Site‐Directed Mutagenesis of Modular Polyketide Synthase Ketoreductase Domains for Altered Stereochemical Control

ChemBioChem ◽  
2020 ◽  
Author(s):  
Erin E. Drufva ◽  
Nolan R. Spengler ◽  
Elijah G. Hix ◽  
Constance B. Bailey
Keyword(s):  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Stereochemical Control ◽  
Modular Polyketide Synthase

scholarly journals Active site labeling of fatty acid and polyketide acyl-carrier protein transacylases

2019 ◽  
Vol 17 (19) ◽  
pp. 4720-4724 ◽  
Author(s):  
Tony D. Davis ◽  
Jennifer M. Michaud ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Fluorescent Probe ◽  
Active Site ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Probe Design ◽  
Directed Mutagenesis ◽  
Carrier Protein

Fluorescent probe design and site-directed mutagenesis unveil new activity-based chemical reporters for fatty acid and polyketide synthase acyl-carrier protein transacylases.

Molecular Cloning, Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from Sargassum binderi (Phaeophyta)

2010 ◽  
Vol 13 (5) ◽  
pp. 845-856 ◽  
Author(s):  
Hariyanti Baharum ◽  
Hiroyuki Morita ◽  
Akifumi Tomitsuka ◽  
Fong-Chin Lee ◽  
Kim-Yong Ng ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Sargassum Binderi

Manipulation of macrolide ring size by directed mutagenesis of a modular polyketide synthase

1995 ◽  
Vol 117 (35) ◽  
pp. 9105-9106 ◽  
Author(s):  
Camilla M. Kao ◽  
Guanglin Luo ◽  
Leonard Katz ◽  
David E. Cane ◽  
C. Khosla
Keyword(s):  
Polyketide Synthase ◽  
Directed Mutagenesis ◽  
Ring Size ◽  
Modular Polyketide Synthase

scholarly journals Alternative method for site-directed mutagenesis of complex polyketide synthase in Streptomyces albus JA3453

2008 ◽  
Vol 40 (4) ◽  
pp. 319-326 ◽  
Author(s):  
Danfeng Song ◽  
Jane Coughlin ◽  
Jianhua Ju ◽  
Xiufen Zhou ◽  
Ben Shen ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Alternative Method ◽  
Streptomyces Albus

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 Structure and mechanism of a dehydratase/decarboxylase enzyme couple involved in polyketide β-methyl branch incorporation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Asha V. Nair ◽  
Alice Robson ◽  
Thomas D. Ackrill ◽  
Marisa Till ◽  
Matthew J. Byrne ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Type Systems ◽  
Site Directed Mutagenesis ◽  
Methyl Branch ◽  
New Paradigm ◽  
X Ray Crystallography ◽  
Polyketide Chain ◽  
Modular Polyketide Synthase

Abstract Complex polyketides of bacterial origin are biosynthesised by giant assembly-line like megaenzymes of the type 1 modular polyketide synthase (PKS) class. The trans-AT family of modular PKSs, whose biosynthetic frameworks diverge significantly from those of the archetypal cis-AT type systems represent a new paradigm in natural product enzymology. One of the most distinctive enzymatic features common to trans-AT PKSs is their ability to introduce methyl groups at positions β to the thiol ester in the growing polyketide chain. This activity is achieved through the action of a five protein HCS cassette, comprising a ketosynthase, a 3-hydroxy-3-methylglutaryl-CoA synthase, a dehydratase, a decarboxylase and a dedicated acyl carrier protein. Here we report a molecular level description, achieved using a combination of X-ray crystallography, in vitro enzyme assays and site-directed mutagenesis, of the bacillaene synthase dehydratase/decarboxylase enzyme couple PksH/PksI, responsible for the final two steps in β-methyl branch installation in this trans-AT PKS. Our work provides detailed mechanistic insight into this biosynthetic peculiarity and establishes a molecular framework for HCS cassette enzyme exploitation and manipulation, which has future potential value in guiding efforts in the targeted synthesis of functionally optimised ‘non-natural’ natural products.

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