scholarly journals Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Edward Kalkreuter ◽  
Kyle S. Bingham ◽  
Aaron M. Keeler ◽  
Andrew N. Lowell ◽  
Jennifer J. Schmidt ◽  
...  
Keyword(s):  
Active Site ◽  
Polyketide Synthase ◽  
Polyketide Synthases ◽  
Substrate Selectivity ◽  
Substrate Selection ◽  
Polyketide Biosynthesis ◽  
Erythromycin Biosynthesis ◽  
Malonyl Coa ◽  
Modular Polyketide Synthase ◽  
Dynamics Simulations

AbstractPolyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.

scholarly journals Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase

2020 ◽  
Author(s):  
Edward Kalkreuter ◽  
Kyle S Bingham ◽  
Aaron M Keeler ◽  
Andrew N Lowell ◽  
Jennifer J. Schmidt ◽  
...  
Keyword(s):  
Active Site ◽  
Polyketide Synthase ◽  
Substrate Selectivity ◽  
Substrate Selection ◽  
Erythromycin Biosynthesis ◽  
Malonyl Coa ◽  
Extender Unit ◽  
Modular Polyketide Synthase ◽  
Dynamics Simulations

ABSTRACTAcyltransferases (ATs) of modular polyketide synthases catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. Subsequently, AT domains have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, critically limiting the efficiency and scope of AT engineering. Here, molecular dynamics simulations were used to prioritize ~50 mutations in the active site of EryAT6 from erythromycin biosynthesis. Following detailed in vitro studies, 13 mutations across 10 residues were identified to significantly impact extender unit selectivity, including nine residues that were previously unassociated with AT specificity. Unique insights gained from the MD studies and the novel EryAT6 mutations led to identification of two previously unexplored structural motifs within the AT active site. Remarkably, exchanging both motifs in EryAT6 with those from ATs with unusual extender specificities provided chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy is expected to advance our ability to engineer PKSs towards designer polyketides.

scholarly journals Type II Thioesterase ScoT, Associated with Streptomyces coelicolor A3(2) Modular Polyketide Synthase Cpk, Hydrolyzes Acyl Residues and Has a Preference for Propionate

2008 ◽  
Vol 75 (4) ◽  
pp. 887-896 ◽  
Author(s):  
Magdalena Kotowska ◽  
Krzysztof Pawlik ◽  
Aleksandra Smulczyk-Krawczyszyn ◽  
Hubert Bartosz-Bechowski ◽  
Katarzyna Kuczek
Keyword(s):  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Hybrid Systems ◽  
Active Site ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Polyketide Synthases ◽  
Type Ii ◽  
Starter Unit ◽  
Modular Polyketide Synthase

ABSTRACT Type II thioesterases (TE IIs) were shown to maintain the efficiency of polyketide synthases (PKSs) by removing acyl residues blocking extension modules. However, the substrate specificity and kinetic parameters of these enzymes differ, which may have significant consequences when they are included in engineered hybrid systems for the production of novel compounds. Here we show that thioesterase ScoT associated with polyketide synthase Cpk from Streptomyces coelicolor A3(2) is able to hydrolyze acetyl, propionyl, and butyryl residues, which is consistent with its editing function. This enzyme clearly prefers propionate, in contrast to the TE IIs tested previously, and this indicates that it may have a role in control of the starter unit. We also determined activities of ScoT mutants and concluded that this enzyme is an α/β hydrolase with Ser90 and His224 in its active site.

scholarly journals Erythromycin Biosynthesis:  Kinetic Studies on a Fully Active Modular Polyketide Synthase Using Natural and Unnatural Substrates

Biochemistry ◽  
1996 ◽  
Vol 35 (31) ◽  
pp. 10248-10248 ◽  
Author(s):  
Rembert Pieper ◽  
Susanne Ebert-Khosla ◽  
David Cane ◽  
Chaitan Khosla
Keyword(s):  
Polyketide Synthase ◽  
Kinetic Studies ◽  
Erythromycin Biosynthesis ◽  
Modular Polyketide Synthase

Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes

2005 ◽  
Vol 23 (9) ◽  
pp. 1171-1176 ◽  
Author(s):  
Hugo G Menzella ◽  
Ralph Reid ◽  
John R Carney ◽  
Sunil S Chandran ◽  
Sarah J Reisinger ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
De Novo ◽  
De Novo Design ◽  
Polyketide Biosynthesis ◽  
Modular Polyketide Synthase

scholarly journals Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones

2013 ◽  
Vol 288 (23) ◽  
pp. 16484-16494 ◽  
Author(s):  
Kuppan Gokulan ◽  
Seán E. O'Leary ◽  
William K. Russell ◽  
David H. Russell ◽  
Mallikarjun Lalgondar ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Polyketide Synthase ◽  
Biological Function ◽  
Type Iii ◽  
Optimal Activity ◽  
Malonyl Coa ◽  
Hydrophobic Tunnel ◽  
Reaction In Solution

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.

scholarly journals Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis

2002 ◽  
Vol 367 (3) ◽  
pp. 781-789 ◽  
Author(s):  
Nobutaka FUNA ◽  
Yasuo OHNISHI ◽  
Yutaka EBIZUKA ◽  
Sueharu HORINOUCHI
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Type Iii Polyketide Synthase ◽  
Melanin Biosynthesis ◽  
Type Iii ◽  
Malonyl Coa

RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys138, His270 and Asn303 of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr224 is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr224 by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala305 mutants and identification of their products showed that the substitution of Ala305 by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr224 (important for starter substrate selection) and Ala305 (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

scholarly journals Dissecting how modular polyketide synthase ketoreductases interact with acyl carrier protein-attached substrates

2017 ◽  
Vol 53 (83) ◽  
pp. 11457-11460 ◽  
Author(s):  
Luisa Moretto ◽  
Steven Vance ◽  
Brennan Heames ◽  
R. William Broadhurst
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Prosthetic Group ◽  
Correct Orientation ◽  
Interaction Studies ◽  
Modular Polyketide Synthase

Interaction studies show that KR domains possess a generic binding site for ACP domains and provide evidence that the 5′-phosphopantetheine prosthetic group plays a key role in delivering acyl substrates to the active site in the correct orientation.

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