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1.
Stimulation of the production of hypericins by mannan in Hypericum perforatum shoot cultures 总被引:2,自引:0,他引:2
Shoot organ cultures were established from callus derived from anthers of Hypericum perforatum flowers and the effect of elicitors on hypericin and pseudohypericin production in shoot organ cultures was investigated. Mannan stimulated pseudohypericin production up to four fold (0.82 mg/g dry wt) and hypericin production up to two fold (0.04 mg/g dry wt.) beta-1,3-glucan and pectin slightly stimulated pseudohypericin production (ca. two fold), but had no effect on hypericin production. On the other hand, yeast extract showed no stimulatory effect, on either hypericin or pseudohypericin production. 相似文献
2.
Kirakosyan A. B. Vardapetyan R. R. Charchoglyan A. G. Yamamoto H. Hayashi H. Inoue K. 《Russian Journal of Plant Physiology》2001,48(6):816-819
The stimulating effect of cork pieces on hypericin and pseudohypericin biosyntheses was studied in cells of shoots regenerated from the callus cultures of St. John's wort (Hypericum perforatumL.). The addition of the cork matrix slightly stimulated shoot growth and enhanced pseudohypericin biosynthesis about threefold (to 0.4 mg/g dry wt). Pseudohypericin production increased proportionally with the amount of cork material added (from 1 to 4 mg/ml of growth medium). Further increase in the amount of cork pieces inhibited both pseudohypericin production and shoot growth. Organic and aqueous extracts of cork pieces did not affect the production of these substances. 相似文献
3.
Erin L. Westman David J. McNally Armen Charchoglyan Dyanne Brewer Robert A. Field Joseph S. Lam 《The Journal of biological chemistry》2009,284(18):11854-11862
The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an
unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA). wbpB, wbpE, and wbpD
are thought to encode oxidase, transaminase, and N-acetyltransferase
enzymes. To characterize their functions, recombinant proteins were
overexpressed and purified from heterologous hosts. Activities of
His6-WbpB and His6-WbpE were detected only when both
proteins were combined in the same reaction. Using a direct MALDI-TOF mass
spectrometry approach, we identified ions that corresponded to the predicted
products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE
(UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction.
Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent
with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was
identified. Preparative quantities of UDP-d-GlcNAc3NA and
UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds
were purified by high-performance liquid chromatography, and their structures
were elucidated by NMR spectroscopy. This is the first report of the
functional characterization of these proteins, and the enzymatic synthesis of
UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.Gram-negative organisms such as Pseudomonas aeruginosa produce
lipopolysaccharide
(LPS)4 as an essential
component of the outer leaflet of the outer membrane. LPS can be conceptually
divided into three parts: lipid A, which anchors LPS into the membrane; core
oligosaccharide, which contributes to membrane stability; and the O-antigen,
which is a polysaccharide that extends away from the cell surface. In P.
aeruginosa, two types of O-antigen are observed: A-band O-antigen, which
is common to most strains, and B-band O-antigen, which is variable and
therefore used as the basis of the International Antigenic Typing Scheme
(1). P. aeruginosa
serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2,
because they all share common backbone sugar structures in their O-antigen
repeat units consisting of two di-N-acetylated uronic acids and one
2-acetamido-2,6-dideoxy-d-galactose
(N-acetyl-d-fucosamine). The minor structural variations
in the O-antigen repeat units that differentiate this serogroup into five
serotypes are: the type of glycosidic linkage between O-units (alpha
versus beta) that is formed by the O-antigen polymerase (Wzy),
isomers present (d-mannuronic or l-guluronic acid), and
acetyl group substituents
(2–4).
The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a
repeating trisaccharide of
2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAmA),
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose
(3).The biosynthesis of the two mannuronic acid derivatives has yet to be fully
understood and has been the subject of investigation by our group. To produce
UDP-d-ManNAc3NAcA, a five-step pathway has been proposed
(Fig. 1) that requires the
products of five genes localized to the B-band O-antigen biosynthesis cluster
(5). The O-antigen biosynthesis
cluster was shown to be identical for all serotypes within serogroup O2, which
further underscores the high similarity between these serotypes
(5). The five genes, including
wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be
essential for B-band LPS biosynthesis, because knockout mutants of each of
these genes are deficient in B-band O-antigen
(6–8).
Homologs of all five of the proteins required for the
UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other
bacterial pathogens, including Bordetella pertussis, Bordetella
parapertussis, and Bordetella bronchiseptica.
Cross-complementation of P. aeruginosa knockout mutants lacking
wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from
B. pertussis could fully restore LPS production in the P.
aeruginosa LPS mutants, suggesting that the genes from B.
pertussis are functional homologs of the wbp genes
(7). Homologs of these genes
could be identified in diverse bacterial species, demonstrating the importance
of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P.
aeruginosa (7).Open in a separate windowFIGURE 1.Proposed pathway for the biosynthesis of UDP-d-ManNAc3NAcA in
P. aeruginosa PAO1. The full names of the sugars are as follows:
GlcNAc, 2-acetamido-2-deoxy-d-glucose; GlcNAcA,
2-acetamido-2-deoxy-d-glucuronic acid; 3-keto-d-GlcNAcA,
2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid; GlcNAc3NA,
2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid; GlcNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-glucuronic acid; ManNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Adapted from Ref.
8.The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway,
WbpA, is a 6-dehydrogenase that converts
UDP-2-acetamido-2-deoxy-d-glucose
(N-acetyl-d-glucosamine; UDP-d-GlcNAc) to
UDP-2-acetamido-2-deoxy-d-glucuronic acid
(N-acetyl-d-glucosaminuronic acid,
UDP-d-GlcNAcA) using NAD+ as a coenzyme
(9)
(Fig. 1). Following this, the
second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an
oxidation reaction catalyzed by WbpB, forming
UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid
(3-keto-d-GlcNAcA), which in turn is used as the substrate for
transamination by WbpE, creating
UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid
(d-GlcNAc3NA).This residue is thought to be the substrate for WbpD, a putative
N-acetyltransferase of the hexapeptide acyltransferase superfamily
(10) that requires acetyl-CoA
as a co-substrate (8). WbpD has
been proposed to synthesize
UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid
(UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of
P. aeruginosa serotype O1. In P. aeruginosa serogroup O2,
the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the
UDP-d-ManNAc3NAcA required for incorporation into B-band LPS
(11). A derivative of
UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen
of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is
thought to be produced through additional modification of
UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase,
which has also been demonstrated to be essential for the production of B-band
O-antigen (12,
13).In the current study, our aim was to define the function of WbpB, WbpE, and
WbpD, because only genetic evidence has previously been given for the
involvement of wbpB and wbpE
(7), and the reaction catalyzed
by WbpD could not be demonstrated due to the unavailability of its presumed
substrate, UDP-d-GlcNAc3NA
(8). The functional
characterization of these proteins is also important for understanding LPS
biosynthesis in B. pertussis, because the genes in the LPS locus of
this species, wlbA, wlbC, and wlbB, could cross-complement
knockouts of wbpB, wbpE, and wbpD, respectively, when
expressed in P. aeruginosa PAO1
(7). Furthermore, these three
proteins form a cassette for the generation of C-3 N-acetylated
hexoses and may be important for the biosynthesis of a variety of other
sugars. Capillary electrophoresis and MALDI-TOF mass spectrometry were used to
analyze reaction mixtures of WbpB and WbpE and showed that the expected
products were produced only when both enzymes were present together. Achieving
the enzymatic synthesis of the product of both enzymes, which was demonstrated
to be UDP-d-GlcNAc3NA by 1H NMR spectroscopy, was a key
breakthrough, because this rare sugar has never before been produced by any
means. UDP-d-GlcNAc3NA was also essential for use as the substrate
of WbpD, which not only allowed us to determine the enzymatic activity of this
protein but also allowed the enzymatic synthesis of
UDP-d-GlcNAc3NAcA to be achieved as well. Although this sugar had
previously been produced through a 17-step chemical synthesis
(11,
14), the 4-step concurrent
enzymatic reaction demonstrates the advantage of linking chemistry with
biology and represents a significant saving of both time and reagents as
compared with chemical synthesis. Finally, our data also showed the success in
reconstituting in vitro the 5-step pathway for the biosynthesis of
UDP-d-ManNAc3NAcA in P. aeruginosa. 相似文献
4.
Differential accumulation of hyperforin and secohyperforin in Hypericum perforatum tissue cultures 总被引:1,自引:0,他引:1
Charchoglyan A Abrahamyan A Fujii I Boubakir Z Gulder TA Kutchan TM Vardapetyan H Bringmann G Ebizuka Y Beerhues L 《Phytochemistry》2007,68(21):2670-2677
Hyperforin is a pharmacologically active constituent of Hypericum perforatum (St. John's wort). In vitro cultures of this medicinal plant were found to contain hyperforin and three related polyprenylated acylphloroglucinol derivatives. The accumulation of these compounds was coupled to shoot regeneration, with secohyperforin being the major constituent in morphogenic cultures. The structure of secohyperforin was elucidated online by LC-DAD, -MS, and -NMR. In multiple shoot cultures, the ratio of hyperforin to secohyperforin was strongly influenced by the phytohormones N6-benzylaminopurine (BAP) and naphthalene-1-acetic acid (NAA). While increasing concentrations of BAP stimulated the formation of hyperforin, increasing concentrations of NAA elevated the level of secohyperforin. No differential stimulation was observed after elicitor treatment. Hyperforin and secohyperforin are proposed to arise from a branch point in the biosynthetic pathway. 相似文献
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