N5-CAIR mutase: role of a CO2 binding site and substrate movement in catalysis

N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N5-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO2 binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the carboxylate or CO2 interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.     

Crystal structure of Escherichia coli PurE, an unusual mutase in the purine biosynthetic pathway

BACKGROUND: Conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxyaminoimidazole ribonucleotide (CAIR) in Escherichia coli requires two proteins - PurK and PurE. PurE has recently been shown to be a mutase that catalyzes the unusual rearrangement of N(5)-carboxyaminoimidazole ribonucleotide (N(5)-CAIR), the PurK reaction product, to CAIR. PurEs from higher eukaryotes are homologous to E. coli PurE, but use AIR and CO(2) as substrates to produce CAIR directly. RESULTS: The 1.50 A crystal structure of PurE reveals an octameric structure with 422 symmetry. A central three-layer (alphabetaalpha) sandwich domain and a kinked C-terminal helix form the folded structure of the monomeric unit. The structure reveals a cleft at the interface of two subunits and near the C-terminal helix of a third subunit. Co-crystallization experiments with CAIR confirm this to be the mononucleotide-binding site. The nucleotide is bound predominantly to one subunit, with conserved residues from a second subunit making up one wall of the cleft. CONCLUSIONS: The crystal structure of PurE reveals a unique quaternary structure that confirms the octameric nature of the enzyme. An analysis of the native crystal structure, in conjunction with sequence alignments and studies of co-crystals of PurE with CAIR, reveals the location of the active site. The environment of the active site and the analysis of conserved residues between the two classes of PurEs suggests a model for the differences in their substrate specificities and the relationship between their mechanisms.     

Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway.

Aminoimidazole riobnucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The Km for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (less than 100 microM), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (less than 100 microM) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and Pi. No dependence on the concentration of HCO3- could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established.     

Treponema denticola PurE Is a bacterial AIR carboxylase

De novo purine biosynthesis proceeds by two divergent paths. In bacteria, yeasts, and plants, 5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxy-AIR (CAIR) by two enzymes: N(5)-carboxy-AIR (N(5)-CAIR) synthetase (PurK) and N(5)-CAIR mutase (class I PurE). In animals, the conversion of AIR to CAIR requires a single enzyme, AIR carboxylase (class II PurE). The CAIR carboxylate derives from bicarbonate or CO(2), respectively. Class I PurE is a promising antimicrobial target. Class I and class II PurEs are mechanistically related but bind different substrates. The spirochete dental pathogen Treponema denticola lacks a purK gene and contains a class II purE gene, the hallmarks of CO(2)-dependent CAIR synthesis. We demonstrate that T. denticola PurE (TdPurE) is AIR carboxylase, the first example of a prokaryotic class II PurE. Steady-state and pre-steady-state experiments show that TdPurE binds AIR and CO(2) but not N(5)-CAIR. Crystal structures of TdPurE alone and in complex with AIR show a conformational change in the key active site His40 residue that is not observed for class I PurEs. A contact between the AIR phosphate and a differentially conserved residue (TdPurE Lys41) enforces different AIR conformations in each PurE class. As a consequence, the TdPurE·AIR complex contains a portal that appears to allow the CO(2) substrate to enter the active site. In the human pathogen T. denticola, purine biosynthesis should depend on available CO(2) levels. Because spirochetes lack carbonic anhydrase, the corresponding reduction in bicarbonate demand may confer a selective advantage.     

Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily

Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2.1 and 2.5 A resolution, respectively. PurK belongs to the ATP grasp superfamily of C-N ligase enzymes. Each subunit of PurK is composed of three domains (A, B, and C). The B domain contains a flexible, glycine-rich loop (B loop, T123-G130) that is disordered in the sulfate-PurK structure and becomes ordered in the MgADP-PurK structure. MgADP is wedged between the B and C domains, as with all members of the ATP grasp superfamily. Other enzymes in this superfamily contain a conserved Omega loop proposed to interact with the B loop, define the specificity of their nonnucleotide substrate, and protect the acyl phosphate intermediate formed from this substrate. PurK contains a minimal Omega loop without conserved residues. In the reaction catalyzed by PurK, carboxyphosphate is the putative acyl phosphate intermediate. The sulfate of the sulfate ion-liganded PurK interacts electrostatically with Arg 242 and the backbone amide group of Asn 245, components of the J loop of the C domain. This sulfate may reveal the location of the carboxyphosphate binding site. Conserved residues within the C-terminus of the C domain define a pocket that is proposed to bind AIR in collaboration with an N-terminal strand loop helix motif in the A domain (P loop, G8-L1). The P loop is proposed to bind the phosphate of AIR on the basis of similar binding sites observed in PurN and PurE and proposed in PurD and PurT, four other enzymes in the purine pathway.     

Biochemical and structural studies of N5-carboxyaminoimidazole ribonucleotide mutase from the acidophilic bacterium Acetobacter aceti

N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) mutase (PurE) catalyzes the reversible interconversion of acid-labile compounds N5-CAIR and 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). We have examined PurE from the acidophilic bacterium Acetobacter aceti (AaPurE), focusing on its adaptation to acid pH and the roles of conserved residues His59 and His89. Both AaPurE and Escherichia coli PurE showed quasi-reversible acid-mediated inactivation, but wt AaPurE was much more stable at pH 3.5, with a > or = 20 degrees C higher thermal unfolding temperature at all pHs. His89 is not essential and does not function as part of a proton relay system. The kcat pH-rate profile was consistent with the assignment of pK1 to unproductive protonation of bound nucleotide and pK2 to deprotonation of His59. A 1.85 A resolution crystal structure of the inactive mutant H59N-AaPurE soaked in CAIR showed that protonation of CAIR C4 can occur in the absence of His59. The resulting species, modeled as isoCAIR [4(R)-carboxy-5-iminoimidazoline ribonucleotide], is strongly stabilized by extensive interactions with the enzyme and a water molecule. The carboxylate moiety is positioned in a small pocket proposed to facilitate nucleotide decarboxylation in the forward direction (N5-CAIR --> CAIR) [Meyer, E., Kappock, T. J., Osuji, C., and Stubbe, J. (1999) Biochemistry 38, 3012-3018]. Comparisons with model studies suggest that in the reverse (nonbiosynthetic) direction PurE favors protonation of CAIR C4. We suggest that the essential role of protonated His59 is to lower the barrier to decarboxylation by stabilizing a CO2-azaenolate intermediate.     

Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N⁵-carboxyaminoimidazole ribonucleotide (N⁵-CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small-molecule inhibitors of N⁵-CAIR synthetase have been published. To remedy this problem, we have conducted high-throughput screening (HTS) against Escherichia coli N⁵-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis–Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicates that these compounds do not follow normal Michaelis–Menten kinetics. Instead, these compounds inhibit N⁵-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N⁵-CAIR synthetase.     

Interrogating the mechanism of a tight binding inhibitor of AIR carboxylase

The enzyme aminoimidazole ribonucleotide (AIR) carboxylase catalyzes the synthesis of the purine intermediate, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Previously, we have shown that the compound 4-nitro-5-aminoimidazole ribonucleotide (NAIR) is a slow, tight binding inhibitor of the enzyme with a Ki of 0.34 nM. The structural attributes and the slow, tight binding characteristics of NAIR implicated this compound as a transition state or reactive intermediate analog. However, it is unclear what molecular features of NAIR contribute to the mimetic properties for either of the two proposed mechanisms of AIR carboxylase. In order to gain additional information regarding the mechanism for the potent inhibition of AIR carboxylase by NAIR, a series of heterocyclic analogs were prepared and evaluated. We find that all compounds are weaker inhibitors than NAIR and that CAIR analogs are not alternative substrates for the enzyme. Surprisingly, rather subtle changes in the structure of NAIR can lead to profound changes in binding affinity. Computational investigations of enzyme intermediates and these inhibitors reveal that NAIR displays an electrostatic potential surface similar to a proposed reaction intermediate. The result indicates that AIR carboxylase is likely sensitive to the electrostatic surface of reaction intermediates and thus compounds which mimic these surfaces should possess tight binding characteristics. Given the evolutionary relationship between AIR carboxylase and N⁵-CAIR mutase, we believe that this concept extends to the mutase enzyme as well. The implications of this hypothesis for the design of selective inhibitors of the N⁵-CAIR mutase are discussed.     

Nucleotide complexes of Escherichia coli phosphoribosylaminoimidazole succinocarboxamide synthetase

Phosphoribosylaminoimidazole-succinocarboxamide synthetase (SAICAR synthetase) converts 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) to 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR). The enzyme is a target of natural products that impair cell growth. Reported here are the crystal structures of the ADP and the ADP.CAIR complexes of SAICAR synthetase from Escherichia coli, the latter being the first instance of a CAIR-ligated SAICAR synthetase. ADP and CAIR bind to the active site in association with three Mg(2+), two of which coordinate the same oxygen atom of the 4-carboxyl group of CAIR; whereas, the third coordinates the alpha- and beta-phosphoryl groups of ADP. The ADP.CAIR complex is the basis for a transition state model of a phosphoryl transfer reaction involving CAIR and ATP, but also supports an alternative chemical pathway in which the nucleophilic attack of l-aspartate precedes the phosphoryl transfer reaction. The polypeptide fold for residues 204-221 of the E. coli structure differs significantly from those of the ligand-free SAICAR synthetase from Thermatoga maritima and the adenine nucleotide complexes of the synthetase from Saccharomyces cerevisiae. Conformational differences between the E. coli, T. maritima, and yeast synthetases suggest the possibility of selective inhibition of de novo purine nucleotide biosynthesis in microbial organisms.     

Structural organization of de novo purine biosynthesis enzymes in plants: 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase cDNAs from Vigna aconitifolia

Nodules of tropical legumes generally export symbiotically fixed nitrogen in the form of ureides that are produced by oxidation of de novo synthesized purines. To investigate the regulation of de novo purine biosynthesis in these nodules, we have isolated cDNA clones encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase from a mothbean (Vigna aconitifolia) nodule cDNA library by complementation of Escherichia coli purE and purC mutants, respectively. Sequencing of these clones revealed that the two enzymes are distinct proteins in mothbean, unlike in animals where both activities are associated with a single bifunctional polypeptide. As is the case in yeast, the mothbean AIR carboxylase has a N-terminal domain homologous to the eubacterial purK gene product. This PurK-like domain appears to facilitate the binding of CO₂ and is dispensable in the presence of high CO₂ concentrations. Because the expression of the mothbean PurE cDNA clone in E. coli apparently generates a truncated polypeptide lacking at least 140 N-terminal amino acids, this N-terminal region of the enzyme may not be essential for its CO₂-binding activity.     

Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE

The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24 h, as assessed by the Bratton–Marshall assay (BMA) for diazotizable amines. The ZnSO₄ stopping reagent is proposed to chelate CAIR, enabling delayed analysis of this acid-labile product by BMA or other HTS methods.     

Isolation and properties of phosphoribosyl-aminoimidazole-succinocarboxyamide-synthestase from Saccharomyces cerevisiae yeasts

An isolation procedure for phosphoribosyl succinocarboxamideaminoimidazole synthetase (SAICAR synthetase) (EC 6.3.2.6) has been developed. Pure SAICAR synthetase was found to be a monomeric protein with the apparent molecular weight of 36 kDa. The Michaelis constant for the three substrates of the reaction are 1.6 microM for CAIR, 14 microM for ATP and 960 microM for aspartic acid. The structural analogs of CAIR, 5-aminoimidazole ribotide and 5-aminoimidazole-4-carboxamide ribotide, act as competitive inhibitors of SAICAR synthetase. GTP and 2'-dATP can substitute for ATP in the reaction, while CTP and UTP inhibit the enzyme. No structural analogs of the aspartic acid were found to have affinity for SAICAR synthetase. The optimal reaction conditions for the enzyme were established to be at pH 8.0 and magnesium chloride concentration around 5 mM.     

Mechanism of action of Escherichia coli phosphoribosylaminoimidazolesuccinocarboxamide synthetase

The conversion of ATP, L-aspartate, and 5-aminoimidazole-4-carboxyribonucleotide (CAIR) to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP, and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represents the eighth step of de novo purine nucleotide biosynthesis. SAICAR synthetase and other enzymes of purine biosynthesis are targets of natural products that impair cell growth. Prior to this study, no kinetic mechanism was known for any SAICAR synthetase. Here, a rapid equilibrium random ter-ter kinetic mechanism is established for the synthetase from Escherichia coli by initial velocity kinetics and patterns of linear inhibition by IMP, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and maleate. Substrates exhibit mutual binding antagonism, with the strongest antagonism between CAIR and either ATP or L-aspartate. CAIR binds to the free enzyme up to 200-fold more tightly than to the ternary enzyme-ATP-aspartate complex, but the latter complex may be the dominant form of SAICAR synthetase in vivo. IMP is a competitive inhibitor with respect to CAIR, suggesting the possibility of a hydrogen bond interaction between the 4-carboxyl and 5-amino groups of enzyme-bound CAIR. Of several aspartate analogues tested (hadacidin, l-malate, succinate, fumarate, and maleate), maleate was by far the best inhibitor, competitive with respect to L-aspartate. Inhibition by IMP and maleate is consistent with a chemical mechanism for SAICAR synthetase that parallels that of adenylosuccinate synthetase.     

The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway

Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively. In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC. We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E. coli purC purine auxotroph. This cDNA encodes AIRc yet is unable to complement an E. coli purE mutant, suggesting functional differences between Drosophila and E. coli AIRc. In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis. In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies. Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated. Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated. Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs.     

Plasticity in the purine-thiamine metabolic network of Salmonella

In Salmonella enterica, 5-aminoimidazole ribonucleotide (AIR) is the precursor of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) pyrophosphate moiety of thiamine and the last intermediate in the common HMP/purine biosynthetic pathway. AIR is synthesized de novo via five reactions catalyzed by the purF, -D, -T, -G, and -I gene products. In vivo genetic analysis demonstrated that in the absence of these gene products AIR can be generated if (i) methionine and lysine are in the growth medium, (ii) PurC is functional, and (iii) 5-amino-4-imidazolecarboxamide ribotide (AICAR) has accumulated. This study provides evidence that the five steps of the common HMP/purine biosynthetic pathway can be bypassed in the synthesis of AIR and thus demonstrates that thiamine synthesis can be uncoupled from the early purine biosynthetic pathway in bacteria.     

Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR

Class I PurE (N5-carboxyaminoimidazole mutase) catalyzes a chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. Solution NMR is a peerless tool for this task but has had limited application to enzymes, most of which are larger than its effective molecular size limit. Solid-state NMR is not subject to this limit. REDOR NMR studies of a 151 kDa complex of uniformly 15N-labeled Acetobacter aceti PurE (AaPurE) and the active site ligand [6-13C]citrate probed a single ionization equilibrium associated with the key histidine (AaPurE His59). In the AaPurE complex, the citrate central carboxylate C6 13C peak moves upfield, indicating diminution of negative charge, and broadens, indicating heterogeneity. Histidine 15N chemical shifts indicate His59 exists in approximately equimolar amounts of an Ndelta-unprotonated (pyridine-like) form and an Ndelta-protonated (pyrrole-like) form, each of which is approximately 4 A from citrate C6. The spectroscopic data are consistent with proton transfers involving His59 Ndelta that are invoked in the class I PurE mechanism.     

Investigation of the ATP binding site of Escherichia coli aminoimidazole ribonucleotide synthetase using affinity labeling and site-directed mutagenesis.

Aminoimidazole ribonucleotide (AIR) synthetase (PurM) catalyzes the conversion of formylglycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and P(i), the fifth step in de novo purine biosynthesis. The ATP binding domain of the E. coli enzyme has been investigated using the affinity label [(14)C]-p-fluorosulfonylbenzoyl adenosine (FSBA). This compound results in time-dependent inactivation of the enzyme which is accelerated by the presence of FGAM, and gives a K(i) = 25 microM and a k(inact) = 5.6 x 10(-)(2) min(-)(1). The inactivation is inhibited by ADP and is stoichiometric with respect to AIR synthetase. After trypsin digestion of the labeled enzyme, a single labeled peptide has been isolated, I-X-G-V-V-K, where X is Lys27 modified by FSBA. Site-directed mutants of AIR synthetase were prepared in which this Lys27 was replaced with a Gln, a Leu, and an Arg and the kinetic parameters of the mutant proteins were measured. All three mutants gave k(cat)s similar to the wild-type enzyme and K(m)s for ATP less than that determined for the wild-type enzyme. Efforts to inactivate the chicken liver trifunctional AIR synthetase with FSBA were unsuccessful, despite the presence of a Lys27 equivalent. The role of Lys27 in ATP binding appears to be associated with the methylene linker rather than its epsilon-amino group. The specific labeling of the active site by FSBA has helped to define the active site in the recently determined structure of AIR synthetase [Li, C., Kappock, T. J., Stubbe, J., Weaver, T. M., and Ealick, S. E. (1999) Structure (in press)], and suggests additional flexibility in the ATP binding region.     

Nucleotide sequence analysis of the purEK operon encoding 5''-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12.

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.     

Analysis of ThiC Variants in the Context of the Metabolic Network of Salmonella enterica

In bacteria, the 4-amino-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine is synthesized from 5-aminoimidazole ribotide (AIR), a branch point metabolite of purine and thiamine biosynthesis. ThiC is a member of the radical S-adenosylmethionine (AdoMet) superfamily and catalyzes the complex chemical rearrangement of AIR to HMP-P. As reconstituted in vitro, the ThiC reaction requires AdoMet, AIR, and reductant. This study analyzed variants of ThiC in vivo and in vitro to probe the metabolic network surrounding AIR in Salmonella enterica. Several variants of ThiC that required metabolic perturbations to function in vivo were biochemically characterized in vitro. Results presented herein indicate that the subtleties of the metabolic network have not been captured in the current reconstitution of the ThiC reaction.     

Two complementary radiometric methods for the measurement of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase)

Two complementary methods have been devised for measuring the activity of 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (SAICAR synthetase, EC 6.3.2.6), a critical enzyme in the pathway of purine biosynthesis. In the first method, l-[4.¹⁴C]aspartic acid is condensed with 5-amino-4-imidazolecarboxylic acid ribonucleotide (AICOR) via the action of SAICAR synthetase. Unreacted l-[4-¹⁴C]aspartic acid is measured by scintillation spectrometry. In the second method, the reverse reaction of SAICAR synthetase is measured; radiactive 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR) is synthetized enzymatically, using a partial purified preparation of SAICAR synthetase from chicken liver. To the purified [¹⁴C]SAICAR is added: sodium arsenate, Tris-HCl buffer containing ADPMgCl₂ or buffer alone, and to initiate the reaction, a 12 000 × g supernatant or other suitable source of enzyme. As a consequence of the arsenolytic cleavage of [¹⁴C]SAICAR, l-[4-¹⁴C]aspartic acid is generated in stoichiometric amounts. The fourth carbon of this amino acid is then detached by selective enzymatic decarboxylation, trapped in 40% KOH and quantitated by scintillation spectrometry. The assays, performed as prescribed, are facile and notably sensitive; using them, the specific activity of SAICAR synthetase has been measured in acetone powders of the livers of representative members of the Vertebrata, and also in the principal viscera of the mouse. Of the livers examined, pigeon liver was the richest source of the investigated enzyme.