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981.
982.
Mark Eugene Mort Christopher P. Randle Priscilla Burgoyne Gideon Smith Ernst Jaarsveld Stephen D. Hopper 《Plant Systematics and Evolution》2009,283(3-4):211-217
Analysis of cpDNA matK sequences for a total of 43 members of the succulent plant family Crassulaceae, including 24 taxa of Crassula, recovered a well-supported clade comprising Crassula species that is sister to the remainder of the family. The resulting topologies do not support the monophyly of the currently recognized subgenera of Crassula, as one member of subgenus Disporocarpa (C. crenulata) is placed as sister to an otherwise monophyletic subgenus Crassula. The major synapomorphy that has been used to recognize the latter subgenus is a base chromosome number of x = 7 versus a base of x = 8 in the other subgenus. We cannot assess the utility of this feature for defining subgenus Crassula because a chromosome count of C. crenulata has yet to be published. The five accessions of the recently resurrected segregate genus Tillaea (of 24 total Crassula species) included here were placed in four separate, well-supported lineages, one of which is greatly removed from the other four accessions. This suggests that this genus is not valid and should not be recognized. An initial examination of the evolution of habit indicates that a perennial habit is ancestral and that the annual habit is a feature that has been derived at least twice in the genus. 相似文献
983.
Daniela Stokmaier Oleg Khorev Brian Cutting Rita Born Daniel Ricklin Thomas O.G. Ernst Fabienne Böni Kathrin Schwingruber Martin Gentner Matthias Wittwer Morena Spreafico Angelo Vedani Said Rabbani Oliver Schwardt Beat Ernst 《Bioorganic & medicinal chemistry》2009,17(20):7254-7264
A series of novel aryl-substituted triazolyl d-galactosamine derivatives was synthesized as ligands for the carbohydrate recognition domain of the major subunit H1 (H1-CRD) of the human asialoglycoprotein receptor (ASGP-R). The compounds were biologically evaluated with a newly developed competitive binding assay, surface plasmon resonance and by a competitive NMR binding experiment. With compound 1b, a new ligand with a twofold improved affinity to the best so far known d-GalNAc was identified. This small, drug-like ligand can be used as targeting device for drug delivery to hepatocytes. 相似文献
984.
Multi-category classification methods were used to detect SNP-mortality associations in broilers. The objective was to select a subset of whole genome SNPs associated with chick mortality. This was done by categorizing mortality rates and using a filter-wrapper feature selection procedure in each of the classification methods evaluated. Different numbers of categories (2, 3, 4, 5 and 10) and three classification algorithms (naïve Bayes classifiers, Bayesian networks and neural networks) were compared, using early and late chick mortality rates in low and high hygiene environments. Evaluation of SNPs selected by each classification method was done by predicted residual sum of squares and a significance test-related metric. A naïve Bayes classifier, coupled with discretization into two or three categories generated the SNP subset with greatest predictive ability. Further, an alternative categorization scheme, which used only two extreme portions of the empirical distribution of mortality rates, was considered. This scheme selected SNPs with greater predictive ability than those chosen by the methods described previously. Use of extreme samples seems to enhance the ability of feature selection procedures to select influential SNPs in genetic association studies. 相似文献
985.
John M. Antos Maximilian Wei-Lin Popp Robert Ernst Guo-Liang Chew Eric Spooner Hidde L. Ploegh 《The Journal of biological chemistry》2009,284(23):16028-16036
Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23). 相似文献
986.
987.
988.
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
989.
990.
Marc F Schetelig Carlos Caceres Antigone Zacharopoulou Gerald Franz Ernst A Wimmer 《BMC biology》2009,7(1):4