1 3. biosynthesis of polyketide und non-ribosomal-peptide ... · the polyketides constitute a large...
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1 3. Biosynthesis of Polyketide und Non-Ribosomal-Peptide Natural Products 3.1 Polyketide Biosynthesis The polyketides constitute a large class of natural products, with widely varying structures. Some are aromatic, others aliphatic, some are cyclic others are acyclic. Polyketide metabolites are found in essentially all organisms, where they have widely differing biological functions, from simple dyestuffs (e.g. in plants) to antibiotics (in microorganisms). The polyketides form one of the largest classes of natural products. Many have been discovered in screening programs aimed at the isolation and discovery of new biologically active compounds, useful in the pharmaceutical industry. Despite the enormous variety of different structures seen whithin the polyketide family, we can nevertheless classify them in two main groups - the aliphatic (or reduced) and the aromatic polyketides, e.g. Aliphatic (reduced) polyketides:
OO O O
O
NaO2C
Me
MeO
Me
HO
MeMe
Me
Me
Me
Me
HO
HO
OH
O
Me
O
Me
OH
Me
Me
Me
O
Me
OO
MeO
HONMe2
Me
O
OMe
Me
OH
Me
H
HO
O
O
O
O
O
O
O
OO
O O
HO
Me
Me
MeMe
Me
MeMe
O
CHO
Monensin A
Erythromycin A
Brevetoxin B
O
O
O
O
OO
Et
OH
HOMe
O
OO
OMe
OMe
HO
H
H
Avermectin A1a
O
O OHO
HO
MeO
N O
OO
OMe
OH
OMe
FK506
S
N
O
OO
OH
O
OH
Epothilon A
2 Aromatic polyketides:
HOOC
Me
OH
O
MeH
H
O
O
OH
OH
COOH
OH
Me
O
O
O
OO
O
H
HMeO
OHOH
O
OH
NMe2
O
NH2
OHOH
6-Methylsalicylic acidActinorhodin
2
Aflatoxin
Oxytetracycline
MeO
O
O
O
O
OH
OH
OH
OMe
NH2OH
Daunorubicin
O
NH
Cl
Cl
OH
HO
Pyoluteorin
Mixed origin There are also many natural products that are of mixed biosynthetic origin, i.e. part of the structure is derived from a polyketide pathway and part from some other pathway (e.g. terpenoid) e.g.
O
OH
C5H11
Tetrahydrocannabinol(Cannabis sativa)
Terpene-Polyketide
O OH
HO OHHO
Chalcone
Shikimic acid-Polyketide
O
O
Me
O
HN
NHO
O
O
O
Me
Cryptophycin-1
Cl
OMe
Polyketide-Aminoacid-Hydroxyacid
O
OH
OH
OHHO
OH
Catechin(a Flavonoid)Shikimic acid-Polyketide
Although the polyketides have widely diverse chemical structures, they have a common biosynthetic origin; they contain a carbon backbone that is constructed from building blocks derived from small fatty acids, such as acetate, propionate, etc. Arthur J. Birch recognized as early as 1957 that many natural products have structures that appear as though they might be produced from simple fatty acids by a process that involves repeated head-to-tail coupling, to produce as an intermeidate a poly-ß-ketide. The poly-ß-ketide then should undergo various plausible transformations to finally give the natural product. When 14C-labelled compounds became available, the Birch Hypothesis could be tested experimentally by feeding the labelled precursors to the producing organisms.
3 For example, the aromatic compound 6-methyl salicylic acid is produced by a fungus (Penicillium griseofulvum) and orsenillic acid is produced by a lichen. When 1-14C-acetate is fed to these organisms, the positions that become labelled are those predicted by the Birch hypothesis:
Me COOH
CO-SX
O
O
O
Me
OH
COOH
MeHO
Me
O
SCoA
Me
O
SCoA
usw
A cell
Orsellinic acid
Me CHOOH
CO-SX
O
O
O
Me
Me
O
SCoA
Me
O
SCoA
usw
A cell
6-Methylsalicylic acid
COOH
Me
OH
There are several key chemical (mechanistic) points to remember about the chemical reactions involved in the assembly of polyketide natural products: 1) The small fatty acid precursors are first converted by enzymes in the cell into coenzyme-A thioesters. Hence, the carboxylic acid must be activated, and for this ATP is used :
O P O P O O
HO OH
N
N
N
N
NH2
O-
O-
O O
P
O-O
O-
R
O
O
ATP
HSN N
H
O
H
O
OH
Me Me
O P
O
P
O
OO
NH2
N
NN
N
OHHO
OOO
R
O
O P O O
HO OH
N
N
N
N
NH2
O-
O
CoA-SH
R-CO-O-AMP
4 2) The building blocks are loaded onto carrier domains, and remain bound to these domains during chain assembly. The loading steps are catalyzed by acyl transferases (AT). These enzymes contain a catalytic triad (Asp-His-Ser) in the active site: 3) In carbon chain assembly, the new C-C-bonds are formed, NOT by Claisen-type condensations (as it might appear), but rather in decarboxylative-condensations using malonyl-coenzyme-A half-thioesters:
CH3
O
SRO
SR
CH3
O
SR
H
CH3
O
SR'
O
SR"
O O
CH3
O
SR'
O
SR"
O O
H
R
CH3
O
SR'
O
SR"
CO O
H
R
H
R
R = H, Me, Et ......
ß-KetoacylSynthase (KS)
!
! ÜZ
CH3 SR"
O O
R
CH3 SR"
O O
Alkyl-Malonyl-Thioester
Claisen-like
The Claisen-type condensation is reversible, whereas the decarboxylative-condensation is not reversible. This is a major difference, since the coupling reaction must be repeated many times to build up a polyketide chain. The product of the coupling step is a ß-ketothioester, catalyzed by a ß-ketoacyl synthase (KS). 4) The keto-group in the ß-ketothioester can be reduced by an NAD(P)H-dependent dehydrogenase to give a ß-hydroxythioester of either configuration (R or S), dependening upon the stereospecificity of the enzyme:
R SR'
O ONADH NAD+
Ketoreductase (KR) 5) The ß-hydroxy group can be lost in a ß-elimination reaction, catalyzed by a dehydratase, to give an α,ß-unsaturated thioester :
R SR'
OH O Dehydratase (DH)
The double bond can in principle have either the E- or Z-configuration, depending on the specificity of the dehydratase. 6) The double bond in the α,ß-unsaturated thioester can be reduced to a fully saturated thioester, again catalyzed by a NAD(P)H-dependent enoyl reductase (dehydrogenase):
5
R SR'
ONADH NAD+
Enolyreductase (ER) With this set of reactions it is possible to install either a keto-group, an alcohol, a double bond, or a fully saturated unit, in the growing polyketide chain:
R
O
SR'
O
SR"
O O
R SR'
O O
R SR'
OH O
R SR'
O
R SR'
OKS KR DH ER
R'"R'" R'" R'" R'"
R'" = H, Me. Et oder ........ 7) As a last step in the chain assembly, the thioester is typically hydrolyzed to a free carboxylic acid, or cyclized to a lactone or lactam, catalyzed by a so-called thioesterase. The thioesterases belong mechanistically to the serine-protease class of enzymes, and so have a catalytic triad in the active site (Asp-His-Ser), with a catalytically important Ser acting as nucleophile :
R SR'
O Thioesterase (TE)
We now have a small library of catalytic activities that can be used for fatty acid and polyketide assembly. These activities can be combined in various ways to generate the large class of polyketide natural products, as shown below. One important example is the multienzyme complex involved in fatty acid biosynthesis. Fatty acid synthases (FASs) function very much like the polyketide synthases (PKSs). 3.2. Fatty acid biosynthesis The biosynthesis of long chain saturated fatty acids (e.g. palmitic and stearic acids) is catalyzed by a large multi-enzyme complex, called the fatty acid synthase (FAS) complex. Typically, the FASs take acetyl-CoA as starter unit, and then extend the chain in a step-wise manner, using malonyl-CoA as extender units. The malonyl-CoA is produced in a biotin-dependent enzymic reaction from acetyl-CoA (see. Biochemistry, 2004, 43, 14035): overall:
CH3
O
S-CoA
Acetyl-CoA Carboxylase
ATPHCO3
ADPPi
O
S-CoA
COO
HN NH
S
O
HN
LYS
O
6 Step 1:
HO
O
O HO
O
O
P
O
O
O
N NH
S
O
HNLYS
O
O
O
ATP
HCO3
ADP
Pi
1-N-Carboxybiotin Step 2:
N NH
S
O
HNLYS
O
O
O
CH2
O
CoA-S
O
S-CoA
COO
C
O
O
N NH
S
O
HNLYS
O
H
CH2
O
CoA-S
C
O
O HN NH
S
O
HNLYS
O The FASs are multienzyme complexes, which catalyze overall the following transformation:
CH3
O
S-CoA
O
S-CoA
COOH
CH3 CH2 CH2 C
O
OH
+ 8 CoA-SH
14 NADP+14 NADPH
+ 77Fatty acid synthase
Multi enzyme complex
All of the intermediates in the assembly process remain bound covalently to the FAS as thioesters linked to a phosphopantetheinyl group attached to the so-called “Acyl-Carrier-Protein (ACP)” :
HSN N
OP
H
O
H
O
OH
Me Me
O
O-
O P
O
O-
OO
N
2-O3PO OH
N
N
N
NH2
HSN N
OP
H
O
H
O
OH
Me Me
O
O-
O CH2
ACP
Ser Phosphopantetheine group in the ACP
Coenzyme-A
7 In plants and most procaryotes 8 distinct proteins are required for fatty acid biosynthesis (the so-called type-II FASs). These 8 proteins work together to assemble fatty acid molecules. The ACP is a small carrier protein containing about 80 amino acids. The active site Ser (modified with the pantetheinyl group) is strictly conserved. Its function is to carry the growing fatty acid chain from one enzyme to the next, in each catalytic cycle. In animals and fungi, Nature has combined these separate proteins into one or two giant proteins, which fold into discrete domains, where each domain then catalyzes one step in the assembly process (the type-I FASs). The complete catalytic cycle is shown below:
HOOC(CH2CH2)nCH3
Thioesterase (TE)
Enoyl-Reductase(ER)
NADP+
NADPH
Dehydratase(DH)
NADP+
NADPH
ß-Ketoacylreductase (KR)
ß-Ketoacyl synthase (KS)
Malonyl-CoAAcetyl-CoA
SH
S.COCH3
SH
S.COCH3
S
S
S
OH
S
S
SCO(CH2CH2)nCH3
CH3
O
O
COOH
O O
Me
Me
O
O
S
Me
O
Me
ACP
ACP
SH
Malonyl-AcetylTransferase(MAT)
ACP
ACP
ACP
ACP
SH
SHACP
ACP
ACP
H2O
CO2
CoASH
CoASH
KS
KS
KS
KS
KS
Malonyl-AcetylTransferase(MAT)
In 2006 the first crystal structures of both mammalian (α2) and fungal FASs (α6ß6) were published (Science 2006, 311, 1263). Later higher resolution structures were published (Science 2008, 321, 1315).
8 3.3. The “aromatic polyketides” (Nat. Prod. Rep. 1999, 16, 425; Accts. Chem. Res. 2009, 42, 631) Only in the past few years have detailed structural and mechanistic studies with PKSs been published. This has become possible through advances in molecular genetics, which provided access to the biosynthetic genes for the individual biosynthetic enzymes. Before this, labelling experiments with intact organisms were possible, which often gave important insights into how a polyketide chain is assembled and cyclized in the producing organisms. The starter unit could be identified in this way, as well as the extender units. Acetyl-CoA is used frequently as starter unit, but in principle any small molecule CoAS-thioester can be used (e.g. benzoic acid-SCoA thioester). As extender units, malonyl-CoA or other malonyl-CoA derivatives may be used. The growing polyketide chains remain bound to the PKS (compare FAS above), and so can never be detected as free intermediates. E.g. three different pathways:
Me COOH
CO-S-PKS
O
OO
Me
OH
COOH
MeHO
CO-S-PKS
O
OOO
O O
Me
O
HO
HO
Me
Me
OO
R
O
SCoACOO
O
SCoA
R
O O
S-PKS
COO
O
S-PKS
hypothetical enzyme bound poly-ß-ketone intermediates
Polyketid-Synthase(PKS)
+
etc
etc
etcOO O S-PKS
O
O O
O
O
OOH
OH
OOH
HO
OOH
OH
OOH
HO
O
O2
By feeding 13C-labelled precursors to the producing organism, and using 13C NMR spectroscopy to detect sites of enrichment, it was possible to deduce how the polyketide chain is assembled. Of special interest is the use of doubly labelled precursors, like 13C2-acetate (each C-Atom 99% 13C), whose intact incorporation into the end product can be detected through an analysis of 13C-13C couplings e.g.:
H3C COONa
Helminthosporium turcicum
O
O
OH
OH
Me
OH
Islandicin
[1,2-13C2]Na-Acetat
9 In the cell, doubly labelled 13C2-acetyl-CoA is produced, but is diluted with excess unlabelled acetyl-CoA made during normal metabolism. For this reason, during polyketide biosynthesis, it is very unlikely that two labelled building blocks will be directly coupled to one another. In the natural product, made by the organism, the 13C2-acetyl-CoA will be incorporated at every possible position, so the substance isolated will be composed of a mixture of molecules that differ in the sites of labelling. Nevertheless, all the possible sites of labelling can be represented on one structure, as follows:
H3C COONa
O
O
OH
OH
Me
OH
[1,2-13C2]Na-AcetateO O O
HO
O O O
O
S-X
Me
Acetyl-CoA+ Malonyl-CoA
OH OH OH
Me
PolyketideSynthase
The 13C{1H}-NMR spectrum of the natural product: 13C-NMR-
Spektrum
200 0 ppm
usw
This labelling approach can be used to distinguish between two or more plausible modes of polyketide chain cyclization, e.g.:
SR
Me
OO
O
O O O
O
Me
OO
O
O
O
O
O
SR
O Me
OOHOH
MeO
Rubrofurasin
In order to characterize a biosynthetic pathway, it is necessary to isolate and identify all of the free intermediates. For examples, starting with an intact plant, or microbial culture, the cellular material can be extracted with organic solvents and fractionated by HPLC, prior to NMR and MS analyses of the pure components. Sometimes this is quite difficult, if the intermediates accumulate to only low concentrations under noraml conditions, are are not very stable. Once identified, potential biosynthetic intermediates can be synthesized in a labelled form (e.g. containing 14C or 13C) and fed to the intact organism, to show that they can be taken up and incorporated into the end product of the biosynthetic pathway.
10 Microorganisms have a distinct advantage here, because they can be genetically modified. For examples, mutants can be sought, in which one of the steps in the biosynthetic pathway is inoperative, due to a mutation of one of the biosynthetic enzymes, which renders it non-functional. In such mutants, the accumulation of biosynthetic intermediates might (should) then occur: Through screening of blocked mutants it is often possible to isolate and identify biosynthetic intermediates. In recent years, the microbiological (genetic) approach has become a very powerful tool in the study of biosynthetic pathways, especially in microorganisms that can be genetically manipulated. Molecular genetic approaches can provide access to the genes for the biosynthetic enzymes, which are usually clustered all together in one (relatively) small region of the chromosome. Once one gene has been isolated, the other biosynthetic genes can be found in the flanking DNA, which is easy to isolate and sequence. Once the biosynthetic genes are available, then the biosynthetic enzymes can normally be overproduced by standard recombinant DNA techniques, and this opens the way for detailed structural and mechanistic studies in vitro. How to proceed ? Consider one of the very first examples of the cloning of a biosynthetic gene cluster - that for the polyketide actinorhodin. Actinorhodin Actinorhodin is a polyketide produced by one species of gram-positive soil microrganism called Streptomyces coelicolor. The production of this natural product was simple to detect, because the compound is a bright blue color at slightly basic pH.
O
O OH
Me
O
HO
OH
O
MeO
HOCO.SR
O O
O
O
OH
O
OOH
COOH
Me
H
O
OH O
COOH
Me
O
Me
CO2H
OOH
O
OH O
OH
Me
OH H
O
Act VI mutantact I (PKS),act III (KR) act Va, Vb
Actinorhodin
act IV
Aloesaponarin II
Act VIIMutant
8 x Malonyl-CoA
2
act VIact VII
Mutactin
11 The cloning of the actinorhodin biosynthetic genes was made possible because:
• the production of the antibiotic is straightforward to detect (without the need for HPLC, NMR, MS etc.).
• all the genes for the pathway are clustered together in the chromosome, making it possible to isolate a single DNA fragment containing all the genes.
• plasmid cloning vectors had been developed, which allowed cloning experiments in these microorganisms.
• many mutants of the actinorhodin-producing organism were available, each blocked at different steps in the pathway. Below are shown different types of act mutants growing on agar plates :
The mutants could be cultivated in pairs on agar plates. The mutants could be classified as early or late mutants depending upon their ability to cosynthesize acinorhodin; thus a late blocked mutant will produce an intermediate that can be taken up by another mutant (an early blocked mutant) and converted to actinorhodin (blue color): The cloning of the entire biosynthetic pathway could then proceed as follows: Step-1. Starting from whole bacterial cells, the chromosomal DNA is isolated and cleaved into small fragments with a restriction enzyme:
12 The small fragments are then cloned into a plasmid cloning vector, which can be stably maintained in this microorganism. Once ligated into the cloning vector, the library of recombinant plasmids+inserts are introduced into one of the late act mutants. One of the cells will obtain a plasmid containing an intact functional copy of the biosynthetic gene that has been inactivated by mutation. This mutant will then be able to make actinorhodin (see left below): Step-2. From this colony or "clone" the plasmid+insert can be isolated. The insert will contain at least one of the late biosynthetic genes. From two such clones, inserts were isolated that contained different but overlapping pieces of chromosomal DNA. From these, a “cut and paste” strategy could be used to reconstruct a new insert containing a larger and hopefully complete copy of the entire biosynthetic gene cluster: This new plasmid+insert was then introduced into a related microorganism (Streptomyces parvulus) that normally does not make actinorhodin. Only now, once the plasmid+insert was introduced into the cell - it could (see right above). The insert in this plasmid thus contains all the information needed to produce all the enzymes needed for actinorhodin biosynthesis (Nature 1984, 309, 462). In the next step, the DNA insert was sequenced, to reveal the locations and nt sequences of all the biosynthetic genes, and hence the primary sequences of all the biosynthetic enzymes. Through sequence comparisons with data-bases of enzymes with known function, the likely functions of the biosynthetic enzymes could be deduced:
Once the genes had been identified in this way, the enzymes could be produced using recombinant DNA methods, and structural and mechanisitic studies could begin. Most efforts recently have focused on the PKS. The picture that has emerged is described below:
13 The construction of the polyketide backbone requires a malonyl-CoA:ACP transacylase (MAT, also used in fatty acid biosynthesis), an ACP (actI-ORF3), a ß-ketoacylsynthase (KS) and another protein called the Chain-Length-Factor (CLF), encoded in 3 genes, shown in black above. These together constitute a "minimal PKS", which can assemble a polyketide chain starting from malonyl-CoA. Surprisingly, it was shown that the starter unit is also derived from malonyl-CoA, by decarboxylation, catalysed by the KS protein. The malonyl-CoA units are loaded onto the ACP by the MAT (red below), and then transported to a heterodimer formed by the KS+CLF proteins (green and yellow below). The polyketide chain is then transferred onto a cysteine -SH group in the active site of KS (just like in fatty acid biosynthesis). The ACP (blue) then departs to collect another malonyl unit. Once this is docked again onto the KS-CLF complex, the chain elongation can occur (see below). The cycle can then be repeated until a chain of 16 C-atoms has been constructed:
A crystal structure of the PKS (Nat. Struct. Mol. Biol. 2004, 11, 888) shows that the growing polyketide sits in a long tunnel buried in the protein. The tunnel can only accept a chain of 16 C atoms, not longer. If no other enzymes are present this minimal PKS will slowly release the polyketide chain, which spontaneously cyclizes in solution to produce SEK4 and SEK4b. These are normally not produced during actinorhodin biosynthesis (only in this in vitro assay). The shape of the tunnel seems to force the polyketide chain to bend, which leads to a cyclization at C7 (shown above). In the normal biosynthetic pathway, when all the enzymes are present, this cyclized form is transported on the ACP to the next enzyme, a KR (actIII), which reduces the carbonyl group at C9. The remaining steps have not been elucidated in detail, but the following should occur:
14
O
O OH
Me
O
HO
OH
O
MeO
OCO.SR
O O
O
O
O
O
OOH
COOH
Me
H
O
OH O
CO.SR
Me
O
Me
CO2H
OOH
O
OH O
OH
Me
OH H
OH
act III (KR)
Actinorhodin
Aloesaponarin II
8 x Malonyl-CoA
2
Mutactin
min. PKS (actI KS, CLF, ACP)
O
MeO
HOCO.SR
O O
O
OH
onlymin. PKS
min. PKS+ KR
O
O
OH O
COOH
Me
O
act VII (ARO)
min. PKS+ KR + actVII ARO
act IV (CYC2/3)
O
OH
OH O
COOH
Me
act VI (ORF1)
act VI (ORFA)act VI (ORF3)
O
Me
CO2H
OOH
H
act VI (ORF2)act VI (ORF4)
O
Me
CO2H
OOH
H
act VA versch. ORF
O
act VA+VB
O
OHO
O
OH
O
O
OHO
O
O+
SEK34 SEK34b
O
OHO
HO
O
OH
O O
O
OHO
OH
OH
Me
O
SEK4 SEK4b
+
min. PKS+ KR + actVII ARO + ActIV(CYC2/3)
The Biosynthesis of Hybrid-Antibiotics There is now enormous interest in the engineering of novel biosynthetic pathways, by taking genes from different pathways and making new combinations, in an attempt to make novel hybrid natural products. This requires a detailed knowledge of what the individual genes do (i.e. what reactions do the biosynthetic enzymes catalyze?) and the specificity and mechanisms of action of the biosynthetic enzymes. A proof of principle that such experiments are possible came shortly after the cloning of the actinorhodin pathway (Nature, 1985, 314, 642). As shown below, the act genes were introduced into different strains that normally produce granaticin or medermycin. The strains then acquired the ability to generate new natural products:
15
O
Me
CO2H
OOH
OOH
H O
MeOOH
O
HO
NMe2
HO
Me
OH
O
O
MeOOH
O
H
OH
O
O
OHMe
OH
H
OH
O
MeOOH
O
HO
NMe2
HO
Me
OHO
HO
O
MeOOH
O
H
O
OHMe
OH
H
OH
COOH
Medermycin(yellow-brown)2
Actinorhodin(red - blue)
Granaticin(red-purple)
"Mederhodin A"(purple)
"Dihydrogranatirhodin"
(Color pictures supplied by D. A. Hopwood)
16 3.4. The “aliphatic polyketides” A large number of polyketides are constructed from small fatty acid building blocks (acetate, propionate, butyrate, benzoic acid etc.), but contain no aromatic rings. They may be viewed as complex long chain fatty acid derivatives, as illustrated below. Many were discovered in screening programmes, during the search for novel natural products with interesting biological activities (e.g. as antibiotics):
O
O O O
O
NaO2C
Me
MeO
Me
HO
MeMe
Me
Me
Me
Me
HO
HO
OH
O
Me
O
Me
OH
Me
Me
Me
O
Me
OO
MeO
HONMe2
Me
O
OMe
Me
OH
Me
H
HO
O
O
O
O
O
O
O
OO
O O
HO
Me
Me
MeMe
Me
MeMe
O
CHO
Monensin A
Erythromycin A
Brevetoxin B
O
O
O
O
OO
Et
OH
HOMe
O
OO
OMe
OMe
HO
H
H
Avermectin A1a
O
O OHO
HO
MeO
N O
OO
OMe
OH
OMe
S
N
O
O OOH
O
OH
Epothilone A
FK506
O
Me Me Me
OH O
HOOC
OH
OH O OH OH OH O O
O O
NH2
O
Me
HO
NH2
HOCandicidin
Labelling experiments with 13C-labelled precursors, which can be fed to the producing organisms, typically reveal which building blocks are needed to assemble the carbon backbones of these natural products, e.g.:
17
OO O O
O
NaOOC Me
*MeOMe
HO
MeMe
Me
Me
Me
Me
HO
HO
Acetate
Propionate
Butyrate
*Methionine
The biosynthesis of polyketides involves, typically, 1) assembly of the carbon backbone by a polyketide synthase (PKS) multienzyme complex; 2) so-called tailoring reactions, which may involve, oxidation, methylation, glycosylation etc. of the crabon backbone. Methylation reactions can occur, and require the use of the coenzyme S-adenosyl methionine (SAM):
ON
N
N
N
NH2
OH OH
SHOOC O
Me
NH2
N
N
N
N
NH2
OH OH
SHOOC
NH2
S-Adenosylmethionine
!
As a typical example of an aliphatic, or reduced, polyketide, consider the macrolide antibiotic erythromycin A. The building blocks needed for the assembly of the antibiotic can be identified by labelling experiments. The first free intermediate on the pathway, however, is 6-deoxyerythronolide B. Thereafter, multiple "tailoring reactions" finally lead to the natural product:
O
O
O
OH
Me
Me
Me
Me
Me
OH
OH
MeMeOH
O
Me
O
Me
OH
Me
Me
Me
OMe
OO
MeO
HONMe2
Me
O
OMe
MeOH
Me
H
HO
Erythromycin A
eryA eryB, C, D, G, HPropionyl-CoA
+
6 Methylmalonyl-CoA
6-Deoxyerythronolide B
An important question is how does the PKS function? Here a poly-ß-ketide is not produced. After most coupling reactions using methylmalonyl-CoA the resulting ß-ketothioester must be reduced (hence "reduced polyketide"). But in some cases an alcohol is left in the polyketide chain, sometimes a fully saturated unit is formed (e.g. at C7), but sometimes the ß-keto group is not reduced (C9). How are these steps controlled, or programmed ? The construction of the backbone is catalyzed by a multienzyme complex. The PKS catalyzes overall, the following process:
Propionyl-CoA
Methylmalonyl-CoA
Et O
O
Me
OH
Me
O
Me Me
OH
Me
OH
MeEt
O
SCoAMe
COO
O
SCoA
6-Deoxyerythronolide B
NADH NAD+
PKS
18 The process can be broken down into the following steps. Each cycle of chain extension and modification can be analyzed and described separately:
Et
O
SCoA
MeCOO
O
SCoA
1. Cycle
Et
O
S-
MeCOO
O
S-
Et
O
Me
O
S-Et
OH
Me
O
S-
Module-1
2. Cycle
MeCOO
O
SCoA
MeCOO
O
S-
Et
OH
Me
O
S- Et
OH
Me
O O
Me
S- Et
OH
Me
OH O
Me
S-
3. Cycle
MeCOO
O
SCoA
MeCOO
O
S-
Et
OH
Me
OH O
Me
S-
Et
OH
Me
OH O
Me
O
S-
Me
4. Cycle
MeCOO
O
SCoA
Et
OH
Me
OH O
Me
O
S-
Me
MeCOO
O
S-
Et
OH
Me
OH O
Me
O
Me
O
S-
Me
ER
Et
OH
Me
OH O
Me Me
O
S-
Me
etc.
Module-2
Module-3
Module-4
ATKS KR
NADH
AT KS
KR
NADH
ATKS
AT KS
KR
DH
But how is this complex series of steps programmed, so that each step occurs in the correct sequence ? Important insights were obtained once the biosynthetic genes had been cloned and sequenced. From the DNA sequence it was possible to deduce the protein coding sequences (or open reading frames (ORFs)), and from the deduced protein sequences, likely functions could be assigned to most proteins on the basis of sequence comparisons and similarities to other enzymes of known function (e.g. FAS). In this way it was discovered that three large multi-domain, multi-functional proteins are responsible for assembling 6-deoxyerythronolide B, and the "programming" of the assembly process is inherent in the sequence of the proteins! As shown below:
19
see JACS 2009, 131, 15784. By genetic engineering, it was then possible to construct a piece of DNA encoding a protein with just the activities indicated below. When introduced into a suitable bacterium, the "clone" was able to make the lactone shown. Similarly, when a clone was made containing DNA for the enzyme shown below, the clone was found to make the ß-hydroxyacid shown. The success of these and many other related engineering experiments showed that the molecular logic of the assembly proecess (shown top) is indeed correct. The PKS is rather like an assembly line, in which intermediates are passed from one domain to the next, until the end is reached. The product is then released free into solution through the action of the TE domain. Afterwards, the subsequent "tailoring steps" can occur (hydroxylations with P450 enzymes, addition of sugars by glycosyl transferases etc.).
20
Another example is the Pik PKS from the methymycin/pikromycin biosynthetic pathway. Pik produces the aglycones 10-deoxymethynolide (10-Dml) and narbonolide (Nbl). Another interesting example is seen in the pathway to the polyether antibiotic monensin A. The monensin PKS is currently thought to assemble the reduced polyketide chain shown below. Note the positions and configurations of the double bonds !
Me
HOOH
Me
MeMe
HOMe
CO2-
CO2-
CO2-
Me
Me
MeMe
Enz-S-OC
O
Me
O
SCoA
SCoA
MeO
SCoA
O
EtSCoA
O
O
Polyketid-SynthaseMultienzym-Komplex
Three monooxygenases then act to produce a triepoxide. It has been shown that four 18O atoms are incorporated into monensin, when the producing bacterium is grown under 18O2. The sites of labelling were determined by 13C NMR spectroscopy. The sites of labelling are consistent with the following cascade
21 cyclization process:
Me
MeOMeO
O O O
O
NaO2C
Me
Me
HO
Me
Me
Me
Me
HO
HO
Me
HOOH
Me
MeMe
HO
Me
Me
MeMe
X.OC
O
Me
O
O
O
O
Monensin
O2
Each cyclization steps occurs stereospecifically with inversion of configuration. The configuration of the triepoxide shown then leads to the correct relative and absolute configuration in monensin A. Similar schemes can be drawn to account for the formation of several of the ether rings (shown in blue) in other polyether antibiotics, such as those shown below, as well as in the complex marine natural product brevetoxin (see page-1):
COOH
O
Me
H HO
MeMe
H
OMe
OOH
OH
Et
Me
Et
O
Me
OH
Me
Et
Me
OH
Narasin
Me
OH
COOH
Me
OH
Me
O
Et
OO
Me
EtH
Et
Me
H
OH
Lasalocid A
OOOOOO
Me
HOOC
Me
Me
MeH
OH
Me
Me H
O Me Me
H H
Me Me
OH OH
O
H
MeO
Me
Dianemycin
OO O O
O
H
MeO
MeMe H
Me
H
Me
Me
HO Me
OMe
H H
H
H
O
OMe
MeO
OH
HOOCMe
OHMe
MeOMe
Septamycin