biosynthesis of natural products nonribosomal peptides 5-19-2011
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Biosynthesis of Natural ProductsNonribosomal Peptides
5-19-2011
Peptide Natural Products• Ribosomally Derived
Walsh, JACS 2010
Nisin – a lantibiotic
Highly stable multicyclic peptide antibiotics. Precursor peptide undergoes posttranslational side chain modification reaction involving amino acid modifications and removal of leader peptide.
thiopeptides
Formation of Lantibiotics
Van der donk, Nat. Chem. Biol. 2010
Formation of Thiopeptides
Van der donk, Nat. Chem. Biol. 2010
Nonribosomal Peptides
O
O
O
Cl
OHCl
NH
HN
O
OHOHHO
HN
ONH
O
H
HO OHN
NH
OHN
O
O
H2NHOOC
O
OHOH
OHOO
OH NH2
vancomycin
N
S
COOHO
HNO
R
penicillins
O
HN
NH
O
O
HN
NH
OO
ONH
O
NH
OOH
NH
O
HN O
HN
O
OH
OHN
O
HN
O
HN
O
NHON
ONH2
OHO
O
H2N OHO
OOH
NH2
daptomycin
O
NHO
NHH2N
OHN O
HO
NH
OHN
OH2N
O
HN
OH2N
O
HNN
HO
N
O
NH
tyrocidine A
cyclosporin
Nonribosomal Peptides (NRPs)• A majority of peptidyl natural products are nonribosomally derived – they
are not synthesized from the protein translational machinery. Instead they are assembled via the assembly-line like actions of nonribosomal peptide synthetases (NRPS)
• NRPs can include many nonproteinogenic amino acids, including hydroxyl acid, N-methylated aa, and D-aa; whereas ribosomally derived peptides are restricted to 20 naturally occurring amino acids.
• The biosynthetic logic of NRPS parallels that of modular PKSs: template-directed, colinearity, assembly-line like.
• NRPS can also be coupled to PKS assembly lines (both emply thioester acyl intermediates) to give hybrid compounds.
Basic Enzymatic Units of NRPS
Marahiel, Science 2008
C A PCP TESurfactin NRPSModule 7 (144 kDa)
condensation
adenylation
peptidyl carrier protein
thioesteraseSH
PCP
Basic Biochemistry – PCP domainThe PCP protein is structurally and functionally analogous to the ACP domain.The apo form of PCP must be modified at a serine side chain to become holo form. The holo form contains a phosphopantetheinyl arm and a nucleophilic thiol, which is the position at which the peptidyl groups will be attached.Sometime also referred to as the thiolation (T) domain.
SHHNO
HNO
HO O P
O
OH
O 3'-5'-ADP
CoA-SH
HO
Apo PCP
3',5'-ADP
SHHNO
HNO
HO O P
O
OH
O
PhosphopantetheineFlexible, 18-20 A
SH
Holo PCP
PCP PCP
PPTase
Konig, Science 2006
PCP
Basic Biochemistry – A domainThe A (adenylation) domain selects and activates the building blocks.The building blocks are mostly amino acids, both proteinogenic and nonproteinogenic. They can also be other organic acids.Two step activation: 1) adenylation with ATP; 2) transfer to PCP domain.The adenylation step is identical to that of aminoacyl-tRNA synthase.
R
NH2
OH
OATP PPi
R
NH2
O
O
AMP
A
1) Adenylation
2) Acyltransfer
R
NH2
O
O
AMP NH2
S
O
R
A
PCP
SH
Aminoacyl-S-PCP
A domain specificityRepresentative nonproteinogenic amino acids selected by the NRPS A domain
OH
O
H2N
OH
O
NH2
OH
O
O
O NH2
OH
O
NH2
OH
O
OH
NH2
OH
O
OH
O
OH
NH2 NH2
OH
O
HO OH
H2NOH
O
OH
H2NOH
O
H2N
NH2
OH
O
NH2
O OH
O
O
O
Cl
OHCl
NH
HN
O
OHOHHO
HN
ONH
O
H
HO OHN
NH
OHN
O
O
H2NHOOC
O
OHOH
OHOO
OH NH2
A-Domain Specificity
Stachelhaus, Chem Biol, 1999
C A PCPn+1
Basic Biochemistry – C domainCatalyzes formation of peptide bonds between growing NRP and aminoacyl-S-PCP.Upstream peptidyl-S-PCP is the acyl donor.Downstream, intramodular aminoacyl-S-PCP as the nucleophile acceptor.Peptide-bond formation is unidirectional.Functionally analogous to the KS domain in PKSs and makes C-N bonds.
PCPn
PCPn C A PCPn+1
H2N
R1
O
SH2N
R2
O
S
HN
R2
O
S
O
R1
H2NSH
Samel, Structure 2007
SH SO
N O
RNH2
SH SO
N
NH2
R
O
SH SO
N
NH2
R
OHO
BH
SH SO
HN OH
O
NH2
R
S SO
H2N OH
O
H2NR
Accessory Domains – Cyclization (Cy) and Oxidation (Ox)
Cy A PCPPCP Cy A PCPPCP Cy A PCPPCP
Cy A PCPPCP
condensation cyclization
dehydration
Cy domains are responsible for the formation of heterocycles in NRPs.Cy domains are variants of C domains and also catalyze peptide bond formation.Cy domains then catalyze the attack of b-nucleophile on the upstream amide carbonyl, and form five membered rings that dehydrates.b-nucleophile: serine (hydroxyl) oxazoline; cysteine (thiol) thiazoline.Further oxidation by the Ox domain can lead to oxazole or thiazoles
N
S
O
OH
OOHO
epothiloneCy APCP
Oxidation FAD dependent
Ox PCP
SO
H2N
H
Accessory Domain – Epimerization (E)
A PCP E
D-amino acids occur commonly in NRPs.Most of the D-amino acids arise from the action of the epimerase (E) domain.The L-amino acid is first selected and tethered to the PCP domain.The aminoacyl-S-PCP is then subjected to epimerization via extraction of the Ca-proton. The epimerization passes through the planar, resonance-stablized Ca carboanion.The downsteam C domain has stereoselectivity in choosing the epimerized substrate.
SO
H2N
H
A PCP Eepimerization
PCP S
O
NH3
N
NH
H R
XH
E
PCP
HN
NH
XH
ES
O R
NH3
SO
OH
SO
HN
Cy A
Accessory Domain – N-methylation (MT)
Cy A MT PCP
S
O
S
NH
N
SOH
MeS
RAd
MT PCP
S
O
S
N
N
SOHMe H
OHS
O
S
N
N
SOH
PchF
OH
O
S
N
N
SOH
Pyochelin
N-methyltransferase domains are embedded between the A and the PCP domains.The substrate for methylation is S-adenosylmethionine (SAM), which provides an electrophilic CH3
+ equivalent.The product of the reaction is N-Me-aminoacyl-S-PCP, before the next condensation.
Cy A MT PCP
Patel, Biochemistry, 2003
C A PCP C A MT PCP PCP TE
SAMHO
O
H2N
O
3X O
ON
O
O O
N
OO
O
NO
enniatin
Basic Biochemistry – TE domainTE is the chain terminating domain, and is usually attached to the last PCP domain.It has the same role as the TE in the PKSs and serves to disconnet the covalent thioester linkage between the full length peptidyl chain and the most downstream PCP domain.TE can function both as a hydrolytic enzyme and as a macrocyclization enzyme.
C A PCP TE C A PCP TE
S
O
R1
HN
O
NH
R2
O
HN R3
O
R4
NH
O
R5
HN
O
R6
NH
OR7
NH2
HO
R1HN
O
NH
R2
O
HN R3
O
R4
NH
O
R5
HN
O
R6
NH
OR7
NH2
O OSH
OH H
ab
c
Adapted from Walsh, Antibiotics
ab c
R1
HN
O
NH
R2
O
HN R3
O
R4
NH
O
R5
HN
O
R6
NH
OR7
NH
O
Macrolactam
R1
HN
O
NH
R2
O
HN R3
O
R4
NH
O
R5
HN
O
R6
NH
ONH2
R7
O
Macrolactam/Macrolactone
R1
HN
O
NH
R2
O
HN R3
O
R4
NH
O
R5
HN
O
R6
NH
ONH2
R7
OH
O
Linear peptide
C
Putting it all together – NRPS modulesInitiation
A PCP
Elongation
C/Cy A Ox MT PCP E
optional
A PCP TE
Termination
Konig, NPR 2008
The principle of NRPSs is elucidated using the synthetic machinery of the branched cyclic decapeptide bacitracin A. A total of twelve modules distributed over three NRPSs (BacA, BacB and BacC) process the growing peptide chain along the protein template. During synthesis, the elongation intermediates remain covalently tethered as thioesters to the cofactors of the NRPSs. After the linear peptide has reached the final module it is released by macrocyclization.
Schwarzer, NPR 2003
Example – ACV SynthetaseA single polypeptide NRPS from Acremonium chrysogenum that synthesizes the acyclic precursor of the penicillin and cephalosporins.ACV synthetase has 10 domains in a 450-kDa protein.Uses three building blocks (d-amino adipic acid, cysteine and valine, ACV).
AAad PCP C ACys PCP C AVal PCP E TE
COOH
H2N S
OH2N
S
O
HS
H2NS
OO
OHN
O
SHHN
OCOOH
H2N
d-amino adipic acid (C6-COOH is activated) L-cysteine
L-valine, Epimerized to D form
hydrolysis
O O
HN
O
SHHN
OCOO
H3N
ACV Isopenicillin N
NO
HN
OCOO
H3N S
COO
Penicillins
IPN SynthetaseN
O
HN
OCOO
H3N S
COO
Deacetoxycephalosporin C (DAOC)
DAOC Synthetase
Both IPN and DAOC synthetases are nonheme ferrous iron dioxygenases
Cephalosporins
Example - Vancomycin
Satterly, NPR, 2008
Post NRPS modificationOxidative Coupling
Glycosylation
PKS-NRPS hybridsMany natural products are hybrids of nonribosomal peptides and polyketides. Colinearity rule still applies to these mosaic assembly line.All intermediates are still shuttled via pPant arms of ACP or PCP domains.
Epothilone biosynthetic gene cluster
Tang and Khosla, Science 2000
O
HN
NH
O
O
HN
NH
OO
ONH
O
NH
OOH
NH
O
HN O
HN
O
OH
OHN
O
HN
O
HN
O
NHON
ONH2
OHO
O
H2N OHO
OOH
NH2
Case Study - Daptomycin
D-Ala8
Asp9
Asp7
Orn6
Gly5Thr4
Kyn13
MeGlu12
D-Ser11
Gly10
Asp3
D-Asn2Trp1R
DptA
DptBC
DptD
Isolated from Streptomyces roseosporus in 1987, Daptomycin is an acidic cyclic lipopeptide antibiotic approved for treatment of infections caused by Gram-positive pathogens, including Staphylococcus aureus strains resistant to other antibiotics.
Structurally, daptomycin is composed of two portions: NRP core and a tethered fatty acyl chain. The NRP core contains thirteen amino acids, including three D-amino acids (D-Asn2, D-Ala8, D-Ser11) and three non-proteinogenic amino acid (ornithine6, (2S, 3R)-3-methylglutamic acid12, kynurenine13). The decanoyl moiety is linked to the N-terminus of the first residue tryptophan.
Mode of action
MRSA MSSA
Straus and Hancock, BBA
Staphylococcus aureus is a facultatively anaerobic, gram positive coccus and is the most common cause of staph infections. Methicillin-sensitive Staphylococcus aureus (MSSA) refers to all of the antibiotic(beta-lactam)-sensitive strains of Staph aureus. Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several difficult-to-treat infections in humans. MRSA is, by definition, any strain of Staphylococcus aureus bacteria that has developed resistance to beta-lactam antibiotics, which include the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the cephalosporins
In a first step, daptomycin aggregates in solution in the presence of a minimum of 1:1 calcium to daptomycin molar ratio. Then, daptomycin inserts into the membrane, a process facilitated by calcium, which binds strongly to phosphatidylglycerol headgroups. At this point, daptomycin induces membrane depolarization without cell lysis. As a result, loss of membrane potential leads to inhibition of protein, DNA, RNA synthese and finally death of cells.
dpt gene cluster and NRPS organization
Truncated depiction of the daptomycin assembly line illustrating modules 1 and 13.Nolan and Walsh, CBC
Related Lipopeptides
Baltz, Methods Enzymol and Miao, Chem. Biol.
Combinatorial Biosynthesis
Baltz, Curr. Opin. Chem. Biol.