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Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein
Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein
During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and H-1 NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

fatty-acid synthase, streptomyces-coelicolor A3(2), coa-acp transacylase, escherlchia-coli, engineered biosynthesis, heterologus expression, nucleotide-sequence, coenzyme-a, actinorhodin, purification
0006-2960
15414-15421
Arthur, Christopher J.
03ba599b-a014-4547-b407-09ed40d7df66
Szafranska, Anna
5a6caa29-3019-4b28-8e95-b1fbe6a83a80
Evans, Simon
c04150d0-d74c-4ca8-accb-451cad66ed52
Findlow, Stuart C.
0a7193ad-45cf-4613-a35e-3fd019a92ebe
Burston, Steve
37d28f0d-f33c-47a4-bf9a-4a26e515a185
Clark-Lewis, Ian
c2eb3a00-9550-4a21-9354-9562937ba43a
Simpson, Thomas J.
17c403f6-38f4-4c81-9267-319f2d441183
Crosby, John
38dff705-47dc-4406-be2b-cce3599a776b
Crump, Matthew P.
ed31b5fd-23f6-434c-a38c-da3cb7b27402
Arthur, Christopher J.
03ba599b-a014-4547-b407-09ed40d7df66
Szafranska, Anna
5a6caa29-3019-4b28-8e95-b1fbe6a83a80
Evans, Simon
c04150d0-d74c-4ca8-accb-451cad66ed52
Findlow, Stuart C.
0a7193ad-45cf-4613-a35e-3fd019a92ebe
Burston, Steve
37d28f0d-f33c-47a4-bf9a-4a26e515a185
Clark-Lewis, Ian
c2eb3a00-9550-4a21-9354-9562937ba43a
Simpson, Thomas J.
17c403f6-38f4-4c81-9267-319f2d441183
Crosby, John
38dff705-47dc-4406-be2b-cce3599a776b
Crump, Matthew P.
ed31b5fd-23f6-434c-a38c-da3cb7b27402

Arthur, Christopher J., Szafranska, Anna, Evans, Simon, Findlow, Stuart C., Burston, Steve, Clark-Lewis, Ian, Simpson, Thomas J., Crosby, John and Crump, Matthew P. (2005) Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein. Biochemistry, 44 (46), 15414-15421. (doi:10.1021/bi051499i).

Record type: Article

Abstract

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and H-1 NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.

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Published date: 2005
Keywords: fatty-acid synthase, streptomyces-coelicolor A3(2), coa-acp transacylase, escherlchia-coli, engineered biosynthesis, heterologus expression, nucleotide-sequence, coenzyme-a, actinorhodin, purification

Identifiers

Local EPrints ID: 24178
URI: https://eprints.soton.ac.uk/id/eprint/24178
ISSN: 0006-2960
PURE UUID: 044ae2b7-05c1-48eb-b930-f5186674eb6b

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Date deposited: 27 Mar 2006
Last modified: 17 Jul 2017 16:14

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Contributors

Author: Christopher J. Arthur
Author: Anna Szafranska
Author: Simon Evans
Author: Stuart C. Findlow
Author: Steve Burston
Author: Ian Clark-Lewis
Author: Thomas J. Simpson
Author: John Crosby
Author: Matthew P. Crump

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