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991.
Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus
fumigatus was cloned and expressed in insect cells. Recombinant
10R-DOX oxidized 18:2n-6 to
(10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid
(10R-HPODE; ∼90%), (8R)-hydroperoxylinoleic acid
(8R-HPODE; ∼10%), and small amounts of
12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic
acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by
site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase
(7,8-LDS), which forms ∼98% 8R-HPODE and ∼2%
10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in
homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and
Val-388/Leu-334, respectively) and at the distal site of the heme
(Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A
of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%,
respectively, as measured by liquid chromatography-tandem mass spectrometry
analysis. The stereospecificity was also decreased, as L384A formed the
R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio.
Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L
mutant augmented the formation of 10R-HPODE 3-fold. Replacement of
Val-388 in 10R-DOX with leucine and phenylalanine increased the
formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of
7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little
influence on the epoxyalcohol synthase activity. Our results suggest that
Leu-384 and Val-388 of 10R-DOX control oxygenation of
18:2n-6 at C-10 and C-8, respectively. The two homologous positions
of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the
position and stereospecificity of the cyclooxygenase reaction.Linoleate diol synthases
(LDS)2 and linoleate
10R-DOX are fungal fatty acid dioxygenases of the myeloperoxidase
gene family
(1-3).
LDS have dual enzyme activities and transform 18:2n-6 sequentially to
8R-HPODE in an 8R-dioxygenase reaction and to 5,8-, 7,8-, or
8,11-DiHODE in hydroperoxide isomerase reactions. These oxylipins affect
sporulation, development, and pathogenicity of Aspergilli
(4-6).
Fatty acid dioxygenases of the myeloperoxidase gene family also occur in
vertebrates, plants, and algae
(7-9).
The most thoroughly investigated vertebrate enzymes are ovine PGHS-1 and mouse
PGHS-2 with known crystal structures
(10-12).
PGHS transforms 20:4n-6 to PGG2 in a cyclooxygenase and
PGG2 to PGH2 in a peroxidase reaction. Aspirin and other
nonsteroidal anti-inflammatory drugs inhibit the cyclooxygenase reaction. This
is of paramount medical importance
(13,
14), and PGHS-1 and -2 are
commonly known as COX-1 and -2
(15). α-DOX occur in
plants and algae, and biosynthesis of α-DOX in plants is elicited by
pathogens (7). α-DOX
oxidizes fatty acids to unstable (2R)-hydroperoxides, which readily
break down nonenzymatically to fatty acid aldehydes and CO2
(7).LDS, 10R-DOX, PGHS, and α-DOX oxygenate fatty acids to
different products, but their oxygenation mechanisms have mechanistic
similarities. Sequence alignment shows that many critical amino acid residues
for the cyclooxygenase reaction are conserved in LDS, 10R-DOX, and
α-DOX. These include the proximal histidine heme ligand, the distal
histidine, and the catalytic important tyrosine (Tyr-385) of PGHS-1. The
latter is oxidized to a tyrosyl radical, which initiates the cyclooxygenase
reaction by abstraction of the pro-S hydrogen at C-13 of
20:4n-6 (16). In
analogy, LDS and 10R-DOX catalyze stereospecific abstraction of the
pro-S hydrogen at C-8 of 18:2n-6
(3), whereas α-DOX
abstracts the pro-R hydrogen at C-2 of fatty acids
(17). Site-directed
mutagenesis of the conserved tyrosine homologues of Tyr-385 and proximal heme
ligands abolishes the dioxygenase activities of 7,8-LDS and α-DOX
(17,
18). The orientation of the
substrate at the dioxygenation site differs. The carboxyl groups of fatty
acids are positioned in a hydrophobic grove close to the tyrosine residue of
α-DOX (19). In contrast,
the ω ends of eicosanoic fatty acids are buried deep inside the
cyclooxygenase channel so that C-13 lies in the vicinity of Tyr-385
(20). Several observations
suggest that 18:2n-6 may also be positioned with its ω end
embedded in the interior of 7,8-LDS of Gaeumannomyces graminis
(18).7,8-LDS of G. graminis and Magnaporthe grisea and 5,8-LDS
of Aspergillus nidulans have been sequenced
(5,
8,
21). Gene targeting revealed
the catalytic properties of 5,8-LDS, 8,11-LDS, and 10R-DOX in
Aspergillus fumigatus and A. nidulans
(3). Homologous genes can be
found in other Aspergilli spp. Alignment of the two 7,8-LDS amino
acid sequences with 5,8-LDS, 8,11-LDS, and 10R-DOX sequences of five
Aspergilli revealed several conserved regions with single amino acid
differences between the enzymes with 8R-DOX and 10R-DOX
activities, as illustrated by the selected sequences in
Fig. 1. Leu-306, Leu-384, and
Val-388 of 10R-DOX are replaced in 5,8- and 7,8-LDS by valine,
valine, and leucine residues, respectively. Whether these amino acids are
important for the oxygenation mechanism is unknown, and this is one topic of
the present investigation. The predicted secondary structure of
10R-DOX suggests that Leu-384 of 10R-DOX can be present in
an α-helix with Val-388 close to its border. This α-helix is
homologous to helix 6 of PGHS-1, which contains Val-349 and Ser-353 at the
homologous positions of Leu-384 and Val-388
(Fig. 1).Open in a separate windowFIGURE 1.Alignments of partial amino acid sequences of five heme containing fatty
acid dioxgenases and a comparison of the predicted secondary structure of
10R-DOX with ovine PGHS-1. A, top, amino acids residues
at the presumed peroxidase and hydroperoxide isomerase sites. The last two
residues, His and Asn, are conserved in all myeloperoxidases
(1). Middle and
bottom, amino acid residues of the presumed dioxygenation sites are
shown. Conserved residues in all sequences are in boldface, and
mutated residues of 10R-DOX and/or 7,8-LDS are marked by an
asterisk. B, alignment of partial amino acid sequences of
10R-DOX with ovine PGHS-1, and a secondary structure prediction of
the 10R-DOX sequence. The secondary structure of 10R-DOX was
predicted by PSIPRED (43) and
the secondary structure of ovine PGHS-1 from its crystal structure (Protein
Data Bank code 1diy; cf. Ref
19). In short, our first
strategy for site-directed mutagenesis was to switch hydrophobic residues
between the enzymes with 10R- and 8R-DOX activities and to
assess the effects on the DOX and hydroperoxide isomerase activities
(10R-DOX/7,8-LDS: Leu-306/Val-256, Leu-384/Val-330, Val-388/Leu-334,
and Ala-426/Ile-375) and to switch one hydrophobic/charged residue
(Ala-435/Glu-384). Only catalytically active pairs would provide clear
information on their importance for the position of dioxygenation
(e.g. L384V of 10R-DOX and V330L of 7,8-LDS, both of which
were active). Unfortunately, replacements of 7,8-LDS often led to inactivation
or very low activity (e.g. V330A, V330M, I375A, E384A). Our second
strategy was to study replacements in two homologous positions of ovine PGHS-1
(Val-349 and Ser-353) with smaller and larger hydrophobic residues,
i.e. at Leu-384 and Val-388 of 10R-DOX. Abbreviations used
are as follows: oCOX-1, ovine cyclooxygenase-1; Af, A.
fumigatus; Gg, G. graminis. The GenBank™ protein sequences
were derived from , P05979, EAL89712, AAD49559, and EAL84400. The
amino acid sequences were aligned with the ClustalW algorithm (DNAStar).The overall three-dimensional structures of myeloperoxidases are conserved.
It is therefore conceivable that important residues for substrate binding in
the cyclooxygenase channel of PGHS could be conserved in LDS and
10R-DOX. The three-dimensional structure of ovine PGHS-1 shows that
Val-349 and Ser-353 are close to C-3 and C-4 of 20:4n-6, and residues
in these positions can alter both position and stereospecificity of
oxygenation
( ACL1417722-24).
Replacement of Val-349 of PGHS-1 with alanine increased the biosynthesis of
11R-HETE, whereas V349L decreased the generation of
11R-H(P)ETE and increased formation of
15(R/S)-H(P)ETE
(23,
25). V349I formed
PGG2 with 15R configuration
(22,
24). Replacement of Ser-353
with threonine reduced cyclooxygenase and peroxidase activities by over 50%
and increased the biosynthesis of 11R-HPETE and 15S-HPETE
4-5 times (23).There is little information on the hydroperoxide isomerase and peroxidase
sites of LDS (18,
26), but the latter could be
structurally related to the peroxidase site of PGHS. PGG2 and
presumably 8R-HPODE bind to the distal side of the heme group, which
can be delineated by hydrophobic amino acid residues
(27). Val-291 is one of these
residues, which form a dome over the distal heme side of COX-1. The V291A
mutant retained cyclooxygenase and peroxidase activities
(27). 5,8- and 7,8-LDS also
have valine residues in the homologous position, whereas 8,11-LDS and
10R-DOX have leucine residues
(Fig. 1). Whether these
hydrophobic residues are important for the peroxidase activities is
unknown.In this study we decided to compare the two catalytic sites of
10R-DOX of A. fumigatus and 7,8-LDS (EC 1.13.11.44) of
G. graminis (18). Our
first aim was to find a robust expression system for 10R-DOX of
A. fumigatus. The second objective was to determine whether
C16 and C20 fatty acid substrates enter the oxygenation
site of 10R-DOX “head” or “tail” first.
Unexpectedly, we found that 10R-DOX oxygenated 20:4n-6 by
hydrogen abstraction at both C-13 and C-10 with formation of two nonconjugated
and four cis-trans-conjugated HPETEs. Our third objective was to
investigate the structural differences between 10R-DOX and 7,8-LDS of
G. graminis, which could explain that oxygenation of 18:2n-6
mainly occurred at C-10 and at C-8, respectively. The strategy for
site-directed mutagenesis of 10R-DOX and 7,8-LDS is outlined in the
legend to Fig. 1; an alignment
of the amino acid sequences of 10R-DOX and 7,8-LDS is found in
supplemental material. 相似文献
992.
Marc F Schetelig Carlos Caceres Antigone Zacharopoulou Gerald Franz Ernst A Wimmer 《BMC biology》2009,7(1):4
Background
The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness. 相似文献993.
994.
995.
996.
997.
Nancy Lewis Ernst Brian Panicucci Jason Carnes Kenneth Stuart 《RNA (New York, N.Y.)》2009,15(5):947-957
Mitochondrial RNAs in trypanosomes are edited by the insertion and deletion of uridine (U) nucleotides to form translatable mRNAs. Editing is catalyzed by three distinct editosomes that contain two related U-specific exonucleases (exoUases), KREX1 and KREX2, with the former present exclusively in KREN1 editosomes and the latter present in all editosomes. We show here that repression of KREX1 expression leads to a concomitant reduction of KREN1 in ∼20S editosomes, whereas KREX2 repression results in reductions of KREPA2 and KREL1 in ∼20S editosomes. Knockdown of KREX1 results in reduced cell viability, reduction of some edited RNA in vivo, and a significant reduction in deletion but not insertion endonuclease activity in vitro. In contrast, KREX2 knockdown does not affect cell growth or editing in vivo but results in modest reductions of both insertion and deletion endonuclease activities and a significant reduction of U removal in vitro. Simultaneous knockdown of both proteins leads to a more severe inhibition of cell growth and editing in vivo and an additive effect on endonuclease cleavage in vitro. Taken together, these results indicate that both KREX1 and KREX2 are important for retention of other proteins in editosomes, and suggest that the reduction in cell viability upon KREX1 knockdown is likely a consequence of KREN1 loss. Furthermore, although KREX2 appears dispensable for cell growth, the increased inhibition of editing and parasite viability upon knockdown of both KREX1 and KREX2 together suggests that both proteins have roles in editing. 相似文献
998.
Ya-Yi Chang Hildegard T. Greinix Anne M. Dickinson Daniel Wolff Graham H. Jackson Reinhard Andreesen Ernst Holler Gerhard C. Hildebrandt 《Cytokine》2009,48(3):218-225
Pro-inflammatory and dendritic cell-activating properties of macrophage migration inhibitory factor (MIF) suggest a potentially important role for MIF in alloantigen-specific immune responses after allogeneic stem cell transplantation (allo-SCT). We tested whether MIF −173 G/C gene polymorphism of donor or patient had impacts on the outcomes after allo-SCT. Four hundred and fifty-four donor–patient pairs were genotyped and mortality, relapse, and development of complications were analyzed. Patient but not donor MIF −173*C allele was associated with improved overall survival (OS) (5 years: 60.8% versus 46.3%, p = 0.042) and disease free survival (DFS) (5 years: 55.4% versus 39.5%; p = 0.014) due to a reduction in relapse (day 2000: 22.8% versus 42.0% p = 0.006) but not due to decreased transplantation-related mortality (TRM) (p = 0.44). Multivariate analysis proved patient −173*C allele as an independent factor for reducing relapse after allo-SCT (p = 0.023). Subgroup analysis showed a clear MIF −173*C allele-related reduction in relapse for those patients who did not receive T cell depleted (TCD) SCT (p = 0.01) in contrast to patients receiving TCD SCT (p = 0.20). In summary, patient MIF −173*C allele may be linked to specific, yet unrevealed functions in tumor biology and graft versus leukemia and lymphoma effects and potentially presents a novel prognostic marker for patient-tailored counseling and therapy in allo-SCT. 相似文献
999.
Background
Risk-taking behavior is a major cause of morbidity and mortality in adolescence. In the context of decision theory and motivated (goal-directed) behavior, risk-taking reflects a pattern of decision-making that favors the selection of courses of action with uncertain and possibly harmful consequences. We present a triadic, neuroscience systems based model of adolescent decision-making.Method
We review the functional role and neurodevelopmental findings of three key structures in the control of motivated behavior, i.e., amygdala, nucleus accumbens and medial/ventral prefrontal cortex. We adopt a cognitive neuroscience approach to motivated behavior that uses a temporal fragmentation of a generic motivated action. Predictions about the relative contributions of the triadic nodes to the three stages of a motivated action during adolescence are proposed.Results
The propensity during adolescence for reward-novelty seeking in the face of uncertainty or potential harm might be explained by a strong reward system (nucleus accumbens), a weak harm avoidant system (amygdala) and/or an inefficient supervisory system (medial/ventral prefrontal cortex). Perturbations in these systems may contribute to the expression of psychopathology, illustrated here with depression and anxiety.Conclusions
A triadic model, integrated in a temporally organized map of motivated behavior, can provide a helpful framework that suggests specific hypotheses of neural bases of typical and atypical adolescent behavior. 相似文献1000.