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.

scholarly journals Structural Modeling and Site-Directed Mutagenesis of the Actinorhodin β-Ketoacyl-Acyl Carrier Protein Synthase

2000 ◽  
Vol 182 (9) ◽  
pp. 2619-2623 ◽  
Author(s):  
Min He ◽  
Mustafa Varoglu ◽  
David H. Sherman
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Three Dimensional Model ◽  
Catalytic Active Site ◽  
Ketoacyl Synthase

ABSTRACT A three-dimensional model of the Streptomyces coelicolor actinorhodin β-ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the relatedEscherichia coli fatty acid synthase condensing enzyme β-ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. 3H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.

scholarly journals The Structure of (3R)-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ) fromPseudomonas aeruginosa

2004 ◽  
Vol 279 (50) ◽  
pp. 52593-52602 ◽  
Author(s):  
Matthew S. Kimber ◽  
Fernando Martin ◽  
Yingjie Lu ◽  
Simon Houston ◽  
Masoud Vedadi ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Isomerization Reaction ◽  
Antibacterial Drug ◽  
Carrier Protein ◽  
Type Ii ◽  
Active Site Residues ◽  
Acid Biosynthesis

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by β-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP totrans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization oftrans-2- tocis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogenPseudomonas aeruginosaat 2.5 Å of resolution.PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

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 Activity Mapping the Acyl Carrier Protein - Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis

2020 ◽  
Author(s):  
Jeffrey T. Mindrebo ◽  
Laetitia E. Misson ◽  
Caitlin Johnson ◽  
Joseph P. Noel ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Chain Extension ◽  
Carrier Protein ◽  
Type Ii ◽  
Multiple Partner ◽  
Carbon Carbon Bond

ABSTRACTElongating ketosynthases (KSs) catalyze carbon-carbon bond forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution detail of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region delete variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of entire regions significantly compromising activity. Structure and sequence analysis of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.

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.

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA

2018 ◽  
Vol 74 (5) ◽  
pp. 383-393 ◽  
Author(s):  
Tanja Küssau ◽  
Marion Flipo ◽  
Niel Van Wyk ◽  
Albertus Viljoen ◽  
Vincent Olieric ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Crystal Structures ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Rational Drug Design ◽  
Carrier Protein ◽  
Cofactor Binding

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP+-bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP+-bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

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