tRNA pseudouridine synthase I (⌿SI) catalyzes the conversion of uridine to ⌿ at positions 38, 39, and͞or 40 in the anticodon loop of tRNAs. ⌿SI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNA Phe containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and ⌿SI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for ⌿S.enzyme mechanism ͉ 5-fluorouridine hydrate P seudouridine synthase (⌿S) catalyzes the conversion of uridine residues in RNA to pseudouridine (⌿), an unusual nucleoside with a carbon-carbon glycoside bond (1-3). The minimal mechanism for the reaction involves cleavage of the N-glycosidic bond of the target Urd, movement of the cleaved uracil to juxtapose C5 of the pyrimidine and C1Ј of the ribosyl moiety of RNA, and formation of the C1Ј-C5 carbon-carbon bond.The earliest proposed mechanism for ⌿S was based on chemical considerations and analogies with enzymes such as thymidylate synthase (4). This involved nucleophilic attack of the thiol of a Cys residue of the enzyme at C6 of the pyrimidine to form a 5,6-dihydropyrimidine (as in Scheme 1, R ϭ H, except that Cys replaces the Asp residue). The formation of a 5,6-dihydropyrimidine adduct could facilitate all of the reactions necessary for the subsequent conversion to products. It would (i) enhance the lability of the N-glycosidic bond (5), (ii) provide an axis for a 180°rotation of the pyrimidine ring to juxtapose the C1Ј and C5 positions, and (iii) activate the 5-carbon of the pyrimidine toward electrophilic attack by C1Ј and facilitate subsequent ␤-elimination to give product and unchanged enzyme (6).Evidence for an essential Cys in ⌿S was provided by the observation that enzyme activity was inhibited by sulfhydryl reagents (7). By analogy with thymidylate synthase, m 5 U-tRNA methyl transferase (MTase) and m 5 C-DNA MTase, such covalent adducts may form via initial attachment of a Cys thiol to the 6-position of the target 5-fluorouracil (FUra) (6,8). Further, the involvement of an enzyme nucleophile was indicated by isolation of a covalent adduct between ⌿SI and FUra-tRNA (tRNA Phe containing FUra in place of Ura) (9). However, sequence comparison of putative and proven ⌿Ss did not reveal a conserved Cys residue that could serve as t...

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The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

A family of RNA m 5 C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m 5 C 967 MTase, Fmu, as an initial probe. The RNA m 5 C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m 5 C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m 5 U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m 5 C MTase uses a different Cys as a catalytic nucleophile than the DNA m 5 C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m 5 C MTases remains unknown.nzymes that catalyze 5-methylation (or hydroxymethylation) of pyrimidines use the thiol of a Cys residue to attack the 6 position of the heterocycle to activate the 5 position toward the one-carbon transfer (1, 2). Scheme 1 depicts the mechanism for methylation of cytosine nucleotides. Subsequent to one-carbon transfer, the 5-proton of the 5,6-dihydropyrimidine intermediate IIA is removed, and ␤-elimination provides the product and free enzyme. The covalent 5,6-dihydropyrimidine intermediate IIA has been identified by biochemical studies as well as by structures of stable covalent complexes formed between enzymes and 5-f luoropyrimidine substrate analogs that act as mechanism-based inhibitors (e.g., Scheme 1, IIB). In the latter, the stable carbon-f luorine bond in IIB prevents ␤-elimination of the enzyme and hence provides stability to the complexes.The Escherichia coli fmu gene product Fmu was shown recently to be the 16S rRNA m . Because all of the RNA m 5 C MTases contained the ProCys dipeptide in motif IV and were highly homologous to the well characterized, functionally similar DNA m 5 C MTases, it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys of motif IV (4).In the present work, we show that, contrary to our expectations, the Cys of the ProCys dipeptide in Fmu is not the catalytic nucleophile in RNA m 5 C methylation; rather, the conserved Cys in motif VI serves this function. Materials and MethodsMaterials. Plasmids pWK1 and pWK1.3 used for preparation of E. coli 16S rRNA and the 56-mer corresponding to nucleotides 927-982 of 16S rRNA, respectively, have been reported (3). T7 RNA polymerase was prepared and purified as described (9). [ 3 H-Me]S-Adenosyl-L-met...

The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster,epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.