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Michael D. Burkart - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis for interactions between an acyl Carrier Protein and a ketosynthase.
Nature Chemical Biology, 2019Co-Authors: Jacob C. Milligan, Michael D. Burkart, D. John Lee, David R. Jackson, Andrew J. Schaub, Joris Beld, Jesus F. Barajas, Joseph J. Hale, Ray Luo, Shiou-chuan TsaiAbstract:Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl Carrier Protein (AcpP) and β-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how Protein–Protein interactions can regulate the fatty acid profile in E. coli. A combination of crosslinking, X-ray crystallography, NMR, and mutagenesis provide a detailed visualization of the interactions between an acyl Carrier Protein and β-ketoacyl-ACP-synthase I in the Escherchia coli fatty acid synthase complex.
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Trapping of the Enoyl-Acyl Carrier Protein Reductase-Acyl Carrier Protein Interaction.
Journal of the American Chemical Society, 2016Co-Authors: Lorillee Tallorin, Joris Beld, Kara Finzel, Quynh G. Nguyen, James J. La Clair, Michael D. BurkartAbstract:An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These Carrier Protein-dependent pathways require fundamental Protein–Protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl Carrier Proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes.
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Resin supported acyl Carrier Protein labeling strategies
RSC Adv., 2014Co-Authors: Michael Rothmann, Nicolas M. Kosa, Michael D. BurkartAbstract:The post-translational modifying enzymes phophopantetheinyl transferase and acyl Carrier Protein hydrolase show utility in the functional modification of acyl Carrier Proteins. Here we develop these tools as immobilized biocatalysts on agarose supports. New utility is imparted through these methods, enabling rapid and label-independent Protein purification. Immobilization of acyl Carrier Protein is also demonstrated for rapid activity assays of these 4′-phosophopantetheine modifying enzymes, displaying a particular advantage in the case of phosphopantetheine removal, where few orthogonal techniques have been demonstrated. These tools further enrich the suite of functional utility of 4′-phosophopantetheine chemistry, with applications to Protein functionalization, materials, and natural product biosynthetic studies.
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A mechanism based Protein crosslinker for acyl Carrier Protein dehydratases
Bioorganic & Medicinal Chemistry Letters, 2010Co-Authors: Jordan L. Meier, Robert W. Haushalter, Michael D. BurkartAbstract:Abstract Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl Carrier Protein (ACP), a small (∼9 kDa) Protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4′-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl Carrier Protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP–DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these Carrier Protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work.
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The ubiquitous Carrier Protein--a window to metabolite biosynthesis.
Natural product reports, 2007Co-Authors: Andrew C. Mercer, Michael D. BurkartAbstract:Nature has developed a remarkable strategy to isolate metabolites from the milieu of the cell for chemical modification through the use of Carrier Proteins. Common to both primary and secondary metabolic pathways, acyl-Carrier Proteins constitute a conserved Protein architecture which mediate the biosynthesis of a variety of metabolic products. Analogies have been made between the Carrier Protein and solid phase resin for chemical synthesis, as both entities provide a mechanism to separate compounds of interest from complex mixtures for selective chemical modification. However, there is significantly more to the Carrier Protein than an attachment point. In this review, we aim to systematically characterize the role of Carrier Proteins in various metabolic pathways and outline their utility in biosynthesis and biotechnology; 185 references are cited.
Pr Adiga - One of the best experts on this subject based on the ideXlab platform.
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Riboflavin Carrier Protein from carp (C. carpio) eggs: comparison with avian riboflavin Carrier Protein
Biochemistry international, 1991Co-Authors: Pooja Malhotra, Anjali A. Karande, Tk Prasadan, Pr AdigaAbstract:A Protein exhibiting immunological cross-reactivity with the chicken egg-white riboflavin Carrier Protein was detected by radioimmunoassay in the eggs and serum of the fresh water fish Cyprinus carpio and subsequently purified to homogeneity by use of affinity chromatography. Fish riboflavin Carrier Protein resembled chicken riboflavin Carrier Protein with respect to most of its physicochemical characteristics. The major epitopes of chicken riboflavin Carrier Protein were shown to be conserved in the fish Protein as probed with monoclonal antibodies to the avian vitamin Carrier.
Christopher T. Walsh - One of the best experts on this subject based on the ideXlab platform.
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Carrier Protein recognition in siderophore-producing nonribosomal peptide synthetases.
Biochemistry, 2002Co-Authors: C. Gary Marshall, Michael D. Burkart, Robin K. Meray, Christopher T. WalshAbstract:Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing Carrier Proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor−Carrier Proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize “Carrier Protein-free” pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor−Carrier Protein. When tested with four noncognate Carrier Proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point ...
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characterization of sfp a bacillus subtilis phosphopantetheinyl transferase for peptidyl Carrier Protein domains in peptide synthetases
Biochemistry, 1998Co-Authors: Luis E N Quadri, Paul H Weinreb, Michiko M Nakano, Peter Zuber, Christopher T. WalshAbstract:The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl Carrier Protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16−17-kDa peptidyl Carrier Protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56−104 min-1 and Km values of 1.3−1.8 μM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant Proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl Carrier Protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl Carrier Protein (...
Namita Surolia - One of the best experts on this subject based on the ideXlab platform.
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Isothermal unfolding studies on the apo and holo forms of Plasmodium falciparum acyl Carrier Protein
FEBS Journal, 2007Co-Authors: Rahul Modak, Sharmistha Sinha, Namita SuroliaAbstract:The unfolding pathways of the two forms of Plasmodium falciparum acyl Carrier Protein, the apo and holo forms, were determined by guanidine hydrochloride-induced denaturation. Both the apo form and the holo form displayed a reversible two-state unfolding mechanism. The analysis of isothermal denaturation data provides values for the conformational stability of the two Proteins. Although both forms have the same amino acid sequence, and they have similar secondary structures, it was found that the – $\Delta G$ of unfolding of the holo form was lower than that of the apo form at all the temperatures at which the experiments were done. The higher stability of the holo form can be attributed to the number of favorable contacts that the 4'-phosphopantetheine group makes with the surface residues by virtue of a number of hydrogen bonds. Furthermore, there are several hydrophobic interactions with 4'-phosphopantetheine that firmly maintain the structure of the holo form. We show here for the first time that the interactions between 4'-phosphopantetheine and the polypeptide backbone of acyl Carrier Protein stabilize the Protein. As Plasmodium acyl Carrier Protein has a similar secondary structure to the other acyl Carrier Proteins and acyl Carrier Protein-like domains, the detailed biophysical characterization of Plasmodium acyl Carrier Protein can serve as a prototype for the analysis of the conformational stability of other acyl Carrier Proteins.
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Analyses of co‐operative transitions in Plasmodium falciparumβ‐ketoacyl acyl Carrier Protein reductase upon co‐factor and acyl Carrier Protein binding
FEBS Journal, 2006Co-Authors: Krishanpal Karmodiya, Namita SuroliaAbstract:The type II fatty acid synthase pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials because of its intrinsic differences from the type I pathway operating in humans. β-Ketoacyl-acyl Carrier Protein reductase is the only enzyme of this pathway that has no isoforms and thus selective inhibitors can be developed for this player of the pathway. We report here intensive studies on the direct interactions of Plasmodiumβ-ketoacyl-acyl Carrier Protein reductase with its cofactor, NADPH, acyl Carrier Protein, acetoacetyl-coenzyme A and other ligands in solution, by monitoring the intrinsic fluorescence (λmax 334 nm) of the Protein as a result of its lone tryptophan, as well as the fluorescence of NADPH (λmax 450 nm) upon binding to the enzyme. Binding of the reduced cofactor makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate, acetoacetyl-coenzyme A, by 16-fold. The binding affinity of acyl Carrier Protein to the enzyme also increases by approximately threefold upon NADPH binding. Plasmodiumβ-ketoacyl-acyl Carrier Protein reductase exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in the presence of acyl Carrier Protein. Acyl Carrier Protein increases the accessibility of NADPH to β-ketoacyl-acyl Carrier Protein reductase, as evident from the increase in the accessibility of the tryptophan of β-ketoacyl-acyl Carrier Protein reductase to acrylamide, from 81 to 98%. In the presence of NADP+, the reaction proceeds in the reverse direction (Ka = 23.17 µm−1). These findings provide impetus for exploring the influence of ligands on the structure–activity relationship of Plasmodiumβ-ketoacyl-acyl Carrier Protein reductase.
Udo Seedorf - One of the best experts on this subject based on the ideXlab platform.
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Sterol Carrier Protein-2
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2000Co-Authors: Udo Seedorf, Peter Ellinghaus, Jerzy Roch NoferAbstract:Abstract The compartmentalization of cholesterol metabolism implies target-specific cholesterol trafficking between the endoplasmic reticulum, plasma membrane, lysosomes, mitochondria and peroxisomes. One hypothesis has been that sterol Carrier Protein-2 (SCP2, also known as the non-specific lipid transfer Protein) acts in cholesterol transport through the cytoplasm. Recent studies employing gene targeting in mice showed, however, that mice lacking SCP2 and the related putative sterol Carrier known as SCPx, develop a defect in peroxisomal β-oxidation. In addition, diminished peroxisomal α-oxidation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) in these null mice was attributed to the absence of SCP2 which has a number of properties supporting a function as Carrier for fatty acyl-CoAs rather than for sterols.
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defective peroxisomal catabolism of branched fatty acyl coenzyme a in mice lacking the sterol Carrier Protein 2 sterol Carrier Protein x gene function
Genes & Development, 1998Co-Authors: Udo Seedorf, Peter Ellinghaus, Martin Raabe, Frank Kannenberg, Manfred Fobker, Thomas Engel, Simone Denis, Fred S Wouters, Karel W A Wirtz, Ronald J A WandersAbstract:Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol Carrier Protein-2 (SCP2; a peroxisomal lipid Carrier) and sterol Carrier Protein-x (SCPx; a fusion Protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(˛/˛) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 Protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx Protein.