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1.  Biosynthesis of Squalene from Farnesyl Diphosphate in Bacteria: Three Steps Catalyzed by Three Enzymes 
ACS Central Science  2015;1(2):77-82.
Squalene (SQ) is an intermediate in the biosynthesis of sterols in eukaryotes and a few bacteria and of hopanoids in bacteria where they promote membrane stability and the formation of lipid rafts in their hosts. The genes for hopanoid biosynthesis are typically located on clusters that consist of four highly conserved genes—hpnC, hpnD, hpnE, and hpnF—for conversion of farnesyl diphosphate (FPP) to hopene or related pentacyclic metabolites. While hpnF is known to encode a squalene cyclase, the functions for hpnC, hpnD, and hpnE are not rigorously established. The hpnC, hpnD, and hpnE genes from Zymomonas mobilis and Rhodopseudomonas palustris were cloned into Escherichia coli, a bacterium that does not contain genes homologous to hpnC, hpnD, and hpnE, and their functions were established in vitro and in vivo. HpnD catalyzes formation of presqualene diphosphate (PSPP) from two molecules of FPP; HpnC converts PSPP to hydroxysqualene (HSQ); and HpnE, a member of the amine oxidoreductase family, reduces HSQ to SQ. Collectively the reactions catalyzed by these three enzymes constitute a new pathway for biosynthesis of SQ in bacteria.
In determining the functions of three highly conserved genes in bacterial hopanoid gene clusters, we discover that each participates in a new pathway for biosynthesis of squalene from farnesyl diphosphate.
PMCID: PMC4527182  PMID: 26258173
2.  Synthesis of Methylerythritol Phosphate Analogues and Their Evaluation as Alternate Substrates for IspDF and IspE from Agrobacterium tumefaciens 
The Journal of Organic Chemistry  2014;79(19):9170-9178.
The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts d-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. d-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five d-MEP analogues—d-erythritol-4-phosphate (EP), d-3-methylthrietol-4-phosphate (MTP), d-3-ethylerythritol-4-phosphate (EEP), d-1-amino-3-methylerythritol-4-phosphate (NMEP), and d-3-methylerythritol-4-thiolophosphate (MESP)—and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes.
PMCID: PMC4184463  PMID: 25184438
3.  Determination of kinetics and crystal structure of a novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase from Streptococcus pneumoniae 
Isopentenyl diphosphate dimethylallyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein and is found in eukaryotes, while the type-2 isoform (IDI-2) is a flavoenzyme found in bacteria and completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in E. coli. Steady state kinetic studies of the enzyme indicated that FMNH2 (KM= 0.3 μM) bound before isopentenyl diphosphate (KM= 40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 Å resolution was obtained for the holo-enzyme, in the closed conformation with reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against closely sequence and structure related pathogens such as E. faecalis or S. aureus.
PMCID: PMC4215930  PMID: 24910111
X-ray structure; steady-state kinetics; bisubstrate; flavoprotein
4.  Crystal structure of type 2 Isopentenyl Diphosphate Isomerase from Thermus thermophilus in complex with inorganic pyrophosphate† 
Biochemistry  2008;47(35):9051-9053.
The N-terminal region is stabilized in the crystal structure of T. thermophilus type 2 IPP isomerase in complex with inorganic pyrophosphate; providing new insights about the active site and the catalytic mechanism of the enzyme. The PPi moiety is located near the conserved residues, H10, R97, H152, Q157, E158 and W219, and the flavin cofactor. The putative active site of IDI-2 provides interactions for stabilizing a carbocationic intermediate similar to those that stabilize the intermediate in the well-established protonation/deprotonation mechanism of IDI-1.
Graphical abstract
PMCID: PMC4459642  PMID: 18693754
5.  Sandwich Antibody Arrays Using Recombinant Antibody-Binding Protein L 
Langmuir  2014;30(22):6629-6635.
Antibody arrays are a useful for detecting antigens and other antibodies. This technique typically requires a uniform and well-defined orientation of antibodies attached to a surface for optimal performance. A uniform orientation can be achieved by modification of antibodies to include a single site for attachment. Thus, uniformly oriented antibody arrays require a bioengineered modification for the antibodies directly immobilization on the solid surface. In this study, we describe a “sandwich-type” antibody array where unmodified antibodies are oriented through binding with regioselectively immobilized recombinant antibody-binding protein L. Recombinant proL-CVIA bearing C-terminal CVIA motif is post-translationally modified with an alkyne group by protein farnesyltransferase (PFTase) at the cysteine residue in the CVIA sequence to give proL-CVIApf, which is covalently attached to an azido-modified glass slide by a Huisgen [3 + 2] cycloaddition reaction. Slides bearing antibodies bound to slides coated with regioselectively immobilized proL-CVIApf gave stronger fluorescence outputs and those where the antibody-binding protein was immobilized in random orientations on an epoxy-modified slide. Properly selected capture and detection antibodies did not cross-react with immobilized proL-CVIApf in sandwich arrays, and the proL-CVIApf slides can be used for multiple cycles of detected over a period of several months.
PMCID: PMC4059220  PMID: 24841983
6.  δ-Deuterium Isotope Effects as Probes for Transition-State Structures of Isoprenoid Substrates 
The Journal of Organic Chemistry  2014;79(8):3572-3580.
The biosynthetic pathways to isoprenoid compounds involve transfer of the prenyl moiety in allylic diphosphates to electron-rich (nucleophilic) acceptors. The acceptors can be many types of nucleophiles, while the allylic diphosphates only differ in the number of isoprene units and stereochemistry of the double bonds in the hydrocarbon moieties. Because of the wide range of nucleophilicities of naturally occurring acceptors, the mechanism for prenyltransfer reactions may be dissociative or associative with early to late transition states. We have measured δ-secondary kinetic isotope effects operating through four bonds for substitution reactions with dimethylallyl derivatives bearing deuterated methyl groups at the distal (C3) carbon atom in the double bond under dissociative and associative conditions. Computational studies with density functional theory indicate that the magnitudes of the isotope effects correlate with the extent of bond formation between the allylic moiety and the electron-rich acceptor in the transition state for alkylation and provide insights into the structures of the transition states for associative and dissociative alkylation reactions.
PMCID: PMC4004232  PMID: 24665882
7.  Tetartohedral twinning in IDI-2 from Thermus thermophilus: crystallization under anaerobic conditions 
Crystals of T. thermophilus IDI-2 obtained under anaerobic conditions (<5 p.p.m. O2) will be useful in understanding the active-site conformation of the reduced flavin cofactor.
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.
PMCID: PMC3944699  PMID: 24598924
isopentenyl diphosphate isomerase; IDI-2; flavoprotein; anaerobic; isoprenoid; twinning
8.  Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces 
Bioconjugate Chemistry  2014;25(2):269-275.
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.
PMCID: PMC3983139  PMID: 24437976
9.  The Tyrosine O-Prenyltransferase SirD Catalyzes S-, C-, and N-Prenylations on Tyrosine and Tryptophan Derivatives 
ACS chemical biology  2013;8(12):10.1021/cb400691z.
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.
PMCID: PMC3870192  PMID: 24083562
10.  Predicting the Functions and Specificity of Triterpenoid Synthases: A Mechanism-Based Multi-intermediate Docking Approach 
PLoS Computational Biology  2014;10(10):e1003874.
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.
Author Summary
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.
PMCID: PMC4191879  PMID: 25299649
11.  Dependence of the product chain-length on detergents for long-chain E-polyprenyl diphosphate synthases 
Biochemistry  2013;52(29):10.1021/bi400681d.
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.
PMCID: PMC3855619  PMID: 23802587
12.  Regioselective Covalent Immobilization of Recombinant Antibody Binding Proteins A, G, and Protein L for Construction of Antibody Arrays 
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.
PMCID: PMC3716362  PMID: 23746333
13.  Tetartohedral twinning in IDI-2 from Thermus thermophilus: crystallization under anaerobic conditions 
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.
PMCID: PMC3944699  PMID: 24598924
isopentenyl diphosphate isomerase; IDI-2; flavoprotein; anaerobic; isoprenoid; twinning
14.  Regioselective Covalent Immobilization of Catalytically Active Glutathione S-Transferase on Glass Slides 
Bioconjugate chemistry  2013;24(4):571-577.
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).
PMCID: PMC3644561  PMID: 23458569
15.  Multi-Site Prenylation of 4-Substituted Tryptophans by Dimethylallyltryptophan Synthase 
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.
PMCID: PMC3593668  PMID: 23301871
16.  Inhibition of IspH, a [4Fe-4S]2+ enzyme involved in the biosynthesis of isoprenoids via the MEP pathway 
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.
PMCID: PMC3644560  PMID: 23316732
17.  Novel route to chaetomellic acid A and analogues: serendipitous discovery of a more competent FTase inhibitor 
Bioorganic & medicinal chemistry  2012;21(1):348-358.
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.
PMCID: PMC3761967  PMID: 23182215
Chaetomellic acid A; Farnesyl pyrophosphate; FTase inhibitors; Modelling studies; Radical cyclization
18.  Mutagenesis of Isopentenyl Phosphate Kinase to Enhance Geranyl Phosphate Kinase Activity 
ACS chemical biology  2012;7(7):10.1021/cb300106e.
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.
PMCID: PMC3856688  PMID: 22533411
19.  Inhibition Studies on Enzymes Involved in Isoprenoid Biosynthesis: Focus on Two Potential Drug Targets: DXR and IDI-2 Enzymes 
Current enzyme inhibition  2011;7(2):10.2174/157340811796575317.
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.
PMCID: PMC3856697  PMID: 24339799
DXR; IDI; isomerase; isopentenyl; isoprenoid; MEP; mevalonate; MVA; reductoisomerase
20.  Characterization of Thermophilic Archaeal Isopentenyl Phosphate Kinases 
Biochemistry  2010;49(1):10.1021/bi9017957.
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.
PMCID: PMC3856865  PMID: 19928876
21.  Enantioselective Inhibition of Squalene Synthase by Aziridine Analogues of Presqualene Diphosphate 
The Journal of organic chemistry  2010;75(14):4769-4777.
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.
PMCID: PMC3781220  PMID: 20545375
23.  The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for Archaeal isopentenyl phosphate kinase 
Biochemistry  2012;51(4):917-925.
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.
PMCID: PMC3273622  PMID: 22148590
24.  Type-2 Isopentenyl Diphosphate Isomerase. Evidence for a Stepwise Mechanism 
Journal of the American Chemical Society  2011;133(47):19017-19019.
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.
PMCID: PMC3237117  PMID: 22047048
25.  The Enzyme Function Initiative† 
Biochemistry  2011;50(46):9950-9962.
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, 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.
PMCID: PMC3238057  PMID: 21999478

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