Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency.

Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child's cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child's cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.

Asparagine synthetase: Function, structure, and role in disease.

Asparagine synthetase (ASNS) converts aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. ASNS is present in most, if not all, mammalian organs, but varies widely in basal expression. Human ASNS activity is highly responsive to cellular stress, primarily by increased transcription from a single gene located on chromosome 7. Elevated ASNS protein expression is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia. There is evidence that ASNS expression levels may also be inversely correlated with asparaginase efficacy in certain solid tumors as well. Children with mutations in the ASNS gene exhibit developmental delays, intellectual disability, microcephaly, intractable seizures, and progressive brain atrophy. Thus far, 15 unique mutations in the ASNS gene have been clinically associated with asparagine synthetase deficiency (ASD). Molecular modeling using the Escherichia coli ASNS-B structure has revealed that most of the reported ASD substitutions are located near catalytic sites or within highly conserved regions of the protein. For some ASD patients, fibroblast cell culture studies have eliminated protein and mRNA synthesis or stability as the basis for decreased proliferation.

Identification and functional characterization of a novel bacterial type asparagine synthetase A: a tRNA synthetase paralog from Leishmania donovani.

Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target.

Asparagine synthetase1, but not asparagine synthetase2, is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots.

Asparagine is synthesized from glutamine by the reaction of asparagine synthetase (AS) and is the major nitrogen form in both xylem and phloem sap in rice (Oryza sativa L.). There are two genes encoding AS, OsAS1 and OsAS2, in rice, but the functions of individual AS isoenzymes are largely unknown. Cell type- and NH4(+)-inducible expression of OsAS1 as well as analyses of knockout mutants were carried out in this study to characterize AS1. OsAS1 was mainly expressed in the roots, with in situ hybridization showing that the corresponding mRNA was specifically accumulated in the three cell layers of the root surface (epidermis, exodermis and sclerenchyma) in an NH4(+)-dependent manner. Conversely, OsAS2 mRNA was abundant in leaf blades and sheathes of rice. Although OsAS2 mRNA was detectable in the roots, its content decreased when NH4(+) was supplied. Retrotransposon-mediated knockout mutants lacking AS1 showed slight stimulation of shoot length and slight reduction in root length at the seedling stage. On the other hand, the mutation caused an approximately 80-90% reduction in free asparagine content in both roots and xylem sap. These results suggest that AS1 is responsible for the synthesis of asparagine in rice roots following the supply of NH4(+). Characteristics of the NH4(+)-dependent increase and the root surface cell-specific expression of OsAS1 gene are very similar to our previous results on cytosolic glutamine synthetase1;2 and NADH-glutamate synthase1 in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4(+) in rice roots.

Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine.

Asparagine Synthetase Deficiency is a recently described cause of profound intellectual disability, marked progressive cerebral atrophy and variable seizure disorder. To date there has been limited functional data explaining the underlying pathophysiology. We report a new case with compound heterozygous mutations in the ASNS gene (NM_183356.3:c. [866G>C]; [1010C>T]). Both variants alter evolutionarily conserved amino acids and were predicted to be pathogenic based on in silico protein modelling that suggests disruption of the critical ATP binding site of the ASNS enzyme. In patient fibroblasts, ASNS expression as well as protein and mRNA stability are not affected by these variants. However, there is markedly reduced proliferation of patient fibroblasts when cultured in asparagine-limited growth medium, compared to parental and wild type fibroblasts. Restricting asparagine replicates the physiology within the blood-brain-barrier, with limited transfer of dietary derived asparagine, resulting in reliance of neuronal cells on intracellular asparagine synthesis by the ASNS enzyme. These functional studies offer insight into the underlying pathophysiology of the dramatic progressive cerebral atrophy associated with Asparagine Synthetase Deficiency.

Metabolomic Profiling of Asparagine Deprivation in Asparagine Synthetase Deficiency Patient-Derived Cells.

The natural amino acid asparagine (Asn) is required by cells to sustain function and proliferation. Healthy cells can synthesize Asn through asparagine synthetase (ASNS) activity, whereas specific cancer and genetically diseased cells are forced to obtain asparagine from the extracellular environment. ASNS catalyzes the ATP-dependent synthesis of Asn from aspartate by consuming glutamine as a nitrogen source. Asparagine Synthetase Deficiency (ASNSD) is a disease that results from biallelic mutations in the ASNS gene and presents with congenital microcephaly, intractable seizures, and progressive brain atrophy. ASNSD often leads to premature death. Although clinical and cellular studies have reported that Asn deprivation contributes to the disease symptoms, the global metabolic effects of Asn deprivation on ASNSD-derived cells have not been studied. We analyzed two previously characterized cell culture models, lymphoblastoids and fibroblasts, each carrying unique ASNS mutations from families with ASNSD. Metabolomics analysis demonstrated that Asn deprivation in ASNS-deficient cells led to disruptions across a wide range of metabolites. Moreover, we observed significant decrements in TCA cycle intermediates and anaplerotic substrates in ASNS-deficient cells challenged with Asn deprivation. We have identified pantothenate, phenylalanine, and aspartate as possible biomarkers of Asn deprivation in normal and ASNSD-derived cells. This work implies the possibility of a novel ASNSD diagnostic via targeted biomarker analysis of a blood draw.

Characterization of FdmV as an amide synthetase for fredericamycin A biosynthesis in Streptomyces griseus ATCC 43944.

Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the findings that (i) inactivation of fdmV in vivo afforded the ΔfdmV mutant strain SB4027 that abolished FDM A and FDM E production but accumulated FDM C, a biosynthetic intermediate devoid of the characteristic amide linkage; (ii) FdmV in vitro catalyzes conversion of FDM C to FDM B, a known intermediate for FDM A biosynthesis (apparent K(m) = 162 ± 67 µM and k(cat) = 0.11 ± 0.02 min(-1)); and (iii) FdmV also catalyzes the amidation of FDM M-3, a structural analog of FDM C, to afford amide FDM M-6 in vitro, albeit at significantly reduced efficiency. Preliminary enzymatic studies revealed that, in addition to the common nitrogen sources (L-Gln and free amine) of class II glutamine amidotransferases (to which AS B belongs), FdmV can also utilize L-Asn as a nitrogen donor. The amide bond formation in FDM A biosynthesis is proposed to occur after C-8 hydroxylation but before the carbaspirocycle formation.

A conserved glutamate controls the commitment to acyl-adenylate formation in asparagine synthetase.

Inhibitor docking studies have implicated a conserved glutamate residue (Glu-348) as a general base in the synthetase active site of the enzyme asparagine synthetase B from Escherichia coli (AS-B). We now report steady-state kinetic, isotope transfer, and positional isotope exchange experiments for a series of site-directed AS-B mutants in which Glu-348 is substituted by conservative amino acid replacements. We find that formation of the ß-aspartyl-AMP intermediate, and therefore the eventual production of asparagine, is dependent on the presence of a carboxylate side chain at this position in the synthetase active site. In addition, Glu-348 may also play a role in mediating the conformational changes needed to (i) coordinate, albeit weakly, the glutaminase and synthetase activities of the enzyme and (ii) establish the structural integrity of the intramolecular tunnel along which ammonia is translocated. The importance of Glu-348 in mediating acyl-adenylate formation contrasts with the functional role of the cognate residues in ß-lactam synthetase (BLS) and carbapenem synthetase (CPS) (Tyr-348 and Tyr-345, respectively), which both likely evolved from asparagine synthetase. Given the similarity of the chemistry catalyzed by AS-B, BLS, and CPS, our work highlights the difficulty of predicting the functional outcome of single site mutations on enzymes that catalyze almost identical chemical transformations.

Genome bias influences amino acid choices: analysis of amino acid substitution and re-compilation of substitution matrices exclusive to an AT-biased genome.

The genomic era has seen a remarkable increase in the number of genomes being sequenced and annotated. Nonetheless, annotation remains a serious challenge for compositionally biased genomes. For the preliminary annotation, popular nucleotide and protein comparison methods such as BLAST are widely employed. These methods make use of matrices to score alignments such as the amino acid substitution matrices. Since a nucleotide bias leads to an overall bias in the amino acid composition of proteins, it is possible that a genome with nucleotide bias may have introduced atypical amino acid substitutions in its proteome. Consequently, standard matrices fail to perform well in sequence analysis of these genomes. To address this issue, we examined the amino acid substitution in the AT-rich genome of Plasmodium falciparum, chosen as a reference and reconstituted a substitution matrix in the genome's context. The matrix was used to generate protein sequence alignments for the parasite proteins that improved across the functional regions. We attribute this to the consistency that may have been achieved amid the target and background frequencies calculated exclusively in our study. This study has important implications on annotation of proteins that are of experimental interest but give poor sequence alignments with standard conventional matrices.

A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase.

Beta-lactam-synthesizing enzymes carbapenam synthetase (CPS) and beta-lactam synthetase (beta-LS) are evolutionarily linked to a common ancestor, asparagine synthetase B (AS-B). These three relatives catalyze substrate acyl-adenylation and nucleophilic acyl substitution by either an external (AS-B) or internal (CPS, beta-LS) nitrogen source. Unlike AS-B, crystal structures of CPS and beta-LS revealed a putative Tyr-Glu dyad (CPS, Y345/E380; beta-LS, Y348/E382) proposed to deprotonate the respective internal nucleophile. CPS and beta-LS site-directed mutagenesis (Y345/8A, Y345/8F, E380/2D, E380/2Q, E380A) resulted in the reduction of their catalytic efficiency, with Y345A, E380A, and E382Q producing undetectable amounts of beta-lactam product. However, [(32)P]PP(i)-ATP exchange assays demonstrated Y345A and E380A undergo the first half-reaction, with the remaining active mutants showing decreased forward commitment to beta-lactam cyclization. pH-rate profiles of CPS and beta-LS supported the importance of a Tyr-Glu dyad in beta-lactam formation and suggested its reverse protonation in beta-LS. The kinetics of CPS double-site mutants reinforced the synergism of Tyr-Glu in catalysis. Furthermore, significant solvent isotope effects on k(cat) ((D)k(cat)) for Y345F (1.9) and Y348F (1.7) maintained the assignment of Y345/8 in proton transfer. A proton inventory on Y348F determined its (D)(k(cat)/K(m)) = 0.2 to arise from multiple reactant-state fractionation factors, presumably from water molecule(s) replacing the missing Tyr hydroxyl. The role of a CPS and beta-LS Tyr-Glu catalytic dyad was solidified by a significant decrease in mutant k(cat) viscosity dependence with respect to the wild-type enzymes. The evolutionary relation and potential for engineered biosynthesis were demonstrated by beta-LS acting as a carbapenam synthetase.

A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase.

The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability.

PVAS3, a class-II ubiquitous asparagine synthetase from the common bean (Phaseolus vulgaris).

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of -5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.

Functional Research on Three Presumed Asparagine Synthetase Family Members in Poplar.

Asparagine synthetase (AS), a key enzyme in plant nitrogen metabolism, plays an important role in plant nitrogen assimilation and distribution. Asparagine (Asn), the product of asparagine synthetase, is one of the main compounds responsible for organic nitrogen transport and storage in plants. In this study, we performed complementation experiments using an Asn-deficient Escherichia coli strain to demonstrate that three putative asparagine synthetase family members in poplar (Populussimonii× P.nigra) function in Asn synthesis. Quantitative real-time PCR revealed that the three members had high expression levels in different tissues of poplar and were regulated by exogenous nitrogen. PnAS1 and PnAS2 were also affected by diurnal rhythm. Long-term dark treatment resulted in a significant increase in PnAS1 and PnAS3 expression levels. Under long-term light conditions, however, PnAS2 expression decreased significantly in the intermediate region of leaves. Exogenous application of ammonium nitrogen, glutamine, and a glutamine synthetase inhibitor revealed that PnAS3 was more sensitive to exogenous glutamine, while PnAS1 and PnAS2 were more susceptible to exogenous ammonium nitrogen. Our results suggest that the various members of the PnAS gene family have distinct roles in different tissues and are regulated in different ways.

Saccharomyces cerevisiae ASN1 and ASN2 are asparagine synthetase paralogs that have diverged in their ability to polymerize in response to nutrient stress.

Recent work has found that many metabolic enzymes have the ability to polymerize in response to metabolic changes or environmental stress. This ability to polymerize is well conserved for the few metabolic enzyme paralogs that have been studied in yeast. Here we describe the first set of paralogs, Asn1p and Asn2p, that have differential assembly behavior. Asn1p and Asn2p both co-assemble into filaments in response to nutrient limitation. However, the ability of Asn2p to form filaments is strictly dependent on the presence of Asn1p. Using mutations that block enzyme activity but have differential effects on Asn1p polymerization, we have found that Asn1p polymers are unlikely to have acquired a moonlighting function. Together these results provide a novel system for understanding the regulation and evolution of metabolic enzyme polymerization.

In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi.

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

Evidence for sugar signalling in the regulation of asparagine synthetase gene expressed in Phaseolus vulgaris roots and nodules.

A cDNA clone, designated as PvNAS2, encoding asparagine amidotransferase (asparagine synthetase) was isolated from nodule tissue of common bean (Phaseolus vulgaris cv. Negro Jamapa). Southern blot analysis indicated that asparagine synthetase in bean is encoded by a small gene family. Northern analysis of RNAs from various plant organs demonstrated that PvNAS2 is highly expressed in roots, followed by nodules in which it is mainly induced during the early days of nitrogen fixation. Investigations with the PvNAS2 promoter gusA fusion revealed that the expression of PvNAS2 in roots is confined to vascular bundles and meristematic tissues, while in root nodules its expression is solely localized to vascular traces and outer cortical cells encompassing the central nitrogen-fixing zone, but never detected in either infected or non-infected cells located in the central region of the nodule. PvNAS2 is down-regulated when carbon availability is reduced in nodules, and the addition of sugars to the plants, mainly glucose, boosted its induction, leading to the increased asparagine production. In contrast to PvNAS2 expression and the concomitant asparagine synthesis, glucose supplement resulted in the reduction of ureide content in nodules. Studies with glucose analogues as well as hexokinase inhibitors suggested a role for hexokinase in the sugar-sensing mechanism that regulates PvNAS2 expression in roots. In light of the above results, it is proposed that, in bean, low carbon availability in nodules prompts the down-regulation of the asparagine synthetase enzyme and concomitantly asparagine production. Thereby a favourable environment is created for the efficient transfer of the amido group of glutamine for the synthesis of purines, and then ureide generation.

Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity.

A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.

Nucleotide sequence and deduced amino acid sequence of a putative asparagine synthetase in the mosquito Aedes aegypti (L.).

A cDNA was cloned from a Aedes aegypti head cDNA library, containing the complete coding sequence for an asparagine synthetase homolog. The predicted polypeptide sequence exhibits high homology with different proteins of the 'purF' glutamine amidotransferase enzyme family. The aminoterminal region, containing Cys-1 which is crucial to perform the glutaminase reaction, was highly conserved among the asparagine synthetase family. Subsequent expression of the cDNA yielded a 54,000 Da protein corresponding to the molecular weight of other asparagine synthetases.

Molecular cloning and characterisation of two genes encoding asparagine synthetase in barley (Hordeum vulgare L.).

Two different cDNA clones encoding asperagine synthetase (AS: EC 6.3.5.4.) were cloned from barley (Hordeum vulgare L. cv. Alexis). The corresponding genes were designated HvAS1 (GenBank no AF307145) and HvAS2 (GenBank no AY193714). Chromosomal mapping using wheat-barley addition lines revealed that the HvAS1 gene is located on the long arm of barley chromosome 5, while the HvAS2 gene maps to the short arm of chromosome 3. Both genes are expressed in barley leaves according to RT-PCR analysis but only the HvAS1 gene expression can be detected in roots. Northern blots show no expression of HvAS1 in plants grown under a normal 16 h light/8 h dark cycle but after 10 h of continuous darkness, transcript appears and mRNA accumulates over a 48-h period of dark treatment. In roots, low-level expression of HvAS1 could be detected and the expression level appears to be unaffected by light. A polyclonal antibody was raised against the HvAS1 protein and used in Western blot analysis. The AS protein accumulated during a 48-h period of dark treatment, following the increase in HvAS1 transcript.

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis.

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.