14 research outputs found

    Mutation of Archaeal Isopentenyl Phosphate Kinase Highlights Mechanism and Guides Phosphorylation of Additional Isoprenoid Monophosphates

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    The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. <i>In vitro</i>, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg<sup>2+</sup>-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0−2.8 Å. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nε<sub>2</sub> nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors

    Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient <i>in Vivo</i> Incorporation of Structurally Divergent Non-native Amino Acids

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    Unnatural amino acids (Uaas) can be translationally incorporated into proteins <i>in vivo</i> using evolved tRNA/aminoacyl-tRNA synthetase (RS) pairs, affording chemistries inaccessible when restricted to the 20 natural amino acids. To date, most evolved RSs aminoacylate Uaas chemically similar to the native substrate of the wild-type RS; these conservative changes limit the scope of Uaa applications. Here, we adapt <i>Methanosarcina mazei</i> PylRS to charge a noticeably disparate Uaa, <i>O</i>-methyl-l-tyrosine (Ome). In addition, the 1.75 Ã… X-ray crystal structure of the evolved PylRS complexed with Ome and a non-hydrolyzable ATP analogue reveals the stereochemical determinants for substrate selection. Catalytically synergistic active site mutations remodel the substrate-binding cavity, providing a shortened but wider active site. In particular, mutation of Asn346, a residue critical for specific selection and turnover of the Pyl chemical core, accommodates different side chains while the central role of Asn346 in aminoacylation is rescued through compensatory hydrogen bonding provided by A302T. This multifaceted analysis provides a new starting point for engineering PylRS to aminoacylate a significantly more diverse selection of Uaas than previously anticipated

    Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases

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    Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis

    Structure, Biochemistry, and Inhibition of Essential 4′-Phosphopantetheinyl Transferases from Two Species of <i>Mycobacteria</i>

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    4′-Phosphopantetheinyl transferases (PPTase) post-translationally modify carrier proteins with a phosphopantetheine moiety, an essential reaction in all three domains of life. In the bacterial genus <i>Mycobacteria</i>, the Sfp-type PPTase activates pathways necessary for the biosynthesis of cell wall components and small molecule virulence factors. We solved the X-ray crystal structures and biochemically characterized the Sfp-type PPTases from two of the most prevalent Mycobacterial pathogens, PptT of <i>M. tuberculosis</i> and MuPPT of <i>M. ulcerans</i>. Structural analyses reveal significant differences in cofactor binding and active site composition when compared to previously characterized Sfp-type PPTases. Functional analyses including the efficacy of Sfp-type PPTase-specific inhibitors also suggest that the Mycobacterial Sfp-type PPTases can serve as therapeutic targets against Mycobacterial infections

    Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase

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    Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is an essential acyltransferase that mediates flux through plant phenylpropanoid metabolism by catalyzing a reaction between <i>p</i>-coumaroyl-CoA and shikimate, yet it also exhibits broad substrate permissiveness <i>in vitro</i>. How do enzymes like HCT avoid functional derailment by cellular metabolites that qualify as non-native substrates? Here, we combine X-ray crystallography and molecular dynamics to reveal distinct dynamic modes of HCT under native and non-native catalysis. We find that essential electrostatic and hydrogen-bonding interactions between the ligand and active site residues, permitted by active site plasticity, are elicited more effectively by shikimate than by other non-native substrates. This work provides a structural basis for how dynamic conformational states of HCT favor native over non-native catalysis by reducing the number of futile encounters between the enzyme and shikimate

    Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase

    No full text
    Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is an essential acyltransferase that mediates flux through plant phenylpropanoid metabolism by catalyzing a reaction between <i>p</i>-coumaroyl-CoA and shikimate, yet it also exhibits broad substrate permissiveness <i>in vitro</i>. How do enzymes like HCT avoid functional derailment by cellular metabolites that qualify as non-native substrates? Here, we combine X-ray crystallography and molecular dynamics to reveal distinct dynamic modes of HCT under native and non-native catalysis. We find that essential electrostatic and hydrogen-bonding interactions between the ligand and active site residues, permitted by active site plasticity, are elicited more effectively by shikimate than by other non-native substrates. This work provides a structural basis for how dynamic conformational states of HCT favor native over non-native catalysis by reducing the number of futile encounters between the enzyme and shikimate

    Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase

    No full text
    Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is an essential acyltransferase that mediates flux through plant phenylpropanoid metabolism by catalyzing a reaction between <i>p</i>-coumaroyl-CoA and shikimate, yet it also exhibits broad substrate permissiveness <i>in vitro</i>. How do enzymes like HCT avoid functional derailment by cellular metabolites that qualify as non-native substrates? Here, we combine X-ray crystallography and molecular dynamics to reveal distinct dynamic modes of HCT under native and non-native catalysis. We find that essential electrostatic and hydrogen-bonding interactions between the ligand and active site residues, permitted by active site plasticity, are elicited more effectively by shikimate than by other non-native substrates. This work provides a structural basis for how dynamic conformational states of HCT favor native over non-native catalysis by reducing the number of futile encounters between the enzyme and shikimate

    Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections

    No full text
    Inferring the historical and biophysical causes of diversity within protein families is a complex puzzle. A key to unraveling this problem is characterizing the rugged topography of sequence–function adaptive landscapes. Using biochemical data from a 29 = 512 combinatorial library of tobacco 5-epi-aristolochene synthase (TEAS) mutants engineered to make the native major product of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary 512 mutant HPS library, we address the question of how product specificity is controlled. These data sets reveal that HPS is far more robust and resistant to mutations than TEAS, where most mutants are promiscuous. We also combine experimental data with a sequence Potts Hamiltonian model and direct coupling analysis to quantify mutant fitness. Our results demonstrate that the Hamiltonian captures variation in product outputs across both libraries, clusters native family members based on their substrate specificities, and exposes the divergent catalytic roles of couplings between the catalytic and noncatalytic domains of TEAS versus HPS. Specifically, we found that the role of the interdomain connectivities in specifying product output is more important in TEAS than connectivities within the catalytic domain. Despite being 75% identical, this property is not shared by HPS, where connectivities within the catalytic domain are more important for specificity. By solving the X-ray crystal structure of HPS, we assessed structural bases for their interdomain network differences. Last, we calculate the product profile Shannon entropies of the two libraries, which showcases that site–site connectivities also play divergent roles in catalytic accuracy

    Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections

    No full text
    Inferring the historical and biophysical causes of diversity within protein families is a complex puzzle. A key to unraveling this problem is characterizing the rugged topography of sequence–function adaptive landscapes. Using biochemical data from a 29 = 512 combinatorial library of tobacco 5-epi-aristolochene synthase (TEAS) mutants engineered to make the native major product of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary 512 mutant HPS library, we address the question of how product specificity is controlled. These data sets reveal that HPS is far more robust and resistant to mutations than TEAS, where most mutants are promiscuous. We also combine experimental data with a sequence Potts Hamiltonian model and direct coupling analysis to quantify mutant fitness. Our results demonstrate that the Hamiltonian captures variation in product outputs across both libraries, clusters native family members based on their substrate specificities, and exposes the divergent catalytic roles of couplings between the catalytic and noncatalytic domains of TEAS versus HPS. Specifically, we found that the role of the interdomain connectivities in specifying product output is more important in TEAS than connectivities within the catalytic domain. Despite being 75% identical, this property is not shared by HPS, where connectivities within the catalytic domain are more important for specificity. By solving the X-ray crystal structure of HPS, we assessed structural bases for their interdomain network differences. Last, we calculate the product profile Shannon entropies of the two libraries, which showcases that site–site connectivities also play divergent roles in catalytic accuracy

    Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections

    No full text
    Inferring the historical and biophysical causes of diversity within protein families is a complex puzzle. A key to unraveling this problem is characterizing the rugged topography of sequence–function adaptive landscapes. Using biochemical data from a 29 = 512 combinatorial library of tobacco 5-epi-aristolochene synthase (TEAS) mutants engineered to make the native major product of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary 512 mutant HPS library, we address the question of how product specificity is controlled. These data sets reveal that HPS is far more robust and resistant to mutations than TEAS, where most mutants are promiscuous. We also combine experimental data with a sequence Potts Hamiltonian model and direct coupling analysis to quantify mutant fitness. Our results demonstrate that the Hamiltonian captures variation in product outputs across both libraries, clusters native family members based on their substrate specificities, and exposes the divergent catalytic roles of couplings between the catalytic and noncatalytic domains of TEAS versus HPS. Specifically, we found that the role of the interdomain connectivities in specifying product output is more important in TEAS than connectivities within the catalytic domain. Despite being 75% identical, this property is not shared by HPS, where connectivities within the catalytic domain are more important for specificity. By solving the X-ray crystal structure of HPS, we assessed structural bases for their interdomain network differences. Last, we calculate the product profile Shannon entropies of the two libraries, which showcases that site–site connectivities also play divergent roles in catalytic accuracy
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