The characterization of mutant Bacillus subtilis adenylosuccinate lyases corresponding to severe human adenylosuccinate lyase deficiencies

Adenylosuccinate lyase is a homotetramer that catalyzes two discrete reactions in the de novo synthesis of purines: the cleavage of adenylosuccinate and succinylaminoimidazole carboxamide ribotide (SAICAR). Several point mutations in the gene encoding the enzyme have been implicated in human disease. Bacillus subtilis adenylosuccinate lyase was used as a model system in which mutations were constructed corresponding to those mutations associated with severe human adenylosuccinate lyase deficiency. Site-directed mutagenesis was utilized to construct amino acid substitutions in B. subtilis adenylosuccinate lyase; Met(10), Ile(123), and Thr(367) were replaced by Leu, Trp, and Arg, respectively, and the altered enzymes were expressed in Escherichia coli. These purified enzymes containing amino acid substitutions were found to have substantial catalytic activity and exhibit relatively small changes in their kinetic parameters. The major deviations from the wild-type-like behavior were observed upon biophysical characterization. All of these enzymes with amino acid replacements are associated with marked thermal instability. I123W adenylosuccinate lyase exhibits notable changes in the circular dichroism spectra, and a native gel electrophoresis pattern indicative of some protein aggregation. T367R also exhibits alterations at the quarternary level, as reflected in native gel electrophoresis. Experimental results, combined with homology modeling, suggest that the altered enzymes are primarily structurally impaired. The enzyme instability was found to be lessened by subunit complementation with the wild-type enzyme, under mild conditions; these studies may have implications for the in vivo behavior of adenylosuccinate lyase in heterozygous patients. Residues Met(10), Ile(123), and Thr(367) appear to be located in regions of the enzyme important for maintaining the structural integrity required for a stable, functional enzyme.     

Gln212, Asn270, and Arg301 are critical for catalysis by adenylosuccinate lyase from Bacillus subtilis

In adenylosuccinate lyase from Bacillus subtilis, Gln(212), Asn(270), and Arg(301) are conserved and located close to the succinyl moiety of docked adenylosuccinate. We constructed mutant enzymes with Gln(212) replaced by Glu and Met, Asn(270) by Asp and Leu, and Arg(301) by Gln or Lys. The wild-type and mutant enzymes were expressed in Escherichia coli and purified to homogeneity. The specific activities of the Q212M and the 270 and 301 mutant enzymes were decreased more than 3000-fold as compared to the wild type. Only Q212E retained sufficient activity for determination of its kinetic parameters: V(max) was decreased approximately 1000-fold, and K(m) was increased 6-fold, as compared to the wild-type enzyme. Adenylosuccinate binding studies of the other mutants revealed greatly weakened affinities that contributed to, but did not account entirely for, the loss of activity. These mutant enzymes did not differ greatly from the wild-type enzyme in secondary structure or subunit association state, as shown by circular dichroism spectroscopy and light-scattering photometry. Incubation of pairs of inactive mutant enzymes led to reconstitution of some functional sites by subunit complementation, with recovery of up to 25% of the specific activity of the wild-type enzyme. Subunit complementation occurs only if the two mutations are contributed to the active site by different subunits. Thus, mixing Q212E with N270L enzyme yielded a specific activity of approximately 20% of the wild-type enzyme, while mixing Q212M with R301K enzyme did not restore activity. As supported by computer modeling, the studies presented here indicate that Gln(212), Asn(270), and Arg(301) are indispensable to catalysis by adenylosuccinate lyase and probably interact noncovalently with the carboxylate anions of the substrates 5-aminoimidazole-4(N-succinylocarboxamide)ribonucleotide and adenylosuccinate, optimizing their bound orientations.     

Evaluation of types of interactions in subunit association in Bacillus subtilis adenylosuccinate lyase

Adenylosuccinate lyase (ASL) of Bacillus subtilis is a homotetramer in which three subunits contribute to each of four active sites. We sought to evaluate the types of interactions responsible for subunit association by studying the enzyme's oligomeric structure at low temperatures as compared to 25 degrees C, in the presence of KBr and after mutagenesis. Analytical ultracentrifugation data reveal that at 25 degrees C ASL is active and exists as 100% tetramer, while at 8 and 4 degrees C, as hydrophobic interactions are weakened, the catalytic activity decreases strikingly and the enzyme dissociates to a mixture of monomer-dimer-trimer, with small amounts of tetramer. In the presence of increasing concentrations of KBr (0.1-2.5 M), which disrupts electrostatic interactions, ASL is dissociated initially to monomer-dimer, with small amounts of trimer-tetramer, and then the monomer species predominates along with small amounts of trimer-tetramer. Very low enzymatic activity was found under these conditions. Accordingly, we postulate that electrostatic interactions are a major source of oligomeric stabilization of B. subtilis ASL. We selected for mutagenesis the closest charged residues (His (299)/Glu (239) and Arg (167)/Asp (217) pairs) located in the subunit interface that has the largest surface area. All of the mutants have low V max values, high K M values, and decreased molecular masses. We conclude that both hydrophobic and electrostatic interactions play roles in maintaining the ASL tetramer and this structure is essential for adenylosuccinate lyase activity.     

Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role

Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.     

Important roles of hydroxylic amino acid residues in the function of Bacillus subtilis adenylosuccinate lyase

Thr(93), Ser(94), Thr(140), and Ser(306) are conserved in all adenylosuccinate lyases (ASL) and are close to other amino acids previously identified by mutagenesis as being in the active site. To test their involvement in the enzyme's function, each of these amino acids was replaced by alanine. All the mutants exhibit circular dichroism spectra which are similar to that of wild-type enzyme, indicating there is no appreciable change in secondary structure. T93A exhibits 0.5% of the V(max) of wild-type ASL with a 10-fold increase in K(m) for adenylosuccinate. S94A has 65% of the V(max) of wild-type ASL with little change in K(m). T140A exhibits 0.03% of the activity of wild-type enzyme with an 11-fold increase in K(m). S306A has 0.4% of the V(max) of wild-type ASL with a sevenfold increase in K(m). Measurements of the pH-V(max) profile reveal a pK(2) value for S94A of 7.83 and S306A of 7.65, in contrast to 8.24 for the wild-type enzyme and 8.42 for T93A. Thr(93) may orient adenylosuccinate optimally for catalysis, while Ser(94) stabilizes protonated His(89), a determinant of pK(2). Thr(140) may, through hydrogen bonding, interact with Asn(270), an amino acid essential for catalysis. Ser(306) may be involved in a hydrogen bond network that ultimately stabilizes protonated His(68), which is probably the general acid in the reaction of enzyme with substrate. The results of this paper demonstrate the importance in the catalytic function of ASL of hydrogen bonds and hydrogen bonding networks involving serine and threonine.     

Crystallization and preliminary structural analysis of Bacillus subtilis adenylosuccinate lyase, an enzyme implicated in infantile autism.

Adenylosuccinate lyase (ASL) from Bacillus subtilis has been crystallized and structural analysis by X-ray diffraction is in progress. ASL is a 200-kDa homotetramer that catalyzes two distinct steps of de novo purine biosynthesis leading to the formation of AMP and IMP; both steps involve the beta-elimination of fumarate. A single point mutation in the human ASL gene has been linked to mental retardation with autistic features. In addition, ASL plays an important role in the bioprocessing of anti-HIV therapeutics. B subtilis ASL, which shares 30% sequence identity and 70% sequence similarity with human ASL, has been crystallized and data to 3.3 A have been collected at 100 K. The space group is P6(1)22 or P6(5)22 with a = b = 129.4 A; the length of the c-axis varies between 275 and 290 A, depending on the crystal. An analysis of solvent content indicates a dimer in the asymmetric unit, although a self-rotation function and an analysis of native Pattersons failed to identify unambiguously the location of any noncrystallographic symmetry axes. Structure determination by isomorphous replacement is in progress.     

Nitro analogs of substrates for adenylosuccinate synthetase and adenylosuccinate lyase

The reactivities of the nitro analogs of the substrates of adenylosuccinate synthetase and adenylosuccinate lyase, the enzymes which catalyze the penultimate and last step, respectively, in the pathway for AMP biosynthesis have been examined. Alanine-3-nitronate, an aspartate analog, was a substrate for the synthetase from Azotobacter vinelandii, having a $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ which was ~30% that for aspartate. The product of this reaction was N⁶-(l-1-carboxy-2-nitroethyl)-AMP. Of nine other substrate analogs tested, only cysteine sulfinate (having 5.5% of the activity of aspartate) was reactive. These results demonstrate the strict requirement of the synthetase for a negatively charged substituent, with a carboxylate-like geometry, at the β-carbon of the α-amino acid substrate. The lyase, purified to homogeneity from brewer's yeast by a new procedure, did not utilize N⁶-(l-1-carboxy-2-nitroethyl)-AMP as a substrate. However, the nitronate form of this analog was a good inhibitor of the lyase ($\text{K}{}_{\mathrm{m}}\text{K}{}_{\mathrm{i}}\text{= 28}$ when compared to adenylosuccinate), suggesting that it mimics a carbanionic intermediate in the reaction pathway. The avid binding of bromphenol blue by the lyase (_i = 0.95 μM) was used for active site titrations and for displacement of the enzyme, in the purification protocol, from blue Sepharose.     

A radiometric technique for the measurement of adenylosuccinate lyase

When radioactive adenylsuccinic acid (AMP-S) is metabolized to AMP and fumaric acid by the enzyme adenylsuccinate lyase (EC 4.3.2.2), a proton is released to the solvent as ³H₂O. This removal is believed to be stereospecifically identical to that catalyzed by the enzyme, l-aspartase [1–5], and therefore entails the loss of a proton from C-3 of the dicarboxylic acid moiety of the nucleotide. Advantage has been taken of this fact in the design of a facile assay for this enzyme. Adenylosuccinic acid, tritiated on C-2 and C-3 of the l-aspartase moiety, is prepared by chemical synthesis. This product is purified, lyophilized to dryness and reconstituted in a solution of unlabelled AMP-S, bringing the final concentration to 5·10^?3 M, and the final specific activity to 8.0 μCi/mol. 5-μl aliquots of this substrate are then incubated at 37°C with 5-μl aliquots of tissue extract; after an appropriate period, any tritium released to the solvent water is distilled at room temperature overnight into a 5 μl droplet of saturated aqueous KOH adherent to the lid of the sealed reaction vessel. The lid is removed and tritium thereon is measured by scintillation spectrometry. The assay, performed as prescribed, is facile, in that it permits the simultaneous estimation of the lyase activity in a large battery of samples, is not interfered with by opalescent or proteinaceous suspensions, is accurate and outstandingly sensitive.     

Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase

Adenylosuccinate lyase (ASL) of Bacillus subtilis contains three conserved histidines, His(68), His(89), and His(141), identified by affinity labeling and site-directed mutagenesis as critical to the intersubunit catalytic site. The pH-V(max) profile for wild-type ASL is bell-shaped (pK (1) = 6.74 and pK (2) = 8.28). Only the alkaline side changes with temperature, characteristic of histidine pKs. To identify determinants of pK (2) in the enzyme-substrate complex, we replaced residues at two positions close to His(68) (but not to His(89) or His(141)) in the structure. Compared with the specific activity of 1.75 mumol adenylosuccinate reacting/min/mg of wild-type enzyme at pH 7.0, mutant enzymes D69E, D69N, R310Q, and R310K exhibit specific activities of 0.40, 0.04, 0.00083, and 0.10, respectively. While D69E has a K (m) for adenylosuccinate similar to that of wild-type ASL, D69N and R310K exhibit modest increases in K (m), and R310Q has an 11-fold increase in K (m). The mutant enzymes show no significant change in molecular weight or secondary structure. The major change is in the pH-V(max) profile: pK (2) is 8.48 for the D69E mutant and is decreased to 7.83 in D69N, suggesting a proximal negative charge is needed to maintain the high pK of 8.28 observed for wild-type enzyme and attributed to His(68). Similarly, R310Q exhibits a decrease in its pK (2) (7.33), whereas R310K shows little change in pK (2) (8.24). These results suggest that Asp(69) interacts with His(68), that Arg(310) interacts with and orients the beta-carboxylate of Asp(69), and that His(68) must be protonated for ASL to be active.     

Epr plays a key role in DegU-mediated swarming motility of Bacillus subtilis

Epr, a minor extracellular protease, is involved in the swarming motility of Bacillus subtilis . It does so by providing essential signals required for swarming. It has also been demonstrated that DegU is required for swarming and that it occurs at very low levels of DegU∼P and is inhibited at high levels of DegU∼P. In this study, we show that maximal epr expression is observed at very low concentrations of DegU∼P, whereas it is repressed at high DegU∼P. A parallel effect of DegU∼P levels on swarming motility is also observed, where very low levels of DegU∼P support swarming and excessive DegU∼P abolishes swarming. We further demonstrate that the defect of swarming motility in a degU ⁻ strain can be rescued, albeit incompletely, by increased expression of an exogenous epr gene. We also show that an additional extracellular factor(s), apart from epr , regulated by DegU, is required for robust swarming.     

Binding of cations in Bacillus subtilis phosphoribosyldiphosphate synthetase and their role in catalysis.

The binding sites for the two cations essential for the catalytic function of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthases have been identified from the structure of the Bacillus subtilis phosphoribosyldiphosphate synthetase (PRPPsase) with bound Cd(2+). The structure determined from X-ray diffraction data to 2.8-A resolution reveals the same hexameric arrangement of the subunits that was observed in the complexes of the enzyme with the activator sulfate and the allosteric inhibitor ADP. Two cation binding sites were localized in each of the two domains of the subunits that compose the hexamer; each domain of the subunit has an associated cation. In addition to the bound Cd(2+), the Cd(2+)-PRPPsase structure contains a sulfate ion in the regulatory site, a sulfate ion at the ribose-5-phosphate binding site, and an AMP moiety at the ATP binding site. Comparison of the Cd(2+)-PRPPsase to the structures of the PRPPsase complexed with sulfate and mADP reveals the structural rearrangement induced by the binding of the free cation, which is essential for the initiation of the reaction. The comparison to the cPRPP complex of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli, a type I phosphoribosyltransferase, provided information about the binding of PRPP. This strongly indicates that the binding of both substrates must lead to a stabilized conformation of the loop region, which remains unresolved in the known PRPPsase complex structures.     

An HPLC-based assay of adenylosuccinate lyase in erythrocytes

ADSL deficiency is a disorder of purine metabolism with a broad clinical spectrum. A rapid and simple HPLC-based assay to measure ADSL activity in erythrocytes was developed. The suitability of DBSs was assessed. ADSL activity was measured in erythrocyte lysates and DBS using succinyl-AMP as the substrate. Detection and quantification were performed using isocratic ion-pairing reversed-phase HPLC with UV-detection. Reference values in erythrocyte lysates were established. The intra- and interassay variations were 2% and 8%, respectively. ADSL deficiency was easily recognized. ADSL activity in DBS was highly unstable, disqualifying DBS for diagnostic procedures.     

Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens

Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K(I) approximately 0.1 microM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.     

Control of key metabolic intersections in Bacillus subtilis

The remarkable ability of bacteria to adapt efficiently to a wide range of nutritional environments reflects their use of overlapping regulatory systems that link gene expression to intracellular pools of a small number of key metabolites. By integrating the activities of global regulators, such as CcpA, CodY and TnrA, Bacillus subtilis manages traffic through two metabolic intersections that determine the flow of carbon and nitrogen to and from crucial metabolites, such as pyruvate, 2-oxoglutarate and glutamate. Here, the latest knowledge on the control of these key intersections in B. subtilis is reviewed.     

The interaction of monofluorofumarate with adenylosuccinate lyase

Monofluorofumarate was tested as an alternate substrate and inhibitor for adenylosuccinate lyase. Monofluorofumarate was found to be a slow reacting substrate when either AMP or AICAR (5-aminoimidazole 4-carboxamide ribonucleotide) were used as substrate acceptor molecules at pH 7.5. There was no indication that monofluorofumarate could induce the inactivation of adenylosuccinate lyase. The initial reaction product when monofluorofumarate was incubated with AMP in the presence of adenylosuccinate lyase has been determined to be 2-fluoro-adenylosuccinate. This molecule lost HF spontaneously, and the subsequent intermediate was rapidly hydrolyzed to oxalacetate and AMP. A similar reaction scheme was also observed when AICAR was utilized as a cosubstrate with monofluorofumarate. The initial reaction rate when 1.0 mM monofluorofumarate and 1.0 mM AMP were used as substrates with adenylosuccinate lyase was only 1.4% of the rate when 1.0 mM fumarate was used. AICAR (1.0 mM) was found to react with monofluorofumarate at 8.9% of the rate that it reacts with fumarate.     

Purification and characterization of extracellular pectate lyase from Bacillus subtilis

Bacillus subtilis strain SO113 secretes a pectate lyase which is produced during the exponential death phase of growth, just before sporulation. This extracellular pectate lyase, which produces unsaturated products from polygalacturonate, was purified 35-fold from the culture supernatant of Bacillus subtilis by a CM Sephadex chromatography. It has an isoelectric point of about 9.6 and an Mr of 42,000. Optimum activity occurred at pH 8.4 and at 42 degrees C. Calcium has a stimulative effect on the enzyme activity while EDTA leads to enzyme inactivation. The pectate lyase has a specific activity of 131 mumol of aldehyde groups per min and per mg of protein. The Km of the purified enzyme for polygalacturonic acid was 0.862 g.l-1 and the Vmax for polygalacturonic acid hydrolysis was 1.475 mumol of unsaturated products per min and per mg of protein. By using monoclonal antibodies raised against Erwinia chrysanthemi 3937 pectate lyases, it was shown that pectate lyases b and c of this strain are immunologically closely related to the Bacillus subtilis pectate lyase.