12/25/2015
1
Amy L. Lane, Ph.D.
Assistant Professor
University of North Florida
Department of Chemistry
Probing the biosynthetic capabilities
of a marine Nocardiopsis sp.
regulation or transportoxidoreductase
nsnR1 R2 I H T1 T2 orf7
cytochrome P450
F L
hypothetical or other
E
nonribosomal peptide synthetase (NRPS)
A B C D G
epoxide hydrolase
lysine cyclodeaminase
R3 R4
orf17-18
polyketide synthase (PKS)
Pacifichem 2015
Raju et al. Org. Lett. 2011;
Tetrahedron 2013; Eur. J. Chem. 2010
Nocardiopsis sp. CMB-M0232: Talented natural product chemist
12/25/2015
2
Nocardiopsins: Newest rapamycin class natural products
• FKBP modulators.
• Nocardiopsinsstructurally distinct from other class members.
FK506
rapamycinnocardiopsin A
Vezina et al. (1975) J. Antibiot.;
Kino et al. (1987) J. Antibiot.;
Raju et al. (2010) Eur. J. Chem.
Nocardiopsins: Newest rapamycin class natural products
FK506
rapamycinnocardiopsin A
Vezina et al. (1975) J. Antibiot.;
Kino et al. (1987) J. Antibiot.;
Raju et al. (2010) Eur. J. Chem.
Macrolide ring closure via 3o C-OH with non-PKS derived hydroxy group
THF group
12/25/2015
3
Nocardiopsin Polyketide Core Assembly
Bis et al. ChemBioChem (2015)
regulation or transportoxidoreductase
nsnR1 R2 I H T1 T2 orf7
cytochrome P450
F L
hypothetical or other
E
nonribosomal peptide synthetase (NRPS)
A B C D G
epoxide hydrolase
lysine cyclodeaminase
R3 R4
orf17-18
polyketide synthase (PKS)
N O
O
O
O
O
OH
OH5
6
N O
O
O
O
O
OH
OH
10
Oxidoreductase and/or P450
(NsnH, NsnI)
nocardiopsin B
6
8
Nonribosomal peptide incorporation and macrolide formation
Bis et al. ChemBioChem (2015)
regulation or transportoxidoreductase
nsnR1 R2 I H T1 T2 orf7
cytochrome P450
F L
hypothetical or other
E
nonribosomal peptide synthetase (NRPS)
A B C D G
epoxide hydrolase
lysine cyclodeaminase
R3 R4
orf17-18
polyketide synthase (PKS)
NH2
COOHH2N
lysine cyclodeaminase
(NsnL)NH
COOH
L-lysine L-pipecolic acid
NRPS,
L-pipecolic
acid
NRPS
(NsnE)
Suggests novel biosynthetic route to prepare molecule for macrolide closure.
nsnG
nsnF
12/25/2015
4
Nocardioazines: Anticancer natural products from a marine Nocardiopsis sp.
• Uniquely prenylatedindole alkaloid diketopiperazine (DKP).
• New class of bridged DKP.
• Reverse drug resistance in cancer cells by inhibiting P-glycoprotein.
Raju et al. (2011) Org. Lett.
Probing the enzymology of the nocardioazinebiosynthetic pathway
Biosynthesis of cyclo(Trp-Trp) core:
Hypothesis 1: Core assembled via nonribosomal peptide synthetase.
Hypothesis 2: Core assembled via cyclodipeptide synthase (CDPS). Two putative CDPSs encoded by genome.
James et al. ACS Synthetic Biology. Articles ASAP.
12/25/2015
5
Cyclodipeptide synthases (CDPSs): Candidates for Biosynthesis of Nocardioazine Core
• First CDPS was characterized by Gondry and coworkers in 2009.
Reviewed by Belin et al. (2012) Nat. Prod. Rep.
CDPS-encoding gene clusters from Nocardiopsis sp.
• The noz cluster encodes CDPS as well as additional enzymes with putative roles in nocardioazine assembly.
• The ncd cluster encodes only a CDPS homolog and no other enzymes homologous to biosynthetic enzymes.
James et al. ACS Synthetic Biology. Articles ASAP.
12/25/2015
6
time (min)
• Expression of either NozA or NcdA in E. coli resulted in biosynthesis of cyclo(Trp-Trp).
• No other DKP products were observed.
• In vitro assays with purified enzymes also supported tryptophanyl-tRNAas sole substrate of both CDPSs.
Evaluating the biosynthetic function of NozA and NcdA
A
James et al. ACS Synthetic Biology.Articles ASAP
Both nozA and ncdA are transcribed by Nocardiopsis sp.
• RT-PCR suggests both NozA and NcdA catalyze production of cyclo(L-Trp-L-Trp) by Nocardiopsis sp.
• NozA and NcdA provide uncommon example of multiple phylogenetically distinct enzymes from one organism yielding the same secondary metabolite.
James et al. ACS Synthetic Biology. Articles ASAP.
RT-PCR Accumulation of cWW in Nocardiopsis cultures
12/25/2015
7
Conclusions
• The unique ensemble of natural products of Nocardiopsis sp. CMB-M0232 is assembled by an equally unique suite of biosynthetic enzymes.
• NozA and NcdA catalyze formation of cyclo(L-Trp-L-Trp) and add to the group of substrate specific CDPSs.
• Both NozA and NcdA are candidates for biosynthesis of cyclo(L-Trp-L-Trp) as a nocardioazine precursor.
• Nocardiopsin assembly includes an unprecedented biosynthetic strategy to prepare the linear PKS-NRPS precursor for macrolide ring closure.
Acknowledgements
Funding:• Research Corporation Cottrell College Science Award• UNF Transformative Learning Opportunities Grants• UNF Faculty Fellowship Award
Lane lab students
Collaborators• Dr. Robert Capon (U Queensland)• Dr. Rajesh Viswanathan (Case
Western Reserve U)• Dr. Jonathan Karty (Indiana U)• Dr. Bryan Knuckley (UNF)
Elle James
Dana Bis
Yang Ban
