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(Last modification:
10. April 2010)
Typ III Polyketidsynthasen
(PKS) in Bakterien
Stilbensynthase (STS) Typ Ring-Faltung
Alkylresorcinole in
Streptomyces griseus
Schlüssel-Publikation: Funabashi et al., 2008
RppA, eine Typ III PKS aus diesem Bakterium, wurde bereits früher in vielen Einzelheiten
charakterisiert (Funa et al., 1999;
Funa et al., 2002a;
Funa et al., 2002b) und ebenso
ein ganz ähnliches Enzym aus
Streptomyces coelicor (Izumikawa
et al., 2003; Austin et al.,
2004; Li et al., 2007). Die Enzyme
katalysieren die Synthese von 1,3,6,8-Tetrahydroxynaphthalen (T4HN):
Mehr…
Bei der Vollendung der genomischen Sequenz von Streptomyces griseus (Ohnishi
et al., 2008) wurde eine weitere Typ III PKS entdeckt. Sie war Teil eines
Operons namens srs (Streptomyces Resorcinol
Synthese). Das Operon enthält drei Gene: srsA, die Typ III PKS,
SrsB, mit Ähnlichkeiten zu Methyltransferasen, und
srsC, mit
Motiven, die man aus Flavoprotein-Hydroxylasen kennt.
Bei der Analyse der Funktion wurden
zunächst Teile des Operons inaktiviert, und dann die Metaboliten-Profile
untersucht, nach Expression der Gene in transgenem
Streptomyces lividans
TK21. Die Zellen mit nur dem leeren Vektor
enthielten gar keine phenolischen Komponenten. Dies war anders mit den
verschiedenen Konstrukten. Sie und die Produkte sind in Fig. 1
zusammengefasst. SrsA allein führte zu
Bildung von Resorcinolen mit einer Methylgruppe am Resorcinol-Ring-System. Die
zusätzliche Expression von srsB resultierte
in methoxylierten Produkten, und wenn dazu noch srsC
exprimiert wurde, fand man Alkylchinone, die durch eine zusätzliche
Hydroxylierung und Oxidation erklärt werden konnten. Die Summe dieser Daten
führte zu dem Schluss, dass die Abfolge der Reaktionen so verlief wie in Fig. 1
dargestellt: Eine Polyketidsynthase (PKS)
zur Synthese des Resorcinol-Grundgerüstes, danach eine
O-Methyltransferase, gefolgt von einer zusätzlichen
Hydroxylierung und noch einer Oxidation.
Fig. 1.
Auf den ersten Blick sah die von SrsA katalysierte PKS-Reaktion sehr
ähnlich aus wie die STS-Typ-Reaktionen mit langkettigen Fettsäuren, die mit
anderen Typ III PKS gefunden wurden, also mit ArsB in dem Bakterium
Azotobacter vinelandii (Biosynthese von langkettigen Alkylresorcinolen, die
in den Cysten deponiert werden:
Mehr…), oder bei der
Biosynthese von Sorgoleone in der Hirse
(Sorghum bicolor (L.) Moench) (Mehr...).
Weiter unten sind noch andere Beispiele für Typ III PKS, die präferentiell mit
langkettigen Fettsäuren arbeiten: Mehr...
Es gab jedoch einen auffallenden Unterschied zu typischen STS-Reaktionen: Das
SrsA Produkt enthielt eine Methylgruppe an dem neu synthetisierten aromatischen
Ringsystem (s. Fig. 1). Das musste erklärt werden, denn die Standard-STS-Typ
Reaktion mit Malonyl-CoA führt keine C-Methylgruppe in den Resorcinol-Ring ein.
Dies wurde mit rekombinantem SrsA
untersucht. Da die Expression in E. coli nicht so gut klappte, wurde das
Enzym in
Streptomyces lividans
produziert. Die Ergebnisse waren ganz interessant; sie sind in
Fig. 2 zusammengefasst. Inkubation des Proteins nur mit Malonyl-CoA ergab
überhaupt kein Produkt. Wenn nur Methylmalonyl-CoA eingesetzt wurde, erhielt man
ein dimethyliertes Triketid-Pyron, welches sich einfach aus zwei Kondensationen
mit Methylmalonyl-CoA erklären lässt. Das in vivo identifizierte Produkt
erhielt man nur dann, wenn sowohl Malonyl-CoA als auch Methylmalonyl-CoA in dem
Enzymtest vorhanden waren. Die Ergebnisse zeigten also, dass SrsA die beiden
Kettenverlängerer in einer strikt kontrollierten Reihenfolge verwendete!
Unglücklicherweise brachten die Autoren in der Original-Publikation die
Reihenfolge durcheinander, aber das wurde in einem Erratum
korrigiert (siehe unten): Die Reihenfolge der
Kondensationen muss sein: Methylmalonyl-CoA,
Malonyl-CoA, und noch einmal
Malonyl-CoA, wie in Fig. 2 dargestellt.
Fig. 2.
R = langkettige Acyl-CoA Ester. Die Farben kennzeichnen die
Kettenverlänger in der Reaktions-Sequenz.
Dies waren wirklich aufregende Ergebnisse: Sie sind einzigartig, als das erste
überzeugende Beispiel, dass eine Typ III PKS diese beiden Kettenverlängerer in
einer genau kontrollierten Abfolge verwendet. Und das Schöne daran ist, dass die
Produkte auch einer physiologischen Rolle zugeordnet werden konnten. Diese
Alkyresorcinole haben vermutlich eine wichtige Rolle für die Zellwand: Sie sind
ein wichtiger Teil der Resistenz der Bakterien gegen Penicillin. Und noch etwas:
Die Suchen der Autoren in anderen Genomen zeigten, dass viele anderen Bakterien
zu srs eng verwandte Operone enthalten; das Supplement zu der Funabashi
et al. (2008) Publikation enthält eine lange und interessante Liste dafür. Es
sieht so aus, als ob solche Alkylresorcinole oder verwandte Substanzen viel
verbreiteter sind als bisher vermutet.
Eine interessante Frage ist, ob dieses Enzym wohl den "aldol switch mechanism"
verwendet, der mit der Stilbensynthase (STS)
aus der Kiefer (Pinus sylvestris) entdeckt wurde:
Mehr...
Das einzige andere Beispiel für die Verwendung verschiedener Kettenverlängerer
scheint die Curcuminoid-Synthase zu sein, die aus Reis (Oryza sativa)
kloniert wurde: Sie verwendet einen Phenylpropanoid-CoA Ester als Starter-Substrat,
bildet mit Malonyl-CoA ein Diketid, und verwendet dieses Diketid als
Kettenverlängerer in einer weiteren Kondensation mit 4-Coumaroyl-CoA als
Substrat: Mehr. Hochinteressant,
wenn auch mit dem kleinen Schönheitsfehler, dass Curcuminoide in Reis noch nie
gefunden wurden.
Es sollte hinzugefügt werden, dass die Verwendung von Methylmalonyl-CoA durch
Typ III PKS nichts Ungewöhnliches oder Neues ist. so etwas wurde bereits vor
langer Zeit mit einem Enzym aus der Kiefer
(Pinus sylvestris)
gezeigt; dieses Protein könnte an der Biosynthese von methylierten
Chalcon-Derivaten beteiligt sein: Mehr…Andere
Arbeiten lassen vermuten, dass Typ III PKS generell in der Lage sind,
Methylmalonyl-CoA als Kettenverlängerer zu akzeptieren (Abe et al.,
2002; Abe et al., 2003;
Abe et al., 2006). Dies scheint
jedoch in der Regel eine reine in vitro Aktivität darzustellen, denn sie
führt nicht zu Produkten, die aus den Pflanzen bekannt sind.
Zum Seitenanfang
Andere Typ
III PKS mit Substrat-Präferenzen für langkettige CoA-Estern
in Pflanzen und in Bakterien
-
-
-
-
Alkylresorcinole
und
langkettige Pyrone in dem Bakterium Azotobacter vinelandii:
Mehr...
-
Alkylresorcinol-
Streptomyces
griseus: Mehr...
-
Pyronsynthasen in dem Bakterium
Mycobacterium tuberculosis und
Bacillus subtilis: Mehr...
-
CsyA: Pyronsynthasen
in dem Pilz Aspergillus oryzae:
Mehr...
Links zu bakteriellen Typ III PKS
Zum Seitenanfang
Zitate
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Funabashi,
M., Funa, N., Horinouchi, S., 2008. Phenolic lipids synthesized by type III
polyketide synthase confer penicillin resistance on Streptomyces griseus.
Journal of Biological Chemistry 283, 13983-13991.
Type III polyketide synthases (PKSs) found in plants, fungi, and bacteria
synthesize a variety of aromatic polyketides. A Gram-positive, filamentous
bacterium Streptomyces griseus contained an srs operon, in
which srsA encoded a type III PKS, srsB encoded a
methyltransferase, and srsC encoded a flavoprotein hydroxylase.
Consistent with this annotation, overexpression of the srs genes in a
heterologous host, Streptomyces lividans, showed that SrsA was
a type III PKS responsible for synthesis of phenolic lipids,
alkylresorcinols, and alkylpyrones, SrsB was a methyltransferase
acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and
SrsC was a hydroxylase acting on the alkylresorcinol methyl ethers.
In vitro SrsA reaction showed that SrsA synthesized alkylresorcinols
from acyl-CoAs of various chain lengths as a starter substrate, one molecule
of methylmalonyl-CoA, and two molecules of malonyl-CoA. SrsA was thus unique
in that it incorporated the extender substrates in a strictly controlled
order of malonyl-CoA, malonyl-CoA, and methylmalonyl-CoA (see erratum
below) to produce alkylresorcinols. An srsA mutant, which produced no
phenolic lipids, was highly sensitive to beta-lactam antibiotics, such as
penicillin G and cephalexin. Together with the fact that the
alkylresorcinols were fractionated mainly in the cell wall fraction, this
observation suggests that the phenolic lipids, perhaps associated with the
cytoplasmic membrane because of their amphiphilic property, affect the
characteristic and rigidity of the cytoplasmic membrane/peptidoglycan of a
variety of bacteria. An srs-like operon is found widely among
Gram-positive and -negative bacteria, indicating wide distribution of the
phenolic lipids.
Protein accession:
YP_001821984
Zum Seitenanfang
Erratum in: J. Biol. Chem. 2008 Sep 5; Vol. 283, page
25104.
On Page
13990, the mode of ring folding of the tetraketide intermediate, leading to
resorcinol formation, in Fig. 5B was incorrect. The correct ring
folding is C-2-C-7 aldol condensation. This error does not change the
conclusions of this study. However, the order of condensation of the
extender
units is
updated as methylmalonyl-CoA, malonyl-CoA, and malonyl-CoA.
Zurück zum Text
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Ohnishi,
Y., Ishikawa, J., Hara, H., Suzuki, H., Ikenoya, M., Ikeda, H., Yamashita,
A., Hattori, M., Horinouchi, S., 2008. Genome sequence of the
streptomycin-producing microorganism Streptomyces griseus IFO 13350.
Journal of Bacteriology 190, 4050-4060.
We
determined the complete genome sequence of Streptomyces griseus IFO
13350, a soil bacterium producing an antituberculosis agent, streptomycin,
which is the first aminoglycoside antibiotic, discovered more than 60 years
ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an
average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA
operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal
inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide
sequence and secondary structure, consisting of several palindromes with a
loop sequence of 5'-GGA-3', are different from those of typical telomeres
conserved among other Streptomyces species. In accordance with the
difference, the chromosome has pseudogenes for a conserved terminal protein
(Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and
Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of
two related species, Streptomyces coelicolor A3(2) and
Streptomyces avermitilis, clarified not only the characteristics of the
S. griseus genome but also the existence of 24 Streptomyces-specific
proteins. The S. griseus genome contains 34 gene clusters or genes
for the biosynthesis of known or unknown secondary metabolites.
Transcriptome analysis using a DNA microarray showed that at least four of
these clusters, in addition to the streptomycin biosynthesis gene cluster,
were activated directly or indirectly by AdpA, which is a central
transcriptional activator for secondary metabolism and morphogenesis in the
A-factor (a gamma-butyrolactone signaling molecule) regulatory cascade in
S. griseus.
Zurück
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Abe, I., Takahashi, Y., Lou, W., Noguchi, H., 2003.
Enzymatic formation of unnatural novel polyketides from alternate starter
and nonphysiological extension substrate by chalcone synthase. Organic
Letters 5, 1277-1280.
In the
chalcone synthase (CHS) enzyme reaction, both the starter molecule and the
extension unit of the poyketide chain elongation reaction were
simultaneously replaced with nonphysiological substrates. When incubated
with benzoyl-CoA and methylmalonyl-CoA as substrates, recombinant CHS from
Scutellaria baicalensis afforded an unnatural novel triketide,
4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide,
4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the
other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as
substrates to produce an unnatural novel triketide,
4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.
Zurück
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Abe, I., Takahashi, Y., Noguchi, H., 2002. Enzymatic
formation of an unnatural C6-C5 aromatic polyketide by
plant type III polyketide synthases. Organic Letters 4, 3623-3626.
Substrate specificities of plant polyketide synthases (PKSs) were
investigated using analogues of malonyl-CoA, the extension unit of the
polyketide chain elongation reactions. When incubated with methylmalonyl-CoA
and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria
baicalensis, stilbene synthase from Arachis hypogaea, and
benzalacetone synthase from Rheum palmatum) afforded an unnatural
C(6)-C(5) aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by
one-step decarboxylative condensation of the two substrates. In contrast,
succinyl-CoA was not accepted as a substrate by the enzymes.
Zurück
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Abe,
T., Noma, H., Noguchi, H., Abe, I., 2006.
Enzymatic
formation of an unnatural methylated triketide by plant type III polyketide
synthases. Tetrahedron Letters 47, 8727-8730.
Octaketide synthase, a novel plant-specific type III polyketide synthase
from
Aloe
arborescens,
efficiently accepted (2RS)-methylmalonyl-CoA
as a sole substrate to produce 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone. On
the other hand, a tetraketide-producing chalcone synthase from
Scutellaria baicalensis
and a diketide-producing benzalacetone synthase from
Rheum
palmatum
also yielded the unnatural methylated C9 triketide pyrone as a single
product by sequential decarboxylative condensations of three molecules of (2RS)-methylmalonyl-CoA.
Zurück
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Austin, M. B., Izumikawa, M., Bowman, M. E., Udwary,
D. W., Ferrer, J.-L., Moore, B. S., Noel, J. P., 2004. Crystal structure of
a bacterial type III polyketide synthase and enzymatic control of reactive
polyketide intermediates. Journal of Biological Chemistry 279, 45162-45174.
In
bacteria, a structurally simple type III polyketide synthase (PKS) known as
1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative
condensation of five CoA-linked malonyl units to form a pentaketide
intermediate. THNS subsequently catalyzes dual intramolecular Claisen and
aldol condensations of this linear intermediate to produce the fused ring
tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed
polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic
triad in an internal active site cavity, is fairly well understood. However,
the mechanistic basis for the unusual production of THN and dual cyclization
of its malonyl-primed pentaketide is obscure. Here we present the first
bacterial type III PKS crystal structure, that of Streptomyces coelicolor
THNS, and identify by mutagenesis, structural modeling, and chemical
analysis the unexpected catalytic participation of an additional THNS-conserved
cysteine residue in facilitating malonyl-primed polyketide extension beyond
the triketide stage. The resulting new mechanistic model, involving the use
of additional cysteines to alter and steer polyketide reactivity, may
generally apply to other PKS reaction mechanisms, including those catalyzed
by iterative type I and II PKS enzymes. Our crystal structure also reveals
an unanticipated novel cavity extending into the "floor" of the traditional
active site cavity, providing the first plausible structural and mechanistic
explanation for yet another unusual THNS catalytic activity: its previously
inexplicable extra polyketide extension step when primed with a long acyl
starter. This tunnel allows for selective expansion of available active site
cavity volume by sequestration of aliphatic starter-derived polyketide
tails, and further suggests another distinct protection mechanism involving
maintenance of a linear polyketide conformation.
Zurück
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Funa, N., Ohnishi, Y., Ebizuka, Y., Horinouchi, S.,
2002a. Alteration of reaction and substrate specificity of a bacterial type
III polyketide synthase by site-directed mutagenesis. Biochemical Journal
367, 781-789.
RppA, which
belongs to the type III polyketide synthase family, catalyses the synthesis
of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of
melanin biosynthesis in the bacterium Streptomyces griseus. The
reaction of THN synthesis catalysed by RppA is unique in the type III
polyketide synthase family, in that it selects malonyl-CoA as a starter
substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in
plant chalcone synthases, as revealed by analyses of active-site mutants
having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA.
Site-directed mutagenesis of the amino acid residues that are likely to form
the active-site cavity revealed that the aromatic ring of Tyr(224) is
essential for RppA to select malonyl-CoA as a starter substrate, since
substitution of Tyr(224) by amino acids other than Phe and Trp abolished the
ability of RppA to accept malonyl-CoA as a starter, whereas the mutant
enzymes Y224F and Y224W were capable of synthesizing THN via the
malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I
was found to produce only a triketide pyrone from hexanoyl-CoA as starter
substrate, although wild-type RppA synthesizes tetraketide and triketide
pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of
Ala(305) mutants and identification of their products showed that the
substitution of Ala(305) by bulky amino acid residues restricted the number
of elongations of the growing polyketide chain. Both Tyr(224) (important for
starter substrate selection) and Ala(305) (important for intermediate
elongation) were found to be conserved in three other RppAs from
Streptomyces antibioticus and Streptomyces lividans.
Zurück
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Funa, N., Ohnishi, Y., Ebizuka, Y., Horinouchi, S.,
2002b. Properties and substrate specificity of RppA, a chalcone synthase-related
polyketide synthase in Streptomyces griseus. Journal of
Biological Chemistry 277, 4628-4635.
RppA, a
chalcone synthase-related polyketide synthase (type III polyketide synthase)
in the bacterium Streptomyces griseus, catalyzes the formation of
1,3,6,8-tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA
(i.e. starter malonyl-CoA + 4 condensations with malonyl-CoA). The Km value
for malonyl-CoA and the kcat value for THN synthesis were determined to be
0.93 +/- 0.1 µM and 0.77 +/- 0.04 min(-1), respectively. RppA accepted
aliphatic acyl-CoAs with the carbon lengths from C4 to C8 as starter
substrates and catalyzed sequential condensation of malonyl-CoA to yield
alpha-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide,
4-hydroxy-6-(2,4,6-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five
molecules of malonyl-CoA, suggesting that the size of the active site cavity
of RppA is larger than any other chalcone synthase-related enzymes found so
far in plants and bacteria. RppA was also found to synthesize a C-methylated
pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the
starter and methylmalonyl-CoA as an extender. Thus, the broad substrate
specificity of RppA yields a wide variety of products.
Zurück
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Funa, N., Ohnishi, Y., Fujii, I., Shibuya, M.,
Ebizuka, Y., Horinouchi, S., 1999. A new pathway for polyketide synthesis in
microorganisms. Nature 400, 897-899.
Chalcone
synthases, which biosynthesize chalcones (the starting materials for many
flavonoids(1,2)), have been believed to be specific to plants. However, the
rppA gene from the Gram-positive, soil-living filamentous bacterium
Streptomyces griseus encodes a 372-amino-acid protein that shows
significant similarity to chalcone synthases'. Several rppA-like genes are
known, but their functions and catalytic properties have not been described.
Here we show that a homodimer of RppA catalyses polyketide synthesis: it
selects malonyl-coenzyme-A as the starter, carries out four successive
extensions and releases the resulting pentaketide to cyclize to
1,3,6,8-tetrahydroxynaphthalene (THN). Site-directed mutagenesis revealed
that, as in other chalcone synthases(4,5), a cysteine residue is essential
for enzyme activity. Disruption of the chromosomal rppA gene in S. griseus
abolished melanin production in hyphae, resulting in 'albino' mycelium. THN
was readily oxidized to form 2,5,7-trihydroxy-1,4- naphthoquinone(flaviolin),
which then randomly polymerized to form various coloured compounds. THN
formed by RppA appears to be an intermediate in the biosynthetic pathways
for not only melanins but also various secondary metabolites containing a
naphthoquinone ring. Therefore, RppA is a chalcone-synthase- related
synthase that synthesizes polyketides and is found in the Streptomyces and
other bacteria.
Zurück
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Izumikawa, M., Shipley, P. R., Hopke, J. N.,
O'Hare, T., Xiang, L., Noel, J. P., Moore, B. S., 2003. Expression and
characterization of the type III polyketide synthase
1,3,6,8-tetrahydroxynaphthalene synthase from Streptomyces coelicolor
A3(2). Journal of Industrial Microbiology and Biotechnology 30, 510-515.
Sequence analysis of the metabolically rich 8.7-Mbp genome of the model
actinomycete Streptomyces coelicolor A3(2) revealed three genes
encoding predicted type III polyketide synthases (PKSs). We report the
inactivation, expression, and characterization of the type III PKS
homologous SCO1206
gene product as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS).
Incubation of recombinant THNS with malonyl-CoA showed THN production, as
demonstrated by UV and HPLC analyses. The K(m) value for malonyl-CoA and the
k(cat) value for THN synthesis were determined spectrophotometrically to be
3.58+/-0.85 µM and 0.48+/-0.03 min(-1), respectively. The C-terminal
region of S. coelicolor THNS, which is longer than most other bacterial and
plant type III PKSs, was shortened by 25 amino acid residues and the
resulting mutant was shown to be slightly more active (K(m)=1.97+/-0.19
µM, k(cat)=0.75+/-0.04 min(-1)) than the wild-type enzyme.
Zurück
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Li, S., Grüschow, S., Dordick, J. S., Sherman, D. H.,
2007. Molecular analysis of the role of tyrosine 224 in the active site of
Streptomyces coelicolor RppA, a bacterial type III polyketide
synthase. Journal of Biological Chemistry 282, 12765-12772.
Streptomyces coelicolor
RppA (Sc-RppA), a bacterial type III polyketide synthase, utilizes
malonyl-CoA as both starter and extender unit substrate to form
1,3,6,8-tetrahydroxynaphthalene (THN) (therefore RppA is also known as THN
synthase (THNS)). The significance of the active site Tyr224 for substrate
specificity has been established previously, and its aromatic ring is
believed to be essential for RppA to select malonyl-CoA as starter unit.
Herein, we describe a series of Tyr224 mutants of Sc-RppA including Y224F,
Y224L, Y224C, Y224M, and Y224A that were able to catalyze a physiological
assembly of THN, albeit with lower efficiency, challenging the necessity for
the Tyr224 aromatic ring. Steady-state kinetics and radioactive substrate
binding analysis of the mutant enzymes corroborated these unexpected
results. Functional examination of the Tyr224 series of RppA mutants using
diverse unnatural acyl-CoA substrates revealed the unique role of
malonyl-CoA as starter unit substrate for RppA, leading to the development
of a novel steric-electronic constraint model.
Zurück
Zum Seitenanfang
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