<p>(A) Orthogonal views of the 7S,8S- stereoisomer of the competitive inhibitor EMDF bound ... more <p>(A) Orthogonal views of the 7S,8S- stereoisomer of the competitive inhibitor EMDF bound to EGS. Hydrogen-bond interactions formed by the EMDF molecule (cyan colored carbons) are represented as magenta dashed lines. The blue-colored contours envelope regions greater than 2.5σ in the initial F<sub>obs</sub>-F<sub>calc</sub> electron-density map. The direction of view used in the right panel (approximately perpendicular to the plane of the nicotinamide ring) is maintained roughly in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g005" target="_blank">figures 5B–D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g006" target="_blank">6B</a>. The chemical structure of EMDF is shown in the inset. (B) Modeled binding of coniferyl acetate to EGS. The atom coloring is the same as in (A), with magenta carbon atoms for the coniferyl acetate. The chemical structures of coniferyl acetate and EMDF are compared in the inset. The close interaction between the EMDF C7-atom and the hydride donor of the nicotinamide (C4) is shown as a yellow dashed line. (C) Binding of EMDF to the Lys132Gln variant of EGS. Hydrogen-bond interactions formed by the EMDF molecule (cyan colored carbons) are represented as magenta dashed lines. Hydrogen bonds involving the side chain of Gln132 are shown as orange dashed lines. The blue-colored contours envelope regions greater than 2.0σ in the initial F<sub>obs</sub>-F<sub>calc</sub> electron-density map. (D) Binding of EMDF to the Lys132Arg variant of EGS (stereo representation). The blue-colored contours envelope regions greater than 2σ in the initial F<sub>obs</sub>-F<sub>calc</sub> electron-density map for the EGS-Arg132/EMDF complex (green). The altered positioning of the Arg132 side-chain and neighboring residues (most notably Phe85, Ile88, and Ile129) and the disordering of the C-terminal tail (residues 310–314) are apparent with respect to the holo-EGS-Arg132 structure (magenta). For comparison, the position of the wild-type Lys132 side chain and the key bridging water molecule shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g005" target="_blank">Figure 5A</a> are also shown (yellow).</p
<div><p>(A) Comparison of the crystal structure of the B. subtilis ferrochelatase bou... more <div><p>(A) Comparison of the crystal structure of the B. subtilis ferrochelatase bound to NMMP to the model of the SynGUN4 core domain bound to Mg-Proto. The SynGUN4 core domain • Mg-Proto model was generated by GOLD [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030151#pbio-0030151-b54" target="_blank">54</a>]. The carboxylic acid moieties of the porphyrin were staggered between the δ-guanido side chains of Arg214 and Arg217. The position of the arginine loop used to tether the carboxyl moieties of the porphyrin bound to ferrochelatase served as the fixed point for the structural alignment of SynGUN4 and ferrochelatase.</p> <p>(B) Close-up view of the structural alignment between Mg-Proto (gold) and NMMP (lavender). Attempts to strictly superimpose all of the atoms of the two porphyrins resulted in at least one corner of the porphyrin scaffold residing out of the plane defined by the flat Mg-Proto complex, because of the pucker of NMMP.</p></div
<p>(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (col... more <p>(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (color coding as shown in inset). For clarity, only the NADP<sup>+</sup> cofactor of EGS is shown. (B) Comparison of NAD(P)-cofactor conformation and substrate-analog binding in EGS and UDP-galactose epimerase. The binding of the EGS competitive inhibitor (7S,8S)-ethyl (7,8-methylene)-dihydroferulate (EMDF) is described in detail in the text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g005" target="_blank">Figure 5</a>. The coloring of the polypeptide-chain segments is the same as in (A). The inset shows the coloring used for the carbon atoms of the nicotinamide cofactors, EMDF bound to EGS, and UDP-glucose bound to UDP-galactose-4-epimerase.</p
<p>Only the EGS polypeptide-chain segments that form direct interactions with the NADP<s... more <p>Only the EGS polypeptide-chain segments that form direct interactions with the NADP<sup>+</sup> cofactor are shown. Hydrogen-bond interactions formed by the cofactor are represented as magenta dashed lines. Atom coloring is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g002" target="_blank">Figure 2A</a>, except that the carbon atoms of the polypeptide-chain segments are green. The blue-colored contours envelope regions greater than 3σ in the NADP-omit electron-density map.</p
Subtle active site changes often result in the redirection of type III polyketide synthase (PKS) ... more Subtle active site changes often result in the redirection of type III polyketide synthase (PKS) reaction pathway intermediates toward different product fates. Complementary insights from two of our recently published structural and mechanistic studies prompt us to revise the existing 'steric modulation' mechanistic model for type III PKS functional divergence and resultant product specificity. Besides allowing for the active control of the reactivity of polyketide intermediates through mechanisms other then pure steric shape-dependent factors, our new 'reaction partitioning' model of functional divergence also recognizes the mechanistic importance of intramolecular chemical features of an extended polyketide that control the intrinsic reactivities of these highly reactive enzyme generated intermediates. This new model better explains how subtle active site changes can alter catalytic steps downstream of alternative reaction pathway branch points. Although formulated based upon work on type III PKS product specificity, the concept of 'reaction partitioning' can be used to describe control mechanisms in many other PKS biosynthetic systems that do not employ complete reduction of the growing polyketide intermediates.
Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Ma... more Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting
This study demonstrates how integration of plants into an artificial carbon cycle is capable of h... more This study demonstrates how integration of plants into an artificial carbon cycle is capable of harmoniously operating with Earth's natural cycles as one means to recycle atmospheric CO2 for economically lucrative green materials.
ABSTRACTElongating ketosynthases (KSs) catalyze carbon-carbon bond forming reactions during the c... more 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 theEscherichia coliFAS 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 bothin vitroandin vivoactivity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interac...
Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesi... more Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursi...
Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce... more Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the first x-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long timescale molecular dynamics (MD) sim...
<p>(A) Orthogonal views of the 7S,8S- stereoisomer of the competitive inhibitor EMDF bound ... more <p>(A) Orthogonal views of the 7S,8S- stereoisomer of the competitive inhibitor EMDF bound to EGS. Hydrogen-bond interactions formed by the EMDF molecule (cyan colored carbons) are represented as magenta dashed lines. The blue-colored contours envelope regions greater than 2.5σ in the initial F<sub>obs</sub>-F<sub>calc</sub> electron-density map. The direction of view used in the right panel (approximately perpendicular to the plane of the nicotinamide ring) is maintained roughly in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g005" target="_blank">figures 5B–D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g006" target="_blank">6B</a>. The chemical structure of EMDF is shown in the inset. (B) Modeled binding of coniferyl acetate to EGS. The atom coloring is the same as in (A), with magenta carbon atoms for the coniferyl acetate. The chemical structures of coniferyl acetate and EMDF are compared in the inset. The close interaction between the EMDF C7-atom and the hydride donor of the nicotinamide (C4) is shown as a yellow dashed line. (C) Binding of EMDF to the Lys132Gln variant of EGS. Hydrogen-bond interactions formed by the EMDF molecule (cyan colored carbons) are represented as magenta dashed lines. Hydrogen bonds involving the side chain of Gln132 are shown as orange dashed lines. The blue-colored contours envelope regions greater than 2.0σ in the initial F<sub>obs</sub>-F<sub>calc</sub> electron-density map. (D) Binding of EMDF to the Lys132Arg variant of EGS (stereo representation). The blue-colored contours envelope regions greater than 2σ in the initial F<sub>obs</sub>-F<sub>calc</sub> electron-density map for the EGS-Arg132/EMDF complex (green). The altered positioning of the Arg132 side-chain and neighboring residues (most notably Phe85, Ile88, and Ile129) and the disordering of the C-terminal tail (residues 310–314) are apparent with respect to the holo-EGS-Arg132 structure (magenta). For comparison, the position of the wild-type Lys132 side chain and the key bridging water molecule shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g005" target="_blank">Figure 5A</a> are also shown (yellow).</p
<div><p>(A) Comparison of the crystal structure of the B. subtilis ferrochelatase bou... more <div><p>(A) Comparison of the crystal structure of the B. subtilis ferrochelatase bound to NMMP to the model of the SynGUN4 core domain bound to Mg-Proto. The SynGUN4 core domain • Mg-Proto model was generated by GOLD [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030151#pbio-0030151-b54" target="_blank">54</a>]. The carboxylic acid moieties of the porphyrin were staggered between the δ-guanido side chains of Arg214 and Arg217. The position of the arginine loop used to tether the carboxyl moieties of the porphyrin bound to ferrochelatase served as the fixed point for the structural alignment of SynGUN4 and ferrochelatase.</p> <p>(B) Close-up view of the structural alignment between Mg-Proto (gold) and NMMP (lavender). Attempts to strictly superimpose all of the atoms of the two porphyrins resulted in at least one corner of the porphyrin scaffold residing out of the plane defined by the flat Mg-Proto complex, because of the pucker of NMMP.</p></div
<p>(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (col... more <p>(A) Superposition of polypeptide-chain backbones of EGS and UDP-galactose epimerase (color coding as shown in inset). For clarity, only the NADP<sup>+</sup> cofactor of EGS is shown. (B) Comparison of NAD(P)-cofactor conformation and substrate-analog binding in EGS and UDP-galactose epimerase. The binding of the EGS competitive inhibitor (7S,8S)-ethyl (7,8-methylene)-dihydroferulate (EMDF) is described in detail in the text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g005" target="_blank">Figure 5</a>. The coloring of the polypeptide-chain segments is the same as in (A). The inset shows the coloring used for the carbon atoms of the nicotinamide cofactors, EMDF bound to EGS, and UDP-glucose bound to UDP-galactose-4-epimerase.</p
<p>Only the EGS polypeptide-chain segments that form direct interactions with the NADP<s... more <p>Only the EGS polypeptide-chain segments that form direct interactions with the NADP<sup>+</sup> cofactor are shown. Hydrogen-bond interactions formed by the cofactor are represented as magenta dashed lines. Atom coloring is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000993#pone-0000993-g002" target="_blank">Figure 2A</a>, except that the carbon atoms of the polypeptide-chain segments are green. The blue-colored contours envelope regions greater than 3σ in the NADP-omit electron-density map.</p
Subtle active site changes often result in the redirection of type III polyketide synthase (PKS) ... more Subtle active site changes often result in the redirection of type III polyketide synthase (PKS) reaction pathway intermediates toward different product fates. Complementary insights from two of our recently published structural and mechanistic studies prompt us to revise the existing 'steric modulation' mechanistic model for type III PKS functional divergence and resultant product specificity. Besides allowing for the active control of the reactivity of polyketide intermediates through mechanisms other then pure steric shape-dependent factors, our new 'reaction partitioning' model of functional divergence also recognizes the mechanistic importance of intramolecular chemical features of an extended polyketide that control the intrinsic reactivities of these highly reactive enzyme generated intermediates. This new model better explains how subtle active site changes can alter catalytic steps downstream of alternative reaction pathway branch points. Although formulated based upon work on type III PKS product specificity, the concept of 'reaction partitioning' can be used to describe control mechanisms in many other PKS biosynthetic systems that do not employ complete reduction of the growing polyketide intermediates.
Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Ma... more Joseph Noel from the Salk Institute on "Metabolic Noise, Vestigial Metabolites or the Raw Material of Ecological Adaptation? Enzymes, Catalytic Promiscuity and the Evolution of Chemodiversity in Nature" on March 26, 2010 at the 5th Annual DOE JGI User Meeting
This study demonstrates how integration of plants into an artificial carbon cycle is capable of h... more This study demonstrates how integration of plants into an artificial carbon cycle is capable of harmoniously operating with Earth's natural cycles as one means to recycle atmospheric CO2 for economically lucrative green materials.
ABSTRACTElongating ketosynthases (KSs) catalyze carbon-carbon bond forming reactions during the c... more 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 theEscherichia coliFAS 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 bothin vitroandin vivoactivity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interac...
Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesi... more Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursi...
Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce... more Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the first x-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long timescale molecular dynamics (MD) sim...
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