Enzymatic formation of an aromatic dodecaketide by engineered plant polyketide synthase

Bioorganic and Medicinal Chemistry Letters

Volumn 21, Issue 7, 2011, Pages 2083-2086

  Wanibuchi, Kiyofumi ^a,b   Morita, Hiroyuki ^a   Noguchi, Hiroshi ^b   Abe, Ikuro ^a  

^a UNIVERSITY OF TOKYO   (Japan)

^b UNIVERSITY OF SHIZUOKA   (Japan)

Author keywords

Chalcone synthase; Octaketide synthase; Pentaketide chromone synthase; Polyketide synthase

Indexed keywords

CHALCONE SYNTHASE; DODECAKETIDE; MALONYL COENZYME A; OCTAKETIDE SYNTHASE; PENTAKETIDE CHROMONE SYNTHASE; POLYKETIDE SYNTHASE; UNCLASSIFIED DRUG;

ALOE; ALOE ARBORESCENS; ARTICLE; CATALYSIS; CRYSTAL STRUCTURE; ELECTROSPRAY MASS SPECTROMETRY; ENZYME ACTIVE SITE; ENZYME ACTIVITY; ENZYME ENGINEERING; ENZYME KINETICS; ENZYME STRUCTURE; LIQUID CHROMATOGRAPHY; NONHUMAN; PROTEIN PURIFICATION; PROTON NUCLEAR MAGNETIC RESONANCE; STEADY STATE; ULTRAVIOLET RADIATION;

CATALYTIC DOMAIN; PLANTS; POLYKETIDE SYNTHASES; PROTEIN ENGINEERING;

ALOE ARBORESCENS; STREPTOMYCES COELICOLOR;

EID: 79952490291     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2011.01.135     Document Type: Article

References (18)

1. Abe, I.; Morita, H. Nat. Prod. Rep. 2010, 27, 809.
2. Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 20, 79.
3. Comprehensive Natural Products Chemistry; Schröder, J., Ed.; Elsevier: Oxford, 1999; 1, p 749.
4. Abe, I.; Oguro, S.; Utsumi, Y.; Sano, Y.; Noguchi, H. J. Am. Chem. Soc. 2005, 127, 12709.
5. Fu, H.; Ebert-Khosla, S.; Hopwood, D. A.; Khosla, C. J. Am. Chem. Soc. 1994, 116, 4166.
6. Fu, H.; Hopwood, D. A.; Khosla, C. Chem. Biol. 1994, 1, 205.
7. Shi, S.-P.; Wanibuchi, K.; Morita, H.; Endo, K.; Noguchi, H.; Abe, I. Org. Lett. 2009, 11, 551.
8. Morita, H.; Kondo, S.; Kato, R.; Wanibuchi, K.; Noguchi, H.; Sugio, S.; Abe, I.; Kohno, T. Acta Crystallogr., Sect. F 2007, 63, 947.
9. Site-directed mutagenesis:The plasmids expressing Aloe arborescens OKS double mutants (F66G/N222G, F66A/N222G, F66V/N222G, F66S/N222G, and F66L/N222G) were constructed with a QuikChange Site-Directed Mutagenesis Kit (Stratagene), according to the manufacturer0s protocol, using the following pairs of primers (mutated codons are underlined) and the previously reported plasmid bearing the N222G OKS mutant as the template: F66G (5′-GGTCGAGCTCAAGAAGAAGGGCGATCGCAT- 3′) and (5′-TGTCTTTTTGCAGATG CGATCGCCCTTCTTCTTGAG-3′), F66A (5′-GGTCGAGCTCAAGAAGAAGGCCGATCGCAT-3′ and 5′- TGTCTTTTTGCAGATGCGATCGGCCTTCTTCTTGAG-3′), F66V (5′- GGTCGAGCTCAAGAAGAAGGTCGATCGCAT-3′) and (5′-TGTCTTTTTGCAGAT GCGATCGACCTTCTTCTTGAG-3′), F66S (5′-GGTCGAGCTCAAGAAGAAGTCGGATC GCAT-3′) and (5′-TGTCTTTTTGCAGATGCGATCCGACTTCTTCTTGAG-3′), and F66L (5′-GGTCGAGCTCAAGAAGAAGTTGGATCGCAT-3′ and 5′-TGTCTTTTT GCAGATGCGATCCAACTTCTTCTTGAG-3′).
10. 600 of 0.6 in LB medium containing 100 μg/mL ampicillin at 37 °C. Subsequently, 1.0 mM isopropylthio-β-D- alactopyranoside was added to induce protein expression, and the cells were further cultured at 23 °C for 16 h. All of the following procedures were performed at 4 °C. The E. coli BL21(DE3)pLysS cells transformed with the OKS mutants were harvested by centrifugation and resuspended in 40 mM potassium phosphate buffer, pH 7.9, containing 0.1 M NaCl and 5 mM imidazole. The cells were disrupted by sonication and centrifuged at 10,000g for 30 min. The supernatant was passed through a Ni Sepharose™ 6 Fast Flow column (GE Healthcare). After the column was washed with 20 mM potassium phosphate buffer, pH 7.9, containing 0.5 M NaCl and 40 mM imidazole, the recombinant enzyme was finally eluted with 15 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 500 mM imidazole. The recombinant PKSs were fairly soluble, and the Ni-chelate affinity column chromatography afforded 3-5 mg of pure enzymes from 1 L of the E. coli culture. The protein concentration was determined by the Bradford method (Protein Assay Dc, Bio-Rad, Hercules, CA, USA) using bovine gamma globulin as the standard.
11. 2O and MeOH, both containing 0.1% TFA: 0-5 min, 30% MeOH; 5-17 min, linear gradient from 30% to 60% MeOH; 17-25 min, 60% MeOH; 25-27 min, linear gradient from 60% to 70% MeOH. Elutions were monitored by a multichannel UV detector (MULTI 340, JASCO) at 280 nm; UV spectra (200-400 nm) were recorded every 0.4 s. Online LC-ESIMS spectra were measured with an Agilent Technologies HPLC 1100 series HPLC coupled to a Bruker Daltonics Esquire4000 ion trap mass spectrometer fitted with an ESI source. HPLC separations were performed under the same conditions as described above. The ESI capillary temperature and the capillary voltage were 320 °C and 4.0 V, respectively. The tube lens offset was set at 20.0 V. All spectra were obtained in the positive mode, over a mass range of 50-600 m/z, and at a range of one scan every 0.2 s. The collision gas was helium, and the relative collision energy scale was set at 30.0% (1.5 eV). The samples for the LC-HRMS were analyzed with an Agilent 1100 series HPLC-microTOF mass spectrometer (Bruker Daltonics) using Electrospray Ionization.
12. 14C]Malonyl-CoA was purchased from GE Healthcare.
13. 14
14. Yu, T.-W.; Shen, Y.; McDaniel, R.; Floss, H. G.; Khosla, C.; Hopwood, D. A.; Moor, B. S. J. Am. Chem. Soc. 1998, 120, 7749.
15. cat values (average of triplicates), using the ENZFITTER software (BIOSOFT).
16. Abe, I.; Utsumi, Y.; Oguro, S.; Morita, H.; Sano, Y.; Noguchi, H. J. Am. Chem. Soc. 2005, 127, 1362.
17. Morita, H.; Kondo, S.; Oguro, S.; Noguchi, H.; Sugio, S.; Abe, I.; Kohno, T. Chem. Biol. 2007, 14, 359.
18. Abe, I.; Morita, H.; Oguro, S.; Noma, H.; Wanibuchi, K.; Kawahara, N.; Goda, Y.; Noguchi, H.; Kohno, T. J. Am. Chem. Soc. 2007, 129, 5976.