scholarly journals Broadening substrate specificity of a chain-extending ketosynthase through a single active-site mutation

2016 ◽  
Vol 52 (54) ◽  
pp. 8373-8376 ◽  
Author(s):  
Annabel C. Murphy ◽  
Hui Hong ◽  
Steve Vance ◽  
R. William Broadhurst ◽  
Peter F. Leadlay
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Polyketide Synthase ◽  
Model System ◽  
Site Mutation ◽  
Ketosynthase Domain ◽  
Substrate Tolerance ◽  
A Chain

An in vitro model system based on a ketosynthase domain of the erythromycin polyketide synthase was used to probe the apparent substrate tolerance of ketosynthase domains of the mycolactone polyketide synthase.

Interaction of a Plasmin A-Chain/t-PA B-Chain Hybrid Enzyme with Plasma Inhibitors In Vivo and In Vitro

1990 ◽  
Vol 63 (03) ◽  
pp. 459-463 ◽  
Author(s):  
S Wilson ◽  
P Chamberlain ◽  
I Dodd ◽  
A Esmail ◽  
J H Robinson
Keyword(s):  
Guinea Pig ◽  
Plasminogen Activator ◽  
Active Site ◽  
Guinea Pigs ◽  
Fibrinolytic Activity ◽  
Pharmacokinetic Profile ◽  
Inhibitory Mechanisms ◽  
A Chain

SummaryA hybrid plasminogen activator consisting of the “A” chain of plasmin linked to the “B” chain of rt-PA was inhibited in vitro in human and guinea pig plasmas 4 to 5-fold more rapidly than its parent activator, two-chain t-PA. Using zymographic and autoradiographic techniques together with the use of immunodepleted plasma the major inhibitor was identified as aIpha-2-antiplasmin. The pharmacokinetic profile of the hybrid in guinea pigs was determined by two different methods: disappearance of fibrinolytic activity and removal of radiolabelled hybrid from the circulation. Fibrinolytic activity was cleared rapidly via inhibitory mechanisms, whilst radiolabelled material was cleared considerably more slowly due to the formation of hybrid-inhibitor complexes. When the active site of the hybrid was reversibly acylated inhibitory mechanisms were evaded and a prolonged pharmacokinetic profile of activity was observed.

scholarly journals Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase

Biochemistry ◽  
2016 ◽  
Vol 55 (32) ◽  
pp. 4476-4484 ◽  
Author(s):  
Thomas Robbins ◽  
Joshuah Kapilivsky ◽  
David E. Cane ◽  
Chaitan Khosla
Keyword(s):  
Active Site ◽  
Assembly Line ◽  
Polyketide Synthase ◽  
Active Site Residues ◽  
Ketosynthase Domain

In Vitro Analysis of Carboxyacyl Substrate Tolerance in the Loading and First Extension Modules of Borrelidin Polyketide Synthase

Biochemistry ◽  
2014 ◽  
Vol 53 (38) ◽  
pp. 5975-5977 ◽  
Author(s):  
Andrew Hagen ◽  
Sean Poust ◽  
Tristan de Rond ◽  
Satoshi Yuzawa ◽  
Leonard Katz ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Substrate Tolerance ◽  
Vitro Analysis ◽  
In Vitro Analysis

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 The DDE Motif in RAG-1 Is Contributed intrans to a Single Active Site That Catalyzes the Nicking and Transesterification Steps of V(D)J Recombination

2001 ◽  
Vol 21 (2) ◽  
pp. 449-458 ◽  
Author(s):  
Patrick C. Swanson
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Active Sites ◽  
Signal Sequence ◽  
Catalytic Triad ◽  
Dna Breaks ◽  
Site Mutation ◽  
Recombination Signal ◽  
Rag 1

ABSTRACT The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first-strand nicking, followed by hairpin formation via direct transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage intrans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.

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