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[ Pobierz całość w formacie PDF ]Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2729–2735
TETRAHEDRON: ASYMMETRY REPORT NUMBER 69
Thermostable carboxylesterases from hyperthermophiles
Haruyuki Atomi and Tadayuki Imanaka
*
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura,
Nishikyo-ku, Kyoto 615-8510, Japan
Received 1 June 2004; accepted 21 July 2004
Available online 11 September 2004
Abstract—This report focuses on the lipolytic enzymes from hyperthermophiles. Most of the enzymes characterized to date are car-
boxylesterases that are structurally related to the hormone-sensitive lipase family, and prefer medium chain (acyl chain length of 6)
p-nitrophenyl substrates. The presence of a GGGX motif in these carboxylesterases suggest the ability of these enzymes to catalyze
the hydrolysis of tertiary alcohol esters. We will also introduce studies that have examined the effects of temperature and organic
solvents on the catalytic e;ciency and enantioselectivity of the thermostable carboxylesterase from Sulfolobus solfataricus. Finally, a
BLAST search of the hyperthermophile genome sequences reveal candidate genes that may encode novel, thermostable esterases.
2004 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . ..................................................... 2729
2. Properties of characterized carboxylesterases from hyperthermophiles. .............. 2730
3. Other candidate carboxylesterase orthologues on the hyperthermophile genomes . ...... 2733
4. Practical advantages in the use of enzymes from hyperthermophiles . . .............. 2734
5. Conclusions. . . ..................................................... 2734
References . . ......................................................... 2734
1. Introduction
to explore a broader range of reaction conditions aimed
to enhance further the selectivity and/or e
;
ciency (turn-
over) of the enzyme reaction. Indeed, much effort has
been spent in order to enhance the stability of enzymes,
through modifying the enzyme itself or its immediate
environment.
5
The dramatic increase in structural infor-
mation of enzymes, along with recently developed tech-
niques (DNA shuDing, high throughput screening
technology, directed evolution), have led to great ad-
vances in enzyme engineering and technology.
4,18,19,24
The application of enzymes in organic synthesis is now a
routine alternative for the organic chemist and process
engineer. The native or engineered enzyme provides
the selectivity, whether it be substrate selectivity, regio-
selectivity, or stereoselectivity, which is desired in the
reaction. Unfortunately, the use of enzymes in many
cases also brings about constraints in the conditions
under which the reaction must be performed. In terms
of stability, not to mention selectivity, the usual enzyme
is far from the ideal catalyst, and in many cases the en-
zyme is more labile than the substrate and product of
the reaction. Enzymes with enhanced stability would
not only allow prolonged usage, but would enable us
Another development that has provided valuable clues
as to how proteins can be made more thermostable or
thermotolerant is the discovery of hyperthermophiles
and studies on their proteins. Hyperthermophiles are
organisms that grow at temperatures above 90C,
1
or
optimally grow at temperatures above 80C.
38
Many
have been found to grow at temperatures above the boil-
ing point of water.
39
Unlike chemical parameters such as
*
Corresponding author. Tel.: +81 75 383 2777; fax: +81 75 383 2778;
e-mail:
0957-4166/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.07.054
2730
H. Atomi, T. Imanaka / Tetrahedron: Asymmetry 15 (2004) 2729–2735
pH, heat cannot be removed or pumped out of the cell,
and consequently, all the biomolecules within a hyper-
thermophiliccell must endure and function at high tem-
perature. Therefore, a single hyperthermophile provides
well over a 1000 different proteins with extreme thermo-
tolerance. This, along with the possibility that hyper-
thermophiles may represent the most primitive forms
of present-day life, has led many to study the protein
structure, physiology, and genome structure of hyper-
thermophiles. Hyperthermophiles have been found to
constitute a diverse group of organisms in terms of en-
ergy and carbon metabolism.
2
Both chemoautotrophs
and heterotrophs are present, with the latter group capa-
ble of utilizing a variety of organic compounds; disac-
charides or polysaccharides with a-orb-1,4-glycosidic
bonds, peptides, amino acids, and organic acids. This
indicates the presence of various enzymes that can con-
vert or degrade these compounds. As expected, a vast
scope of enzymes with an application potential have
been identified from these organisms in the past
years.
15,17,25,29,40
enzymes identified from hyperthermophiles and their
biochemical properties. At present, a lipase has not been
identified from hyperthermophiles, and most of the en-
zymes characterized up till now are carboxylesterases.
Although the number is still very limited, we will also
introduce some initial examples where the application
of hyperthermophilicesterases in organicsynthesis has
been explored.
2. Properties of characterized carboxylesterases from
hyperthermophiles
Thermostable carboxylesterases have been identified
and characterized from Archaeoglobus fulgidus, Pyro-
coccus abyssi, Pyrococcus furiosus, Aeropyrum pernix,
Sulfolobus solfataricus,andPyrobaculum calidifontis
(
Table 1
). Among these, the enzyme from A. fulgidus
(AFEST) is the most characterized; its gene has been
cloned, the recombinant enzyme has been purified and
characterized (AAB89533),
22
and moreover, the crystal
structure of the protein is available at 2.2
˚
resolution.
8
The structure of AFEST should provide valuable infor-
mation for future engineering of the enzyme, and for the
modelling of other esterases from hyperthermophiles.
AFEST is a member of the hormone sensitive lipase
(HSL) family, or Family IV of the prokaryoticlipolytic
enzymes proposed by Arpigny and Jaeger.
3
The HSL
family also includes the carboxylesterase from the
thermophile Alicyclobacillus acidocaldarius (EST2)
7
and Brefeldin A esterase from the mesophilic Bacillus
subtilis (BFAE),
41
whose three-dimensional structures
have been determined. The three structures thus allow
a detailed structural comparison among closely re-
lated enzymes from mesophiles, thermophiles, and
hyperthermophiles. As reported in other structural
comparisons between mesophilic/hyperthermophilic
proteins,
11,15,37,40
(i) an increase in the percentage of
ion pairs, (ii) an increase in cationic-p aromaticinterac-
tions, (iii) a decrease in the surface area occupied by
hydrophobicresidues, and (iv) a reduction in the lengths
Carboxylesterases (EC 3.1.1.1) are a class of lipolytic en-
zymes that hydrolyze water-soluble, ester-containing
molecules. Taking into account this substrate selectivity,
carboxylesterases are distinguished from lipases (EC
3.1.1.3), which prefer water-insoluble long-chain triglyc-
erides and display activation at lipid–water interfaces,
and arylesterases (EC 3.1.1.2), which hydrolyze esters
with aromaticmoieties. Phospholipase A2 (EC
3.1.1.4), lysophospholipase (EC 3.1.1.5), and acetylcho-
line esterase (EC 3.1.1.7) are also representatives of the
abundant number of ester bond hydrolyzing enzymes.
On the other hand, the rapid accumulation of sequence
data in recent years has made possible the classification
of these enzymes in terms of primary structure.
3
Although this structural classification in general agrees
well with the classification based on substrate selectivity,
there are some structurally-related families of enzymes
that include both the traditionally named lipases and
carboxylesterases. This report will focus on the lypolytic
Table 1. Biochemical properties of thermostable esterases from hyperthermophiles
Organism
No of
residues
T
opt
(C) Substrate
(examined temperature,
C)
a
K
m
(lM) k
cat
(s
1
) k
cat
/K
m
(s
1
lM
1
)
Specific
activity
(lmolmin
1
mg
1
)
Refs.
A. fulgidus
311
80
PNP-hexanoate (70)
11 ± 3
1014 ± 38 92.2
Ca. 3200
b
19
S. solfataricus P1 305
95–100 4-Methylumbelliferyl
acetate (80)
450
1000
2.2
1600
c
28
S. solfataricus MT4 305
P90
PNP-valerate (60)
NR
NR
NR
747
d
23
P. calidifontis VA1 313
90
PNP-caproate (70)
44.4 ± 5.9 2620 ± 90 59
4050
e
13
A. pernix
582
90
PNP-caprylate (70)
NR
NR
NR
0.92
f
9
P. furiosus
NR
100
NR
NR
NR
NR
Crude sample
15
P. furiosus
257
NR
NR
NR
NR
NR
NR
30
P. abyssi
NR
65–74
NR
NR
NR
NR
Crude sample
6
NR, not reported.
a
Temperature at which kinetic analysis was performed, or specific activity measured.
Measured with 0.2mM PNP-hexanoate.
c
Measured with 0.6mM 4-methylumbelliferyl acetate.
d
Measured with 0.3mM PNP-valerate.
e
Measured with 1mM PNP-caproate.
f
Measured with 0.2mM PNP-caprylate.
b
H. Atomi, T. Imanaka / Tetrahedron: Asymmetry 15 (2004) 2729–2735
2731
of loops connecting secondary structures, was ob-
served.
8
Further statistical analyses of single amino acid
replacements among the three aligned proteins have re-
vealed particular trends in residue exchange in the direc-
tion mesophilicto hyperthermophilic.
23
In terms of the
biochemical performance of the enzyme, AFEST was
thermostable with t
1/2
values of 30h (58C), 7.5h
(70C), 60min (85C), 28min (90C), and 26min
(95C). The optimal temperature of the enzyme under
the conditions examined was 80C. The thermostability
and optimal temperature of the enzyme may seem rela-
tively low, as A. fulgidus grows at temperatures up to
95C. There are some examples in which the in vitro
thermostability of an enzyme from a hyperthermophile
is lower than one would expect.
9
There is a possibility
that these intracellular enzymes are further stabilized
in vivo by small intracellular molecules such as
polyamines.
21
be expected to differ from those of mesophilic enzymes.
This is due to the fact that these temperatures are still
below the optimal temperature of hyperthermophilic
enzymes, and therefore these enzymes can be considered
to be in a structurally rigid state, while mesophilic
enzymes, at temperatures above their optimum, are
already in a highly flexible state.
35
In order to examine
the possibilities of enhancing the function of Sso EST1
at suboptimal temperatures by increasing enzyme flexi-
bility, various co-solvents were added to the reaction
mixture using 4-methylumbelliferyl acetate as the sub-
strate. Dimethyl sulfoxide (DMSO) was found to have
an activating effect at concentrations between 1.2%
and 10% (v/v), and the effect was more striking at lower
temperatures. Structural and biochemical analyses at
various temperatures in the presence of co-solvent sug-
gested that the activating effect of DMSO at relatively
lower temperatures could be attributed to an increase
in the structural flexibility of the enzyme at suboptimal
temperatures. The results point out the fact that the
presence of co-solvent, in some cases, may compensate
for the activating effect of temperature, and provide an
alternative to reaction systems with highly stable en-
zymes and thermolabile substrates.
35
Kineticanalyses of AFEST toward various p-nitrophe-
nyl (PNP) esters revealed maximum k
cat
/K
m
values
toward PNP-hexanoate (92.2s
1
lM
1
). Activities
toward long PNP esters were very low, and hydrolysis
of trioleoylglycerol could not be detected. Enantioselec-
tivity of AFEST was examined with several compounds,
and although significant conversion was observed in
short reaction times with high substrate/enzyme ratios,
only moderate enantioselectivity was observed (
Fig. 1
,
60% enantiomericexcess of (R)-6-methyl-5-hepten-2-ol
with hydrolysis of (±)-6-methyl-5-hepten-2-yl buta-
noate).
22
The difference in behavior between hyperthermophilic
and mesophilicenzymes can also be observed through
the effects of temperature on their enantioselectiv-
ity.
19,28,33
The enantiomericratio of an enzyme reaction
is related to the difference in the free energy of activation
of the paths of the two enantiomers (DDG)as
DDG =
RTlnE. DDG can also be expressed by the
differences in activation enthalpy (DDH) and entropy
(DDS)asDDG = DDH
TDDS. When there is no
enantiomericdisrimination, E = 1, and hence
DDG =0, or DDH = TDDS. The temperature at
which enantiomeric discrimination is absent is defined
as the racemic temperature, T
r
.
28
At temperatures below
T
r
, the DDG is dominated by DDH (under enthalpic
control), and the E value will decrease as temperature
is elevated until it reaches 1 at T
r
. At temperatures above
T
r
, the DDG is dominated by TDDS (under entropic
control), and the E value will increase with the increase
in temperature. DDH is due to differences in the steric
binding of the enantiomers to the substrate pocket of
the enzyme through van der Waals or other noncovalent
interactions, while DDS most likely reflects differences
in the rotational motion of the substrate and amino acid
side chains lining the substrate binding pocket. When
substrates bind to the enzyme pocket through strong
interactions such as hydrogen bonds or ionic bonds,
DDH can be expected to be large, resulting in little or
no effect of temperature on the enantioselectivity of
the enzyme. As the substrates for carboxylesterases
and lipases are in many cases lipophilic, and interact
with the enzymes through relatively weak hydrophobic
interactions, the effect of temperature on these enzyme
reactions can be expected to be significant.
28
OCOC
3
H
7
AFEST
HO
H
(
R
)-6-methyl-5-hepten-2-ol
39% (conversion)
ee% = 60
E = 5.7
Figure 1.
Carboxylesterases have been examined from two strains
of S. solfataricus, strains P1
32
and MT4.
26
The strain
whose genome has been sequenced is S. solfataricus
strain P2.
36
In order to avoid misunderstanding, the en-
zyme from strain MT4 (EstA) is 99% identical with the
enzyme from strain P1 (Sso EST1), and both are 91%
identical to a gene on the P2 genome annotated as
lipP-1 lipase. Sso EST1 and EstA (along with lipP-1)
are also members of the HSL family. Sso EST1 exhibits
a surprisingly high optimal temperature between 95 and
100C compared to the optimal growth temperature of
its host (75C). The enzyme prefers PNP-caproate
among the PNP-esters, and displays k
cat
/K
m
values
of 2.2s
1
lM
1
at 80C with 4-methylumbelliferyl
The effects of temperature and various organic co-sol-
vents on the structure and catalytic activity of Sso
EST1 have been examined in detail.
33–35
The conforma-
tional state of hyperthermophilicenzymes at moderately
high temperatures, such as in the range of 50–80C, can
The enantioselectivities of Sso EST1 and the mesophilic
enzymes Candida rugosa lipase (CRL) and Palatase in
the hydrolysis of (RS)-Naproxen methyl ester have been
examined at various temperatures (Scheme of
Fig. 2
).
33
acetate.
32
2732
H. Atomi, T. Imanaka / Tetrahedron: Asymmetry 15 (2004) 2729–2735
H
3
Sso EST1
O
CH
3
OH
O
25% methanol
O
H
3
CO
H
3
CO
(
S
)-Naproxen
8.3% (conversion)
ee% = 92.9
E = 30
Figure 2.
The lnE versus 1/T (K
1
) plot revealed an inverse rela-
tionship between Sso EST1 and the mesophilicenzymes,
the former displaying a decrease in the E value with
higher temperature [>6-fold higher (S)-selectivity at
48.5C than at 70C], while the latter exhibited an
increase in E values [>3-fold higher (S)-selectivity at
55C than at 4C]. The estimated T
r
values were 88.1,
46.3, and 1.1C for Sso EST1, CRL, and Palatase,
respectively. The results clearly reveal that the reactions
are controlled by distinct thermodynamic features; the
CRL and Palastase reactions are under entropic control,
while the Sso EST1 reaction is under enthalpic control.
33
This difference can be related to the different conforma-
tional states of the enzymes mentioned above; at the
examined temperatures the flexibility of the mesophilic
enzymes is su;cient to encourage entropic control,
while the rigidity of thermostable enzymes give rise to
enthalpiccontrol.
2100lmolmg
1
min
1
with 50% (v/v) DMSO,
560lmolmg
1
min
1
with 50% methanol and
300lmolmg
1
min
1
with 50% dimethylformamide
(4000lmolmg
1
min
1
with no co-solvents). Pc-Est pre-
ferred PNP-valerate, PNP-caproate, and PNP-caprylate
among the examined PNP-esters, and displayed only lit-
tle activity against PNP-palmitate. One interesting prop-
erty of Pc-Est is its activity toward esters with branched
alcohols. The enzyme hydrolyzed sec-butyl acetate and
moreover tert-butyl acetate with specific activities of
880 and 270lmolmg
1
min
1
, respectively. Carboxylest-
erases that hydrolyze tertiary alcohol esters are limited
in number; the lipase from Candida rugosa and the lipase
A from Candida antarctica have been shown to exhibit
this activity.
12,13
These enzymes, as well as Pc-Est, har-
bor a GGGX motif located in the active site that con-
tributes to the oxyanion hole. Along with a systematic
examination of the enzyme activities of various
GGGX-type a/b hydrolases, the importance of this mo-
tif structure in allowing the hydrolysis of tertiary alcohol
esters has been revealed by computer modelling,
12,13
indicating that the GGGX motif creates a larger active
site, providing more space for the alcohol. The enzymes
mentioned above from A. fulgidus and S. solfataricus
also harbor this motif, and are therefore also likely to
hydrolyze tertiary alcohol esters.
Possibilities for the application of Sso EST1 in chiral
separations of racemic esters have also been explored.
A strategicselection of esterases from hyperthermo-
philes was carried out for the resolution of 2-arylprop-
ionicesters.
34
The abundant sequence information
available from hyperthermophile genomes was searched
with the sequences of two mesophilic esterases that have
been experimentally proven to exhibit high enantioselec-
tivity in the resolution of Naproxen ester derivatives
(CRL and Carboxylesterase NP from Bacillus subtilis
ThaiI-8). Sso EST1, along with a putative lysophospho-
lipase from P. furiosus, was identified as a potential can-
didate. Sso EST1 proved to be the more effective
enzyme, hydrolyzing the (S)-Naproxen methyl ester with
an enantiomericexcess of over 90 and an enantiomeric
ratio of 24 at 50C. Addition of 25% methanol led to
an increase in the E value from 24 to 30 (
Fig. 2
). The
effects of other co-solvents were also examined and
revealed an inverse relationship between the denatura-
tion capacity of the solvent
20
and the observed enantio-
meric ratio. This can also be attributed to the increase in
flexibility of the enzyme brought about by the solvent,
counteracting with the enantioselectivity of the enzyme
under enthalpiccontrol.
While the enzymes mentioned above are all members of
the HSL family of a/b hydrolases, a structurally distinct
protein with both esterase and acyl amino acid-releasing
enzyme (AARE) activity has been identified and charac-
terized from A. pernix
10
The enzyme was 29% identical
to the AARE from pig liver and 27% identical to the
carboxylesterase from mouse liver. The pentapeptide
motif was found with the sequence G-Y-S-Y-G. The re-
combinant enzyme was extremely thermostable, retain-
ing 60% activity after incubation at 90C for 160h.
Among PNP-esters at a fixed concentration of 2mM,
PNP-caprylate was the most hydrolyzed substrate. The
enzyme also hydrolyzed N-acetylamino acid p-nitroani-
lide derivatives as well as dipeptides.
Other than the enzymes mentioned above, a thermosta-
ble protein with esterase activity has been cloned from
P. furiosus.
16
Unfortunately, sequence information is
not available. The enyzme displayed maximum activity
at 100C under the conditions employed, with a t
1/2
value of 34h at 100C. At a substrate concentration of
625lM, 4-methylumbelliferyl acetate was hydrolyzed
2-fold faster than 4-methylumbelliferyl butyrate. This
enzyme did not hydrolyze peptide substrates. Another
Another HSL carboxylesterase has been characterized
from P. calidifontis (Pc-Est).
14
Pc-Est is extremely
thermostable, with a t
1/2
value of ca. 1h at 110C, with
no apparent decrease in activity after 2h at 100C. The
optimal temperature of the enzyme under the applied
conditions was 90C. The enzyme also retained
activity in the presence of various co-solvents;
H. Atomi, T. Imanaka / Tetrahedron: Asymmetry 15 (2004) 2729–2735
2733
study reports the screening of 160 thermophilic or
hyperthermophilic microorganisms for esterase activ-
ity.
6
Forty seven strains were esterase positive, and elec-
trophoreticprofiles suggested at least three different
classes of esterases were present. Interestingly, the per-
centage of esterase-positive microorganisms increased
with the increase in isolation temperature. The thermo-
stable esterase from P. abyssi was selected for further
examination. At a fixed concentration of PNP-esters,
C4–C6 acyl moieties were hydrolyzed the most e;-
ciently. This esterase was also extremely thermostable,
but sequence information is not available.
encode proteins with esterase activity, we did not ex-
clude genes that were annotated with a different func-
tion, such as a peptidase. Candidates were excluded
only when the GXSXG motif was absent. We would
also like to note that a sequence identified from a
BLAST search is not necessarily a member of the same
Family as the template sequence. A more detailed struc-
tural examination and alignment is recommended before
one initiates experiments with a particular candidate.
With the Family I-2 lipase from Burkholderia glumae,an
open reading frame with notable similarity was found
on the A. fulgidus genome (annotated as 2-hydroxy-6-
oxo-6-phenylhexa-2,4-dienoicacid hydrolase). Interest-
ingly, a further Blast using this sequence did not lead
to genes from other hyperthermophiles, but to mesophi-
lic sequences. The sequence was 26% identical to the b-
ketoadipate enol–lactone hydrolase from Agrobacterium
tumefaciens.
27
Using the Family I-4 sequence of the li-
pase from Bacillus subtilis, a second, rather long (474
amino acid residues) open reading frame from A. fulgi-
dus (annotated as putative lipase) was identified. The
Family IV enzymes (HSL) are found in multiple hyper-
thermophiles, and besides the specific enzymes described
in the previous section, orthologues can also be found
on the S. tokodaii and T. maritima genomes. Using
the Family V sequence from Pseudomonas oleovorans,
multiple open reading frames from A. fulgidus were
3. Other candidate carboxylesterase orthologues on the
hyperthermophile genomes
We performed a BLAST search for serine esterases
against the genome sequences of A. pernix K1, S. solfa-
taricus P2, Pyrobaculum aerophilum IM2, Sulfolobus
tokodaii 7, A. fulgidus DSM4304, Methanococcus janna-
schii DSM2661, Methanopyrus kandleri AV19, P. abyssi
GE5, P. furiosus, P. horikoshii OT3, Aquifex aeolicus
VF5, and Thermotoga maritima MSB8 (
Table 2
). The se-
quences applied to the BLAST search were representa-
tives of each of the (sub)families of lipolytic enzymes
classified by Arpigny and Jaeger.
3
As we intended to
identify as many candidate genes as possible that may
Table 2. BLAST search against hyperthermophile genome sequences using members of the Family I–VIII lipolytic enzymes
BLAST template
Hits
GXSXG
Protein
characterization
Organism
Accession No No of residues
Family I-2 lipase from
Burkholderia glumae (CAA49812)
A. fulgidus AAB89544
a
238
98-GLSMG-102 No
T. maritima AAD35147 364
160-AHSMG-164 No
A. aeolicus BAA80234
570
186-GVSMG-190 No
Family I-4 lipase from
Bacillus subtilis (AAA22574)
A. fulgidus AAB89488
474
134-GHSMG-138 No
Family IV esterase from
Alicyclobacillus acidocaldarius (1EVQ_A)
A. fulgidus AAB89533
b
311
158-GDSAG-162 Yes
S. solfataricus AAK42652
b
311
154-GDSAG-158 No
S. tokodaii BAB65028
b
303
148-GDSAG-152 No
S. solfataricus AAK42629
b
305
149-GDSAG-153 No
c
S. solfataricus AAK42648
b
251
97-GISAG-101
No
T. maritima AAD36236 306
158-GLSAG-162 No
Family V PHA-depolymerase from
Pseudomonas oleovorans (AAA25933)
A. fulgidus AAB88916
247
86-GHSLG-90
No
A. fulgidus AAB90371
266
93-GHSFG-97
No
A. fulgidus AAB89709
251
87-GHSLG-91
No
S. solfataricus AAK40458 231
69-GHSIG-73
No
A. aeolicus AAC07858
207
60-GWSLG-64
No
P. abyssi
CAB50498
259
86-GHSLG-90
No
T. maritima AAD36421 259
84-GHSLG-88
No
S. tokodaii BAB67203
193
92-GASMG-96 No
S. solfataricus AAK43219 310
114-GHSYG-118 No
P. furiosus
AAL80604
257
86-GHSLG-90
Yes
Type VI esterase from Pseudomonas fluorescens
(AAC60403)
T. maritima AAD35127 395
284-GLSMG-288 No
A. pernix
BAA81456
591
449-GGSYG-453 No
a
Also identified in the Family V BLAST.
b
Also identified in the Family VII BLAST (not shown due to redundancy).
c
LipP-1 mentioned in the text.
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