Isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The reaction involves protonation and deprotonation of the isoprenoid unit and proceeds through a carbocationic transition state. Analysis of the crystal structures (2 Å) of complexes of Escherichia coliIPP⅐DMAPPs isomerase with a transition state analogue (N,N-dimethyl-2-amino-1-ethyl diphosphate) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphosphate) indicates that Glu-116, Tyr-104, and Cys-67 are involved in the antarafacial addition/ elimination of protons during isomerization. This work provides a new perspective about the mechanism of the reaction. Isopentenyl diphosphate (IPP)1 :dimethylallyl diphosphate (DMAPP) isomerase (EC 5.3.3.2) catalyzes the rearrangement of IPP (Scheme 1, 1) to its electrophilic allylic isomer DMAPP (2). This is a mandatory activation step of the isoprenoid unit for subsequent prenyl transfer reactions in isoprenoid biosynthesis. Isomerization of the carbon-carbon double bond in IPP involves an antarafacial (1, 3) transposition of hydrogen by a proton addition-elimination mechanism (1, 2). During the reaction, a proton is added to the re-face of the double bond in IPP, and the re-proton at C2 is removed (3).In eukaryotes, IPP is synthesized by the mevalonate pathway and is the exclusive product from phosphorylation and decarboxylation of mevalonic acid. IPP isomerase activity is an essential enzyme for these organisms. In many bacteria and in plant chloroplasts, IPP and DMAPP are both synthesized from 4-hydroxydimethylallyl diphosphate by the methylerythritol phosphate pathway (4). Although IPP isomerase is often found in these bacteria, it is not essential. Multiple sequence alignments indicate that Escherichia coli IPP isomerase is related to the eukaryotic enzymes (5). In addition, a combination of gas chromatography/mass spectrometry and NMR studies with labeled IPP showed that the stereochemistry for the reversible introduction and removal of a proton at C2 during interconversion of IPP and DMAPP is the same for E. coli IPP isomerase and the eukaryotic enzymes (6).Several lines of evidence support the protonation/deprotonation mechanism shown in Scheme 1. These include proton exchange measurements (7), decreased reactivity for a fluorinated analogue of DMAPP, potent noncovalent inhibition by ammonium analogues of the putative carbocationic intermediate (8, 9), and irreversible inhibition by mechanism-based inhibitors containing epoxide moieties (9 -11). Based on the antarafacial stereochemistry for protonation/deprotonation, it has been proposed that IPP isomerase has two active-site bases located on opposite faces of the allylic moiety defined by C2-C3-C4 in IPP. One of these bases is in the conjugate acid form and protonates the double bond. The other assists with the subsequent elimination of a proton (9). Reardon and Abeles (8) presented evidence for involvement of a thiol group during catalysis. The thiol group a...

We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-L-methionine-dependent formation of N 7 -methylguanosine at position 46 (m 7 G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m 7 G46) methyltransferase activity.About 30 different modified nucleosides have been identified in Escherichia coli tRNA. Methylation is one of the most common modifications, and several mutants affected in tRNA methylation have been obtained (5). However, only a few E. coli tRNA methyltransferase (MTase) genes have been cloned and characterized: trmA, trmD, and trmH are involved in the formations of m 5 U54, m 1 G37, and Gm18, respectively (8, 15, 16). Several tRNA MTases have been purified, and the corresponding genes have been mapped on the E. coli chromosome (5, 13), but it has not been convincingly shown which open reading frame (ORF) encodes a given enzyme. On the other hand, evolutionary relationships among various RNA MTase families have been studied and predictions of novel specificities for uncharacterized ORFs have been made (3). Nevertheless, there are still missing links between many known enzymatic activities and predicted RNA MTase genes.As part of a large-scale project aimed at the identification and classification of novel RNA MTases among the uncharacterized or putative proteins in sequence databases, we analyzed the product of the E. coli yggH ORF. This protein exhibits similarity to S-adenosyl-L-methionine (AdoMet)-dependent MTases in the predicted cofactor-binding region but shares no specific amino acid signatures with other families of RNA MTases in the predicted catalytic region, suggesting that it may encode an RNA MTase with a novel specificity. Thus, we selected it for experimental characterization.Amplification and cloning of the yggH ORF. The yggH ORF was PCR amplified from E. coli genomic DNA (strain XL1-Blue) by using Pfu DNA polymerase (Promega). The primers (Table 1) were designed to amplify the yggH ORF with its ribosome binding site. Primers LDB1 and LDB3 were used for the production of a recombinant YggH protein bearing a Cterminal His tag (YggHH6). Primers LDB1 and LDB2 were used for the production of the untagged YggH.The PCR products were cloned into the pCR-BluntII-

Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N1-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N1 atom of a purine residue is discussed.

The gene encoding the bifunctional enzyme MnmC that catalyzes the two last steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U) in tRNA has been previously mapped at about 50 min on the Escherichia coli K12 chromosome, but to date the identity of the corresponding enzyme has not been correlated with any of the known open reading frames (ORFs). Using the protein fold-recognition approach, we predicted that the 74-kDa product of the yfcK ORF located at 52.6 min and annotated as "putative peptidase" comprises a methyltransferase domain and a FAD-dependent oxidoreductase domain. We have cloned, expressed, and purified the YfcK protein and demonstrated that it catalyzes the formation of mnm 5 s 2 U in tRNA. Thus, we suggest to rename YfcK as MnmC.

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon±carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn 2+ or Mg 2+ ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon±carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole± charge interaction.