Protein chips are powerful tools
as analytical and diagnostic devices
for detection of biomolecular interactions, where the proteins are
covalently or noncovalently attached to biosensing surfaces to capture
and detect target molecules or biomarkers. Thus, fabrication of biosensing
surfaces for regio- and chemoselective immobilization of biomolecules
is a crucial step for better biosensor performance. In our previous
studies, a regio- and chemoselective immobilization strategy was demonstrated
on glass surfaces. This strategy is now used to regioselectively attach
proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant
green fluorescent protein (GFP), glutathione S-transferase (GST),
and antibody-binding protein G, bearing a C-terminal CVIA motif,
were prepared and a farnesyl analogue with an ω-alkyne moiety
was attached to the sulfhydryl moiety in the cysteine side chain by
protein farnesyltransferase. The proteins, modified with the bioorthogonal
alkyne functional group, were covalently and regioselectively immobilized
on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen
[3 + 2] cycloaddition reaction with minimal nonspecific binding. A
concentration-dependent increase of fluorescence intensity was observed
in wells treated with GFP on both thiol- and DTC-SAMs. The highly
ordered, densely packed layer allowed for a high loading of immobilized
protein, with a concomitant increase in substrate binding capacity.
The DTC-SAMs were substantially more resistant to displacement of
the immobilized proteins from the gold surface by β-mercaptoethanol
than alkane-thiol SAMs.
The tyrosine O-prenyltransferase SirD in Leptosphaeria maculans catalyzes normal prenylation of the hydroxyl group in tyrosine as the first committed step in the biosynthesis of the phytotoxin sirodesmin PL. SirD also catalyzes normal N-prenylation of 4-aminophenylalanine and normal C-prenylation at C7 of tryptophan. In this study, we found that 4-mercaptophenylalanine and several derivatives of tryptophan are also substrates for prenylation by dimethylallyl diphosphate. Incubation of SirD with 4- mercaptophenylalanine gave normal S-prenylated mercaptophenylalanine. We found that incubation of the enzyme with tryptophan gave reverse prenylation at N1 in addition to the previously reported normal prenylation at C7. 4-Methyltryptophan also gave normal prenylation at C7 and reverse prenylation at N1; whereas 4-methoxytryptophan gave normal and reverse prenylation at C7, and 7-methyltryptophan gave normal prenylation at C6 and reverse prenylation at N1. The ability of SirD to prenylate at three different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is similar to behavior seen for dimethylallyltryptophan synthase. The multiple products produced by SirD suggests it and dimethylallyltryptophan synthase use a dissociative electrophilic mechanism for alkylation of amino acid substrates.
Terpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed.
The rapid growth in the number of protein sequences presents challenges for enzyme function assignment. Computational methods, such as bioinformatics, homology modeling and docking, are becoming increasingly important for predicting of enzyme functions from protein sequences. Terpenoids are one of largest classes of natural products, and many drugs (e.g. taxol) consist of terpenoids or terpenoid derivatives. Understanding the biosynthesis of the terpenoids is of great interest. Terpenoid synthases catalyze the key cyclization steps of the biosynthesis of terpenoids via carbocation rearrangements, generating numerous multiple-ring carbon skeletons. Triterpenoid synthases, as an important class of terpenoid synthases, catalyze the cyclization of either squalene or oxido-squalene into cyclized products such as sterols (e.g. lanosterol). In this work, we propose a computational approach that can be used to predict product specificity of the triterpenoid synthases. Our approach provides insight into the ‘design principles’ of these fascinating enzymes, and may become a practical approach for function prediction and enzyme engineering.
Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and non-ionic detergents, we discovered considerable variability. Examination of a series of non-ionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo. In contrast incubations in buffer with n-butanol, CHAPS, DMSO, n-octyl-β-glucopyranoside, or β-cyclodextrin or in buffer without detergent typically proceeded more slowly and gave a broad range of chain lengths.
Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies are attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody binding proteins are produced in E. coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 ° C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies.
Type-2 isopentenyl diphosphate isomerase (IDI-2) is a key flavoprotein involved in the biosynthesis of isoprenoids. Since fully reduced flavin mononucleotide (FMNH2) is needed for activity, it was decided to crystallize the enzyme under anaerobic conditions in order to understand how this reduced cofactor binds within the active site and interacts with the substrate isopentenyl diphosphate (IPP). In this study, the protein was expressed and purified under aerobic conditions and then reduced and crystallized under anaerobic conditions. Crystals grown by the sitting-drop vapour-diffusion method and then soaked with IPP diffracted to 2.1 Å resolution and belonged to the hexagonal space group P6322, with unit-cell parameters a = b = 133.3, c = 172.9 Å.
isopentenyl diphosphate isomerase; IDI-2; flavoprotein; anaerobic; isoprenoid; twinning
The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His6-tagged glutathione S-transferase (His6-GSTase-CVIA) was posttranslationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His6-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω–alkyne and azide moieties and covalently attached to the surface by a Cu(I)-catalyzed Huisgen [3+2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in A340 upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His6-GSTase-CVIA-PE) or azide (His6-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His6-GSTase-CVIA-F).
The aromatic prenyltransferase dimethylallyltryptophan synthase in Claviceps purpurea catalyzes the normal prenylation of tryptophan at C4 of the indole nucleus in the first committed step of ergot alkaloid biosynthesis. 4-Methyltryptophan is a competitive inhibitor of the enzyme that has been used in kinetic studies. Upon investigation of background activity during incubations of 4-methyltryptophan with dimethylallyl diphosphate, we found that the analogue was an alternate substrate, which gave four products. The structures of three of these compounds were established by 1H NMR and 2D NMR studies and revealed that dimethylallyltryptophan synthase catalyzed both normal and reverse prenylation at C3 of the indole ring and normal prenylation of N1. Similarly, 4-methoxytryptophan was an alternate substrate, giving normal prenylation at C5 as the major product. 4-Aminotryptophan, another alternate substrate, gave normal prenylation at C5 and C7. The ability of dimethylallyltryptophan synthase to prenylate at five different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is consistent with a dissociative electrophilic alkylation of the indole ring where orientation of the substrates within the active site and substituent electronic effects determine the position and type of prenylation. These results suggest a common mechanism for prenylation of tryptophan by all of the members of the structurally related dimethylallyltryptophan synthase family.
The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-2-methylbut-2-enyl 1-diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S]2+ moiety. Here we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methyl-but-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with Ki = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with Ki = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting in the formation of the enzyme-inhibitor (EI) complex.
A new practical route to chaetomellic acid A (ACA), based on the copper catalysed radical cyclization (RC) of (Z)-3-(2,2-dichloropropanoyl)-2-pentadecylidene-1,3-thiazinane, is described. Remarkably, the process entailed: i) a one-pot preparation of the intermediate N-α-perchloroacyl-2-(Z)-alkyliden-1,3-thiazinanes starting from N-(3-hydroxypropyl)palmitamide, ii) a two step smooth transformation of the RC products into ACA and iii) only one intermediate chromatographic purification step. The method offers a versatile approach to the preparation of ACA analogues, through the synthesis of an intermediate maleic anhydride with a vinylic group at the end of the aliphatic tail, a function that can be transformed through a thiol-ene coupling. Serendipitously, the disodium salt of 2-(9-(butylthio)nonyl)-3-methylmaleic acid, that we prepared as a representative sulfurated ACA analogue, was a more competent FTase inhibitor than ACA. This behaviour was analysed by a molecular docking study.
Chaetomellic acid A; Farnesyl pyrophosphate; FTase inhibitors; Modelling studies; Radical cyclization
Isopentenyl phosphate kinase (IPK) catalyzes the ATP-dependent phosphorylation of isopentenyl phosphate (IP) to form isopentenyl diphosphate (IPP) during biosynthesis of isoprenoid metabolites in Archaea. The structure of IPK from the archeaon Thermoplasma acidophilum (THA) was recently reported and guided the reconstruction of the IP binding site to accommodate the longer chain isoprenoid monophosphates geranyl phosphate (GP) and farnesyl phosphate (FP). We created four mutants of THA IPK with different combinations of alanine substitutions for Tyr70, Val73, Val130 and Ile140, amino acids with bulky side chains that limited the size of the side chain of the isoprenoid phosphate substrate that could be accommodated in the active site. The mutants had substantially increased GP kinase activity, with 20 to 200–fold increases in kcatGP and 30–130–fold increases in kcatGP/KMGP relative to that of wild type THA IPK. The mutations also resulted in a 106–fold drop in kcatIP/KMIP compared to wild-type IPK. No significant change in the kinetic parameters for the cosubstrate ATP were observed, signifying that binding between the nucleotide binding site and the IP binding site was not cooperative. The shift in substrate selectivity from IP to GP, and to a lesser extent, FP, in the mutants could act as a starting point for the creation of more efficient GP or FP kinases whose products could be exploited for the chemoenzymatic synthesis of radiolabeled isoprenoid diphosphates.
Isoprenoid compounds constitute an immensely diverse group of acyclic, monocyclic and polycyclic compounds that play important roles in all living organisms. Despite the diversity of their structures, this plethora of natural products arises from only two 5-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This review will discuss the enzymes in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways leading to IPP and DMAPP with a particular focus on MEP synthase (DXR) and IPP isomerase (IDI), which are potential targets for the development of antibiotic compounds. DXR is the second enzyme in the MEP pathway and the only one for which inhibitors with antimicrobial activity at pharmaceutically relevant concentrations are known. All of the published DXR inhibitors are fosmidomycin analogues, except for a few bisphosphonates with moderate inhibitory activity. These far, there are no other candidates that target DXR. IDI was first identified and characterised over 40 years ago (IDI-1) and a second convergently evolved isoform (IDI-2) was discovered in 2001. IDI-1 is a metalloprotein found in Eukarya and many species of Bacteria. Its mechanism has been extensively studied. In contrast, IDI-2 requires reduced flavin mononucleotide as a cofactor. The mechanism of action for IDI-2 is less well defined. This review will describe how lead inhibitors are being improved by structure-based drug design and enzymatic assays against DXR to lead to new drug families and how mechanistic probes are being used to address questions about the mechanisms of the isomerases.
DXR; IDI; isomerase; isopentenyl; isoprenoid; MEP; mevalonate; MVA; reductoisomerase
Archaea synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the essential building blocks of isoprenoid compounds, from mevalonate (MVA). However, an analysis of the genomes of several members of the Archaea failed to identify genes for the enzymes required to convert phosphomevalonate (PM) to IPP in Eukaryotes. The recent discovery of an isopentenyl kinase (IPK) in Methanocaldococcus jannaschii (MJ) suggests a new variation of the MVA pathway where PM is decarboxylated to give isopentenyl phosphate (IP), which is phosphorylated to produce IPP. A blast search using the MJ protein as a probe revealed a subfamily of amino acid kinases that include the fosfomycin resistance protein fomA, which deactivates the antibiotic by phosphorylation of its phosphonate residue in a reaction similar to the conversion of IP to IPP. IPK genes were cloned from two organisms identified in the search, Methanothermobacter thermautotrophicus (MTH) and Thermoplasma acidophilum (THA), and the His-tagged recombinant proteins were purified by Ni-NTA chromatography. The enzymes catalyze the reversible phosphorylation of IP by ATP, Keq = 6.3 ± 1. The catalytic efficiencies (V/K) of the proteins were ~2 × 106 M−1s−1. In the reverse direction, ADP was a substrate inhibitor for THA IPK, KiADP = 58 ± 6 µM but not for MTH IPK. Both enzymes were active over a broad range of pH and temperature. Five compounds, dimethylallyl phosphate, isopentenyl thiolophosphate, 1-butyl phosphate, 3-buten-1-yl phosphate, and geranyl phosphate, were evaluated as alternative substrate for the MTH and THA IP kinases. All of the compounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate.
Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this novel reaction constitutes the first committed step in sterol biosynthesis, there has been considerable interest and research on the stereochemistry and mechanism of the process and in the design of selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as inhibitors of recombinant yeast squalene synthase. The key aziridine-2-methanol intermediates (6-OH, 7-OH, and 8-OH) were obtained by N-alkylations or by an N-acylation–reduction sequence of (±)-, (2R,3S)-, and (2S,3R)-2,3-aziridinofarnesol (9-OH) protected as tert-butyldi-methylsilyl ethers. SN2 displacements of the corresponding methanesulfonates with pyrophosphate and methanediphosphonate anions afforded aziridine 2-methyl diphosphates and methanediphosphonates bearing N-undecyl, N-bishomogeranyl, and N-(α-methylene)bishomogeranyl substituents as mimics for the 2,6,10-trimethylundeca-2,5,9-trienyl side chain of PSPP. The 2R,3S diphosphate enantiomer bearing the N-bishomogeranyl substituent corresponding in absolute stereochemistry to PSPP proved to be the most potent inhibitor (IC50 1.17 ± 0.08 μM in the presence of inorganic pyrophosphate), a value 4-fold less than that of its 2S,3R stereoisomer. The other aziridine analogues bearing the N-(α-methylene)bishomogeranyl and N-undecyl substituents, and the related methanediphosphonates, exhibited lower affinities for recombinant squalene synthase.
Inhibitors; LytB (IspH); MEP pathway; Metalloenzymes; Mössbauer spectroscopy
Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate (IPP) in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK, and H58 and K18 in S. wedmorensis FomA. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase superfamily. We measured the fosfomycin kinase activity of THA IPK, Km = 15.1 ± 1.0 mM and kcat = (4.0 ± 0.1) × 10−2 s−1, resulting in a catalytic efficiency, kcat/Km = 2.6 M−1s−1, that is five orders of magnitude less than the native reaction. Fosfomycin is a competitive inhibitor of IPK, Ki = 3.6 ± 0.2 mM. Molecular dynamics simulation of the IPK•fosfomycin•MgATP complex identified two binding poses for fosfomycin in the IP binding site, one of which results in a complex analogous to the native IPK•IP•ATP complex that it engages H50 and the lysine triangle formed by K5, K14, and K205. The other binding pose leads to a dead-end complex that engages K204 near the IP binding site to bind fosfomycin. Our findings suggest a mechanism for acquisition of FomA-based antibiotic resistance in fosfomycin producing organisms.
Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These two molecules are the building blocks for construction of isoprenoid carbon skeletons in nature. Two structurally unrelated forms of IDI are known. A variety of studies support a proton addition/proton elimination mechanism for both enzymes. During studies with Thermus thermophilus IDI-2, we discovered that the olefinic hydrogens of a vinyl thiomethyl analogue of isopentenyl diphosphate exchanged with solvent when the enzyme was incubated with D2O without concomitant isomerization of the double bond. These results suggest that the enzyme-catalyzed isomerization reaction is not concerted.
The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily-specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include: 1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation); 2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia; 3) computational and bioinformatic tools for using the strategy; 4) provision of experimental protocols and/or reagents for enzyme production and characterization; and 5) dissemination of data via the EFI’s website, enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal and pharmaceutical efforts.
Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a co-substrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of KM3-ClIPP (with DMAPP) and KM3-ClDMAPP (with IPP) were similar to those for IPP and DMAPP, however values of kcat for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.
Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition/abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP, but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1, but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP.
Isoprenoid compounds are ubiquitous in nature, participating in important biological phenomena such as signal transduction, aerobic cellular respiration, photosynthesis, insect communication, and many others. They are derived from the 5-carbon isoprenoid substrates isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In Archaea and Eukarya, these building blocks are synthesized via the mevalonate pathway. However, the genes required to convert mevalonate phosphate (MP) to IPP are missing in several species of Archaea. An enzyme with isopentenyl phosphate kinase (IPK) activity was recently discovered in Methanocaldococcus jannaschii (MJ), suggesting a departure from the classical sequence of converting MP to IPP. We have determined the high-resolution crystal structures of isopentenyl phosphate kinases in complex with both substrates and products from Thermoplasma acidophilum (THA), as well as the IPK from Methanothermobacter thermautotrophicus (MTH), by means of single-wavelength anomalous diffraction (SAD) and molecular replacement. A histidine residue (His50) in THA IPK makes a hydrogen bond with the terminal phosphates of IP and IPP, poising these molecules for phosphoryl transfer through an in-line geometry. Moreover, a lysine residue (Lys14) makes hydrogen bonds with non-bridging oxygen atoms at Pα and Pγ and with the Pβ- Pγ bridging oxygen atom in ATP. These interactions suggest a transition state-stabilizing role for this residue. Lys14 is a part of a newly discovered “lysine triangle” catalytic motif in IPK’s that also includes Lys5 and Lys205. Moreover, His50, Lys5, Lys14, and Lys205 are conserved in all IPK’s and can therefore serve as fingerprints for identifying new homologues.
Incubation of farnesyl diphosphate with recombinant yeast squalene synthase in the absence of NADPH gives a mixture of triterpene hydrocarbons and alcohols, including botryococcene-like compounds with 1’-3 linkages between the farnesyl units. One of these molecules has an unusual cyclopentane structure similar to those recently reported in plant extracts and lakebed sediments.
Organic substances were conceived as those found in living organisms. Although the definition was soon broadened to include all carbon-containing compounds, naturally occurring molecules have always held a special fascination for organic chemists. From these beginnings, molecules from nature were indespensible tools as generations of organic chemists developed new techniques for determining structures, analyzed the mechanisms of reactions, explored the effects conformation and stereochemistry on reactions, and found challenging new targets to synthesize. Only recently have organic chemists harnessed the powerful techniques of organic chemistry to study the functions of organic molecules in their biological hosts, the enzymes that synthesize molecules and the complex processes that occur in a cell. In this Perspective, I present a personal account my entrée into bioorganic chemistry as a physical organic chemist and subsequent work to understand the chemical mechanisms of enzyme-catalyzed reactions, to develop techniques to identify and assign hydrogen bonds in tRNAs through NMR studies with isotopically labeled molecules, and to study how structure determines function in biosynthetic enzymes with proteins obtained by genetic engineering.
Eleven farnesyl diphosphate analogues, which contained ω-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3+2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated as substrates for alkylation of peptide co-substrates by yeast protein farnesyl transferase. Five of the diphosphates were good alternative substrates for FPP. Steady-state kinetic constants were measured for the active compounds, and the products were characterized by HPLC and LC-Mass. Two of the analogues gave steady state kinetic parameters (kcat and KM) very similar to those of the natural substrate.
Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway-specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from the hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His6 tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His6-SQS gave a single band at 42 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [14C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg2+ for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the Km of FPP is 0.97 ± 0.10 μM and the kcat is 1.74 ± 0.04 s−1. Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 ± 13.6 nM.