the saturated analogues [54].
Besides the fatty acid moieties, the other molecular
constituents of the phospholipids also proved to be
important for the substrate turnover. The preference for
monoglycerides and lysophospholipids to the correspond-
ing fatty acid methyl ester (Fig. 5; Table 2) indicates the
recognition of the glycerol backbone. Moreover, the head
group of phospholipids is essential for the hydrolysis by
PLA from Aspergillus oryzae [38].
1
The similarity between the A. ostoyae PLA s and fungal
1
PLBs was additionally confirmed by the identification of
proteolytic peptide fragments with a high degree of simi-
larity to an amino acid sequence, which is unique to the latter
group of enzymes (Fig. 3). Otherwise, PLA -1, PLA -3 and
1
1
PLA -4 clearly differ from the conventional PLBs by a
1
sequence that it is highly similar to the catalytic motifs of
microbial and plant PLA s and esterases (Fig. 3). In fungal
1
phospholipid-cleaving enzymes, a related motif is only
reported for the uncommon PLB from the fungus Anthrodia
cinnamomea [45] and the PLA from Aspergillus oryzae
1
[
38]. Comparable to these two enzymes, the molecular
masses of the A. ostoyae PLA s are much lower
1
(
33–47 kDa) than those of common fungal PLBs, which are
in the range of 63–95 kDa [40–44, 46–48]. Also the insen-
sitivity towards serine- and cysteine-modifying reagents is
uncommon for mammalian and fungal PLBs [49, 50].
PLA -1. The other enzymes do not need this charged group
1
(Fig. 5; Table 2) but also show an effect of the kind of
Instead, the inhibition of PLA -1 and -2 by 4-bromophena-
1
head group on PLA activity (Fig. 6c). This effect may
1
cyl bromide points to the presence of a catalytically
important histidine residue, which has also been shown to be
crucial for the catalytic activity of some other phospholip-
ases [51, 52]. In combination with their strong preference for
the sn-1 position, the Armillaria enzymes are more similar to
arise from a decreased interaction of the acidic PLA1
enzymes with negatively charged lipid aggregates due to
electrostatic repulsion. However, together with the non-
acceptance of triglycerides as substrates, the presence of a
small, hydrophilic and charge-sensitive sn-3 binding
lipase-related PLA s than to conventional PLBs.
1
pocket is suggested for all four PLA s. A similar influence
1
of the phospholipid head group on activity was described
for yeast [43] and mammalian lysophospholipases [55].
Another mode as to how the substrate structure influ-
ences the catalytic efficiency is reflected by the coopera-
tivity of substrate binding as expressed in the Hill
coefficients (Table 2). This cooperative behavior might be
caused by regulatory substrate binding sites, as has been
The Substrate Selectivity of the PLA Enzymes
1
from A. ostoyae Reflects Steric Restraints and Head
Group Recognition
The results described on A. ostoyae PLA s allow us to draw
1
conclusions on the relationship between enzyme activity
and the molecular details of the substrate structure. The
conversion rates of the structurally diverse lipid substrates
discussed for several monomeric PLA s [56, 57] or the
2
formation of enzyme oligomers as described for many
fungal phospholipases [40, 44].
(
Table 2) can be explained by a combined recognition of
the hydrophobic and hydrophilic regions in the respective
substrate molecule.
In conclusion, the PLA s seem to combine two different
1
mechanisms of substrate recognition. Firstly, size-control
mechanisms similar to that which determines the specific-
ity for fatty acid chain length in lipases [58] or esterases
[59] clearly generate (I) the selectivity of the sn-1 site and
(II) the discrimination of substrates bearing bulky acyl
moieties in the sn-2 position. Secondly, the dependence of
The hydrolysis of different lysophospholipids and fatty
acid methyl esters (Fig. 7a, b) provides information about
the role of the binding site of the scissile acyl moiety.
Clearly, this site strongly favors hydrophobic medium
chain acyl substrates. Likewise, the non-acceptance of the
very short chain ester 4-nitrophenyl acetate seems to be
caused by this chain length selectivity, although its turn-
over may also be prevented by the non-natural secondary
alcohol moiety. On the other hand, the state of saturation
of acyl chains only marginally influenced the substrate
conversion. For the docking of a second acyl moiety in
glycerol-based lipids to the corresponding sn-2 site, its
molecular properties are even more significant. Compared
to the monoacyl substrates, the presence of the additional
PLA activity on the phospholipid head group resembles
1
the mechanism of the lipase-related PLA from Strepto-
1
coccus hyicus, which favors phospholipid turnover due to a
small and charged sn-3 binding cavity [60].
Acknowledgments We thank the LIPOID GmbH (Ludwigshafen,
Germany) for the gift of phospholipids, Christa Kuplens and Katja
Fr o¨ hlich for their excellent technical assistance, Gary Sawers for
reading the manuscript and Cornelia Dippe, Frank Dippe and Emil
Hinze for their help in collecting fungal fruiting bodies. The financial
1
23

J Am Oil Chem Soc (2012) 89:1435–1448
1447
support by the State of Sachsen-Anhalt and by the Graduiertenkolleg
1026 (Deutsche Forschungsgemeinschaft, Bonn, Germany) is grate-
fully acknowledged.
17. Laemmli UK (1970) Cleavage of structural proteins during
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