Structures of Mycobacterium tuberculosis isoprenyl diphosphate synthase Rv2173 in substrate-bound forms

Exceptionally high percentage of IPP synthesis by Ginkgo biloba IspH is mainly due to Phe residue in the active site

Farnesyl diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. In plants the presence of farnesyl diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized farnesyl diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl diphosphate over dimethylallyl diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl diphosphate and lavandulyl diphosphate, when incubated with dimethylallyl diphosphate and an additional product, the regular monoterpene geranyl diphosphate, when incubated with isopentenyl diphosphate and dimethylallyl diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.

Dimethylallyl pyrophosphate:aspulvinone dimethylallyltransferase, the prenylation enzyme for the biosynthesis of aspulvinone pigments, has been purified from mycelia of Aspergillus terreus. The transferase catalyzed the transfer of the dimethylallyl moiety from dimethylallyl pyrophosphate to either of the two aromatic rings of aspulvinone E to give the mono- and diprenylated derivatives which were identified with the metabolites aspulvinone I and aspulvinone H, respectively. Aspulvinone G, another fundamental metabolite of this series, also acted as substrate to afford the corresponding diprenylated derivative, which is assumed to be a precursor for aspulvinone C, D, and F. The molecular weight of the enzyme was estimated to be 240 000--270 000 by gel filtration. Since the subunit molecular weight determined by NaDodSO4-polyacrylamide disc gel electrophoresis was 45 000, the native enzyme appears to be a hexomeric protein composed of identical molecular weight subunits. The apparent Km values for aspulvinone E, aspulvinone G, and dimethylallyl pyrophosphate were 13.7, 7.7, and 40.0 micron, respectively. The enzyme shows the maximum activity at pH 7.0, and no metal ion is necessary for the activation. Sulfhydryl blocking agents or mercaptoethanol has no effect. Bromophenol blue binds specifically to the transferase and strongly inhibits the enzyme activity.

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 cosubstrate 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 K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) 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.

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP). The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate. Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling. FPPase mutants containing seven promising modifications were constructed. Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site. These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants.

Crystal structures of a 6-dimethylallyltryptophan synthase, IptA: Insights into substrate tolerance and enhancement of prenyltransferase activity

A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.

6-Pyruvoyl Tetrahydropterin Synthase, An Enzyme With a Novel Type of Active Site Involving Both Zinc Binding and an Intersubunit Catalytic Triad Motif; Site-directed Mutagenesis of the Proposed Active Center, Characterization of the Metal Binding Site and Modelling of substrate Binding

The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway, producing the essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the polar and cramped (and so not easily druggable) substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM.

Structures of a transition metal transporter spanning the entire Mn2+ transport cycle reveal distinct coordination geometries and dynamic polar networks that enable Mn2+ import, and a Cd2+-bound structure helps explain how Cd2+ behaves differently as a substrate.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. DHOase is divided into two types (I and II). Type II DHOase generally contains a binuclear metal center in its active site. Recently, the crystal structure of DHOase domain in human CAD protein (huDHOase) has revealed three metal ions in the protein's active site. However, whether type II DHOase can have the critical third metal ion, as observed in huDHOase, remains unknown. In the present study, the putative third metal binding site in type II enzymes, such as the prokaryotic Salmonella enterica serovar Typhimurium LT2 DHOase (StDHOase) and the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase), was created and identified. StDHOase T198E and ScDHOase T208E mutants had higher activities compared with their wild-type enzymes. The need for a higher DHOase stability and activity may drive creation of the third metal ion binding site in huDHOase, which can be achieved by mutating a highly conserved position T in type II dihydroorotases to E, similar to that in huDHOase.

Metalloenzymes are ubiquitous in nature catalyzing a number of crucial biochemical processes in animal and plant kingdoms. For better adaptation to the relative abundance of different metal ions in different cellular fluids, many of these enzymes exhibit metal promiscuity. The microbial enzyme DapE, an essential enzyme for bacterial growth and survival and a potentially safe target for antibiotics, continues to show enzyme activity when the two zinc ions in its active site are replaced by other transition metal ions. The effect of metal-ion substitution at the second metal-binding site of DapE on its substrate affinity and catalytic efficiency is investigated by QM/MM treatment of the enzyme-substrate complex, by modelling the enzyme with Mn(II), Co(II), Ni(II), or Cu(II) ion in place of Zn(II) at its second metal-binding site, while retaining Zn(II) ion at the first metal-binding site. On the basis of substrate binding energy and activation energy barrier for the chemical catalysis, it is found that Zn-Mn DapE shows poor binding affinity as well as inefficient chemical catalysis. Although Zn-Cu and Zn-Ni DapEs show activation energy barriers comparable to that of wild-type Zn-Zn DapE, their weaker substrate affinity renders these mixed-metal enzymes less efficient. On the other hand, Zn-Co DapE is found to outperform the naturally occurring Zn-Zn DapE, both in terms of substrate affinity and chemical catalysis. The observed metal promiscuity may have played an important role in the survival of bacteria even in those cellular media where Zn ions are in limited supply. Metal nonspecificity in the catalysis of DapE enzyme allows bacteria to thrive in different cellular media.

Isoprene, a C5 hydrocarbon emitted by various land plants, serves as a protective molecule under heat stress. The moss Calohypnum plumiforme isoprene synthase (ISPS) catalyzes isoprene formation from dimethylallyl diphosphate and contains two aromatic residues (Y393 and F615) at the bottom of the active site that restrict the cavity size to accommodate the smaller C5 substrate. Here, we show that these residues are critical determinants of substrate size selectivity. Substitution of the two residues with alanine (Y393A and F615A) expanded the active site and exhibited diterpene synthase activity, generating ent-pimaradienes from ent-copalyl diphosphate. Further mutagenesis of the catalytic motif yielded ent-kaurene, demonstrating stepwise functional conversion from diterpene synthase to ISPS activity. Screening of ISPS genes across mosses identified two structurally unrelated enzyme classes: typical diterpene synthase-type ISPSs (C. plumiforme, Pohlia nutans) and microbial-type terpene synthase (TPS)-like ISPSs (Polytrichum commune, Leucobryum juniperoideum), the latter representing a previously unrecognized ISPS scaffold. Microbial-type TPS-like ISPSs of P. communehave two aromatic residues (W151 and F288), as in the case of C.plumiformeISPS, to restrict the cavity size. Substitution experiments expanding the active site in ISPS of P. commune (W151A and F288A) determined the production of ocimenes as monoterpene synthase activity. These results define the molecular basis of substrate specificity in TPSs and demonstrate that structurally divergent enzyme families can independently acquire ISPS activity.

IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies of IspH using fluoro-substituted substrate analogues to dissect the contributions of several factors to IspH catalysis, including the coordination of the HMBPP C(4)-OH group to the iron-sulfur cluster, the H-bonding network in the active site, and the electronic properties of the substrates, are reported.

Novel strategy to produce prenylated resveratrol by prenyltransferase iacE and evaluation of neuroprotective mechanisms