Characterization of the interaction between an ACP domain and a halogenase in the curacin A polyketide synthetase

Alena E. L. Busche¹, Daniel Gottstein², Christopher Hein³, Nina Ripin⁴, Irina Pader⁵, Liangcai Gu⁶, Christopher T. Walsh⁷, David H. Sherman⁸, Frank Löhr⁹, Peter Güntert¹⁰, Volker Dötsch¹¹

1 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

2 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

3 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

4 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

5 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

6 Department of Genetics, Harvard Medical School Boston, MA 02115 (USA)

7 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

8 Life Sciences Institute, Departments of Medicinal Chemistry, Chemistry, and Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109 (USA)

9 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

10 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

11 Institute of Biophysical Chemistry, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance, Max-von-Laue Str. 9, 60438 Frankfurt am Main, Germany

Abstract

Polyketide (PK)- and non-ribosomal peptide (NRP)-synthetases are large multidomain proteins present in microorganisms that produce bioactive compounds. Curacin A is such a bioactive compound with anti- proliverative activity. In this work we investigate a triplett ACP_I,II,IIIpresent at the C-terminus of CurA. We show that the ACPs are independent of each other even upon dimerization and report the high resolution nuclear magnetic resonance (NMR) structure of the isolated ACP_I loaded with the unlabelled substrate 3-hydroxy-3-methylglutaryl (HMG). Recently, the role, timing and structure of the curacin halogenase (Cur Hal) could be established in the biosynthesis of curacin A^1,2. We identified the interaction surface important for ACP recognition by the halogenase Hal using mutational analysis. These investigations give a new insight into the molecular basis modulating and regulating the specificity of the interaction between two domains of this important mega-synthase.

References

1. Khare D, Wang B, Gu L, Razelun J, Sherman DH, Gerwick WH, Hakansson K, Smith JL: Conformational switch triggered by alpha-ketoglutarate in a halogenase of curacin A biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 2010, 107(32):14099-14104. 2 Gu L, Wang B, Kulkarni A, Geders TW, Grindberg RV, Gerwick L, Hakansson K, Wipf P, Smith JL, Gerwick WH et al: Metamorphic enzyme assembly in polyketide diversification. Nature 2009, 459(7247):731-735.

DOI^®: 10.3288/contoo.paper.1696