Inside Cover: Mutant Malonyl-CoA Synthetases with Altered Specificity for Polyketide Synthase Extender Unit Generation (ChemBioChem 15/2011)

ChemBioChem ◽  
2011 ◽  
Vol 12 (15) ◽  
pp. 2230-2230
Author(s):  
Irina Koryakina ◽  
Gavin J. Williams
Keyword(s):  
Polyketide Synthase ◽  
Malonyl Coa ◽  
Extender Unit

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 Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Huimin Wang ◽  
Junheng Liang ◽  
Qianwen Yue ◽  
Long Li ◽  
Yan Shi ◽  
...  
Keyword(s):  
Parental Strain ◽  
Assembly Line ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Critical Factor ◽  
Catalytic Domains ◽  
Malonyl Coa ◽  
Epothilone D ◽  
Careful Design ◽  
Selection Of

Abstract Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. Conclusions These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

scholarly journals Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria

2018 ◽  
Vol 115 (40) ◽  
pp. 9835-9844 ◽  
Author(s):  
Dongsoo Yang ◽  
Won Jun Kim ◽  
Seung Min Yoo ◽  
Jong Hyun Choi ◽  
Shin Hee Ha ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
High Throughput Screening ◽  
Polyketide Synthase ◽  
Rapid Development ◽  
Type Iii Polyketide Synthase ◽  
E Coli ◽  
Type Iii ◽  
Small Regulatory Rna ◽  
Enhanced Production ◽  
Malonyl Coa

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only inEscherichia coliandSaccharomyces cerevisiaeand require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria:E. coli,Pseudomonas putida, andCorynebacterium glutamicum. RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor inE. coli. Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level inE. coli. These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple differentE. colistrains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA–derived chemicals.

Divergolide congeners illuminate alternative reaction channels for ansamycin diversification

2015 ◽  
Vol 13 (6) ◽  
pp. 1618-1623 ◽  
Author(s):  
Ling Ding ◽  
Jakob Franke ◽  
Christian Hertweck
Keyword(s):  
Structure Elucidation ◽  
Polyketide Synthase ◽  
Ring Closure ◽  
Side Chain ◽  
Neighboring Group ◽  
Ring Contraction ◽  
Reaction Channels ◽  
Extender Unit

Isolation and structure elucidation of six new divergolides reveal unusual ansamycin diversification reactions including formation of the unusual isobutenyl side chain from a branched polyketide synthase extender unit, azepinone ring closure, macrolide ring contraction and formation of a seco variant by a neighboring group-assisted decarboxylation.

scholarly journals The crystal structure of benzophenone synthase from Garcinia mangostana L. pericarps reveals the basis for substrate specificity and catalysis

2020 ◽  
Vol 76 (12) ◽  
pp. 597-603
Author(s):  
Chomphunuch Songsiriritthigul ◽  
Natsajee Nualkaew ◽  
James Ketudat-Cairns ◽  
Chun-Jung Chen
Keyword(s):  
Substrate Specificity ◽  
Polyketide Synthase ◽  
Docking Studies ◽  
Structural Basis ◽  
Garcinia Mangostana ◽  
Type Iii Polyketide Synthase ◽  
Catalytic Function ◽  
Malonyl Coa ◽  
Horizontal Cavity ◽  
Polyketide Chain

Benzophenone synthase (BPS) catalyzes the production of 2,4,6-trihydroxybenzophenone via the condensation of benzoyl-CoA and three units of malonyl-CoA. The biosynthetic pathway proceeds with the formation of the prenylated xanthone α-mangostin from 2,4,6-trihydroxybenzophenone. Structural elucidation was performed to gain a better understanding of the structural basis of the function of Garcinia mangostana L. (mangosteen) BPS (GmBPS). The structure reveals the common core consisting of a five-layer αβαβα fold as found in other type III polyketide synthase enzymes. The three residues Met264, Tyr266 and Gly339 are proposed to have a significant impact on the substrate-binding specificity of the active site. Crystallographic and docking studies indicate why benzoyl-CoA is preferred over 4-coumaroyl-CoA as the substrate for GmBPS. Met264 and Tyr266 in GmBPS are properly oriented for accommodation of the 2,4,6-trihydroxybenzophenone product but not of naringenin. Gly339 offers a minimal steric hindrance to accommodate the extended substrate. Moreover, the structural arrangement of Thr133 provides the elongation activity and consequently facilitates extension of the polyketide chain. In addition to its impact on the substrate selectivity, Ala257 expands the horizontal cavity and might serve to facilitate the initiation/cyclization reaction. The detailed structure of GmBPS explains its catalytic function, facilitating further structure-based engineering to alter its substrate specificity and obtain the desired products.

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.

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