Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates.

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.

A Nanoporous Supramolecular Metal-Organic Framework Based on a Nucleotide: Interplay of the π···π Interactions Directing Assembly and Geometric Matching of Aromatic Tails.

Self-assembly is the most powerful force for creating ordered supramolecular architectures from simple components under mild conditions. π···π stacking interactions have been widely explored in modern supramolecular chemistry as an attractive reversible noncovalent tool for the nondestructive fabrication of materials for different applications. Here, we report on the self-assembly of cytidine 5'-monophosphate (CMP) nucleotide and copper metal ions for the preparation of a rare nanoporous supramolecular metal-organic framework in water. π···π stacking interactions involving the aromatic groups of the ancillary 2,2'-bipyridine (bipy) ligands drive the self-assemblies of hexameric pseudo-amphiphilic [Cu6(bipy)6(CMP)2(µ-O)Br4]2+ units. Owing to the supramolecular geometric matching between the aromatic tails, a nanoporous crystalline phase with hydrophobic and hydrophilic chiral pores of 1.2 and 0.8 nanometers, respectively, was successfully synthesized. The encoded chiral information, contained on the enantiopure building blocks, is transferred to the final supramolecular structure, assembled in the very unusual topology 8T6. These kinds of materials, owing to chiral channels with chiral active sites from ribose moieties, where the enantioselective recognition can occur, are, in principle, good candidates to carry out efficient separation of enantiomers, better than traditional inorganic and organic porous materials.

Unliganded and CMP-Neu5Ac bound structures of human α-2,6-sialyltransferase ST6Gal I at high resolution.

Sialic acid residues found as terminal monosaccharides in various types of glycan chains in cell surface glycoproteins and glycolipids have been identified as important contributors of cell-cell interactions in normal vs. abnormal cellular behavior and are pivotal in diseases such as cancers. In vertebrates, sialic acids are attached to glycan chains by a conserved subset of sialyltransferases with different enzymatic and substrate specificities. ST6Gal I is a sialyltransferase using activated CMP-sialic acids as donor substrates to catalyze the formation of a α2,6-glycosidic bond between the sialic acid residue and the acceptor disaccharide LacNAc. Understanding sialyltransferases at the molecular and structural level shed light into their function. We present here two human ST6Gal I structures, which show for the first time the enzyme in the unliganded state and with the full donor substrate CMP-Neu5Ac bound. Comparison of these structures reveal flexibility of the catalytic loop, since in the unliganded structure Tyr354 adopts a conformation seen also as an alternate conformation in the substrate bound structure. CMP-Neu5Ac is bound with the side chain at C5 of the sugar residue directed outwards at the surface of the protein. Furthermore, the exact binding mode of the sialic acid moiety of the substrate directly involves sialylmotifs L, S and III and positions the sialylmotif VS in the immediate vicinity. We also present a model for the ternary complex of ST6Gal I with both the donor and the acceptor substrates.

Expression system for structural and functional studies of human glycosylation enzymes.

Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

Synthesis and biological evaluation of aryl phosphoramidate prodrugs of fosfoxacin and its derivatives.

Aryl phosphoramidate prodrugs of fosfoxacin derivatives 15a-b and 8a-b were synthesized and investigated for their ability to target bacteria. No growth inhibition was observed neither for Mycobacterium smegmatis nor for Escherichia coli on solid medium, demonstrating the absence of release of the active compounds in the bacterial cells. Investigation of the stability of the prodrugs and their multienzymatic cleavage in abiotic and biotic conditions showed that the use of aryl phosphoramidate prodrug approach to deliver non-nucleotides compounds is not obvious and might not be appropriate for an antimicrobial drug.

C-Methylation Catalyzed by Fom3, a Cobalamin-Dependent Radical S-adenosyl-l-methionine Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration.

Fom3, a cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In this study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be ( S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is ( S)-selective. In addition, Fom3 reaction was performed with ( S)-[2-2H1]HEP-CMP and ( R)-[2-2H1]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. Liquid chromatography-electrospray ionization mass spectrometry analysis of the 5'-deoxyadenosine produced showed that the 2H atom of ( R)-[2-2H1]HEP-CMP was incorporated into 5'-deoxyadenosine but that from ( S)-[2-2H1]HEP-CMP was not. Retention of the 2H atom of ( S)-[2-2H1]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at the C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located on the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.

Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3.

Fom3, the antepenultimate enzyme in the fosfomycin biosynthetic pathway in Streptomyces spp., is a class B cobalamin-dependent radical SAM methyltransferase that catalyzes methylation of (5'-cytidylyl)-2-hydroxyethylphosphonate (2-HEP-CMP) to form (5'-cytidylyl)-2-hydroxypropylphosphonate (2-HPP-CMP). Previously, the reaction of Fom3 with 2-HEP-CMP produced 2-HPP-CMP with mixed stereochemistry at C2. Mechanistic characterization has been challenging because of insoluble expression and poor cobalamin (B12) incorporation in Escherichia coli. Recently, soluble E. coli expression and incorporation of cobalamin into Fom3 were achieved by overexpression of the BtuCEDFB cobalamin uptake system. Herein, we use this new method to obtain Fom3 from Streptomyces wedmorensis. We show that the initiator 5'-deoxyadenosyl radical stereospecifically abstracts the pro- R hydrogen atom from the C2 position of 2-HEP-CMP and use the downstream enzymes FomD and Fom4 to demonstrate that our preparation of Fom3 produces only (2 S)-2-HPP-CMP. Additionally, we show that the added methyl group originates from SAM under multiple-turnover conditions, but the first turnover uses a methyl donor already present on the enzyme; furthermore, cobalamin isolated from Fom3 reaction mixtures contains methyl groups derived from SAM. These results are consistent with a model in which Fom3 catalyzes methyl transfer from SAM to cobalamin and the resulting methylcobalamin (MeCbl) is the ultimate methyl source for the reaction.

Synthesis of a Nonhydrolyzable Nucleotide Phosphoroimidazolide Analogue That Catalyzes Nonenzymatic RNA Primer Extension.

We report the synthesis of guanosine 5'-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5'-phosphoro-2-methylimidazolide (2-MeImpN) analogues designed for mechanistic studies of nonenzymatic RNA primer extension. The addition of a 2-MeImpN monomer to a primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogues do not show catalytic effects under standard mildly basic primer extension conditions. Surprisingly, ICG, which has a pKa similar to that of 2-MeImpG, is a modest catalyst of nonenzymatic primer extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5'-5'-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen-phosphorus and a stable carbon-phosphorus linkage flanking the central imidazole bridge. Cognate RNA primer-template complexes react with this GC-dinucleotide by attack of the primer 3'-hydroxyl on the activated N-P side of the 5'-5'-imidazole bridge. These observations support the hypothesis that 5'-5'-imidazole-bridged dinucleotides can bind to cognate RNA primer-template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5'-5'-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven primer extension.

A Kinetic Model of Nonenzymatic RNA Polymerization by Cytidine-5'-phosphoro-2-aminoimidazolide.

The nonenzymatic polymerization of RNA may have enabled copying of functional sequences during the origin of life. Recent progress utilizing 5'-phosphoro-2-aminoimidazole activation has reinvigorated the possibility of using nonenzymatic RNA polymerization for copying arbitrary sequences. However, the reasons why 2-aminoimidazole (AI) is a superior activation group remain unclear. Here we report that the predominant mechanism of polymerization using cytidine-5'-phosphoro-2-aminoimidazolide (Cp*) involves a 2-aminoimidazolium-bridged dinucleotide (Cp*pC) intermediate. To explore the role of this intermediate, we first identify and quantify four reactions involving the synthesis and breakdown of Cp*pC that occur in the absence of the primer-template duplex. We then analyze the dependence of the rate of polymerization on the concentration of the Cp*pC intermediate in the presence and absence of the competitive inhibitor Cp. We also show that the contribution of the monomer Cp* to the polymerization rate is negligible under our primer extension conditions. Finally, we use the experimentally determined rate constants of these reactions to develop a kinetic model that helps explain the changing rate of nonenzymatic RNA polymerization over time. Our model accounts for the concentration of Cp*pC formed by Cp* under primer extension conditions. The model does not completely account for the decline in polymerization rate observed over long times, which indicates that additional important inhibitory processes have not yet been identified. Our results suggest that the superiority of 2-aminoimidazole over the traditional 2-methylimidazole activation is mostly due to the higher level of accumulation of the imidazolium-bridged intermediate under primer extension conditions.

Active-Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac.

Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m-1 for the H85N variant, compared to 17 000 m-1 for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction.

Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Probing the CMP-Sialic Acid Donor Specificity of Two Human ß-d-Galactoside Sialyltransferases (ST3Gal†I and ST6Gal†I) Selectively Acting on O- and N-Glycosylproteins.

Sialylation of glycoproteins and glycolipids is catalyzed by sialyltransferases in the Golgi of mammalian cells, whereby sialic acid residues are added at the nonreducing ends of oligosaccharides. Because sialylated glycans play critical roles in a number of human physio-pathological processes, the past two decades have witnessed the development of modified sialic acid derivatives for a better understanding of sialic acid biology and for the development of new therapeutic targets. However, nothing is known about how individual mammalian sialyltransferases tolerate and behave towards these unnatural CMP-sialic acid donors. In this study, we devised several approaches to investigate the donor specificity of the human ß-d-galactoside sialyltransferases ST6Gal I and ST3Gal I by using two CMP-sialic acids: CMP-Neu5Ac, and CMP-Neu5N-(4pentynoyl)neuraminic acid (CMP-SiaNAl), an unnatural CMP-sialic acid donor with an extended and functionalized N-acyl moiety.

Cytosine-Cytosine Base-Pair Mismatch and Chirality in Nucleotide Supramolecular Coordination Complexes.

The base-pair sequences are the foundation for the biological processes of DNA or RNA, and base-pair mismatch is very important to reveal genetic diseases and DNA rearrangements. However, the lack of well-defined structural information about base-pair mismatch is obstructing the investigation of this issue. The challenge is to crystallize the materials containing the base-pair mismatch. Engineering the small-molecule mimics or model is an effective strategy to solve this issue. Here, six cytidine-5'-monophosphate (CMP) and 2'-deoxycytidine-5'-monophosphate (dCMP) coordination polymers were reported containing cytosine-cytosine base-pair mismatch (i-motif), and their single-crystal structures and chiralities were studied. The precise control over the formation of the i-motif was demonstrated, in which the regulating of supramolecular interactions was achieved based on molecular design. In addition, the chiralities of these coordination polymers were investigated according to their crystal structures and solution- and solid-state circular dichroism spectroscopy.

Improved workflow for the efficient preparation of ready to use CMP-activated sialic acids.

Natural and synthetically modified cytidine monophosphate activated sialic acids (CMP-Sias) are essential research assets in the field of glycobiology: among other applications, they can be used to probe glycans, detect sialylation defects at the cell surface or carry out detailed studies of sialyltransferase activities. However, these chemical tools are notoriously unstable because of hydrolytic decomposition, and are very time-consuming and costly to obtain. They are nigh impossible to store with satisfactory purity, and their preparation requires multiple laborious purification steps that usually lead to heavy product loss. Using in situ time-resolved 31P phosphorus nuclear magnetic resonance (31P NMR), we precisely established the kinetics of formation and degradation of a number of CMP-Sias including CMP-Neu5Ac, CMP-Neu5Gc, CMP-SiaNAl and CMP-SiaNAz in several experimental conditions. 31P NMR can be carried out in undeuterated solvents and is a sensitive and nondestructive technique that allows for direct in situ monitoring and optimization of chemo-enzymatic syntheses that involve phosphorus-containing species. Thus, we showed that CMP-sialic acid derivatives can be robustly obtained in high yields using the readily available Neisseria meningitidis CMP-sialic acid synthase. This integrated workflow takes less than an hour, and the freshly prepared CMP-Sias can be directly transferred to sialylation biological assays without any purification step.

Conformational and electrostatic analysis of SN1 donor analogue glycomimetic inhibitors of ST3Gal-I mammalian sialyltransferase.

Mammalian sialyltransferases play a role in the metastasis of various cancers in humans. Inhibitors of these enzymes will in principle be able to directly inhibit aberrant sialylation in cancer. Inhibitors of ST3Gal-I resembling the donor component of SN1 Transition State structures were previously evaluated as part of a kinetics study. Here, using classical dynamics simulations and free energy perturbation calculations, we rationalize the performance of three of these donor analogue ST3Gal-I enzyme inhibitors. We find to inhibit the mammalian ST3Gal-I enzyme a donor analogue requires configurationally limited functionality. This is mediated by the binding of the inhibitor to the enzyme. The inhibitor's ability to interact with Y194 and T272 through a charged group such as a carboxylate is especially important. Furthermore, a conformational rigid form approximating the donor substrate is central. Here this is achieved by an intramolecular hydrogen bond formed between the carboxylate group and one of the ribose hydroxyl groups of the cytidine monophosphate (CMP) leaving group. This intramolecular interaction results in the donor substrate conformer complimenting the form of the catalytic binding site. Finally the carboxylate charge is essential for electrostatic pairing with the binding site. Substituting this group for an alcohol or amide results in severe weakening of the ligand binding. The carboxylate thus proves an to be an irreplaceable functional group and an essential pharmacophore.

Structure of the N-glycosidase MilB in complex with hydroxymethyl CMP reveals its Arg23 specifically recognizes the substrate and controls its entry.

5-Hydroxymethylcytosine (5hmC) is present in T-even phage and mammalian DNA as well as some nucleoside antibiotics, including mildiomycin and bacimethrin, during whose synthesis 5hmC is produced by the hydrolysis of 5-hydroxymethyl cytidine 5'-monophosphate (hmCMP) by an N-glycosidase MilB. Recently, the MilB-CMP complex structure revealed its substrate specificity for CMP over dCMP. However, hmCMP instead of CMP is the preferred substrate for MilB as supported by that its KM for CMP is ∼27-fold higher than that for hmCMP. Here, we determined the crystal structures of MilB and its catalytically inactive E103A mutant in complex with hmCMP. In the structure of the complex, Phe22 and Arg23 are positioned in a cage-like active site resembling the binding pocket for the flipped 5-methylcytosine (5mC) in eukaryotic 5mC-binding proteins. Van der Waals interaction between the benzene ring of Phe22 and the pyrimidine ring of hmCMP stabilizes its binding. Remarkably, upon hmCMP binding, the guanidinium group of Arg23 was bent ∼65° toward hmCMP to recognize its 5-hydroxymethyl group, inducing semi-closure of the cage-like pocket. Mutagenesis studies of Arg23 and bioinformatics analysis demonstrate that the positively charged Arg/Lys at this site is critical for the specific recognition of the 5-hydroxymethyl group of hmCMP.

NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase.

Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase-primer-template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3'-terminal CMP to generate the dinucleoside tetraphosphate products Ap(4)C, Up(4)C, and Cp(4)C, respectively. Pre-steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3'deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

Kinetico-Mechanistic Studies of Nucleoside and Nucleotide Substitution Reactions of Co(III) Complexes of Fully Alkylated Cyclen.

The solution chemistry of complex [Co{(Me)2(µ-ET)cyclen}(H2O)2](3+) containing a fully substituted tetraammine ligand designed for the avoidance of base-conjugated substitution mechanisms in the 6-8 pH range has been studied. The study should shed some light on the possible involvement of such Co(III) skeleton in inert interactions with biomolecules. The reactivity and speciation of the complex has been found similar to that of the parent cyclen derivative with the presence of mono- and bis-hydroxo-bridged species; at pH < 7.1, all reactivity has been found to be related to the aqua/hydroxo monomeric complexes. Under these pH conditions, the substitution reactions of the aqua/hydroxo ligands by chloride, inorganic phosphate, thymidine, cytidine 5'-monophosphate (5'-CMP), and thymidine-5'-monophosphate (5'-TMP) have been studied at varying conditions; ionic strength has been kept at 1.0 NaClO4 due to the high concentration of 2-(N-morpholino)ethanesulfonic acid (MES) or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) used to ensure buffering. Except for chloride, the process occurs neatly in a one or two step process, showing dissociatively activated substitution mechanisms, having in general large ΔH(⧧), positive ΔS(⧧), and values of ΔV(⧧) close to those corresponding to the liberation of an aqua ligand to the reaction medium. The actuation of noticeable encounter-complex formation equilibrium constants has been found to be the determinant for the reactions with nucleosides and nucleotides, a clear indication of the relevance of hydrogen-bonding interactions in the reactivity of these molecules, even in this highly ionic strength medium. For the substitution of the active aqua/hydroxo ligands with 5'-TMP, the first substitution reaction produces an Nthymine-bound 5'-TMP complex that evolves to a bis-5'-TMP with an Nthymine,Ophosphate-bonding structure. The formation of outer-sphere complexes between the dangling phosphate group of the Nthymine-bound 5'-TMP and the thymine moiety of another entering 5'-TMP has been found to be responsible for this fact, which leaves only the phosphate group for coordination available.

The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization.

We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.