Regulation of O-acetylserine sulfhydrylase B by L-cysteine in Salmonella typhimurium.

A technique based on resistance to azaserine was used to isolate mutants lacking O-acetylserine sulfhydrylase B, one of two enzymes in Salmonella typhimurium capable of synthesizing L-cysteine from O-acetyl-L-serine and sulfide. The mutant locus responsible for this defect has been designated cysM, and genetic mapping suggests that cysM is very close to and perhaps contiguous with cysA. Strains lacking either O-acetylserine sulfhydrylase B or the second sulfhydrylase, O-acetylserine sulfhydrylase A (coded for by cysK), are cysteine prototrophs, but cysK cysM double mutants were found to require cysteine for growth. O-Acetylserine sulfhydrylase B was depressed by growth on a poor sulfur source, and depression was dependent upon both a functional cysB regulatory gene product and the internal inducer of the cysteine biosynthetic pathway, O-acetyl-L-serine. Furthermore, a cysBc strain, in which other cysteine biosynthetic enzymes cannot be fully repressed by growth on L-cystine, was found to be constitutive for O-acetylserine sulfhydrylase B as well. Thus O-acetylserine sulfhydrylase B is regulated by the same factors that control the expression of O-acetylserine sulfhydrylase A and other activities of the cysteine regulon. It is not clear why S. typhimurium has two enzymes whose physiological function appears to be to catalyze the same step of L-cysteine biosynthesis.     

Structure, mechanism, and conformational dynamics of O-acetylserine sulfhydrylase from Salmonella typhimurium: comparison of A and B isozymes

O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final step in the cysteine biosynthetic pathway in enteric bacteria and plants, the replacement of the beta-acetoxy group of O-acetyl-l-serine by a thiol to give l-cysteine. Two isozymes are found in Salmonella typhimurium, with the A-isozyme expressed under aerobic and the B-isozyme expressed under anaerobic conditions. The structure of O-acetylserine sulfhydrylase B has been solved to 2.3 A and exhibits overall a fold very similar to that of the A-isozyme. The main difference between the two isozymes is the more hydrophilic active site of the B-isozyme with two ionizable residues, C280 and D281, replacing the neutral residues S300 and P299, respectively, in the A-isozyme. D281 is above the re face of the cofactor and is within hydrogen-bonding distance to Y286, while C280 is located about 3.4 A from the pyridine nitrogen (N1) of the internal Schiff base. The B-isozyme has a turnover number (V/Et) 12.5-fold higher than the A-isozyme and an approximately 10-fold lower Km for O-acetyl-l-serine. Studies of the first half-reaction by rapid-scanning stopped-flow indicate a first-order conversion of the internal Schiff base to the alpha-aminoacrylate intermediate at any concentration of O-acetyl-l-serine. The Kd values for formation of the external Schiff base with cysteine and serine, obtained by spectral titration, are pH dependent and exhibit a pKa of 7.0-7.5 (for a group that must be unprotonated for optimum binding) with values, above pH 8.0, of about 3.0 and 30.0 mM, respectively. In both cases the neutral enolimine is favored at high pH. Failure to observe the pKa for the alpha-amines of cysteine and serine in the pKESB vs pH profile suggests a compensatory effect resulting from titration of a group on the enzyme with a pKa in the vicinity of the alpha-amine's pKa. The pH dependence of the first-order rate constant for decay of the alpha-aminoacrylate intermediate to give pyruvate and ammonia gives a pKa of about 9 for the active site lysine (K41), a pH unit higher than that of the A-isozyme. The difference in pH dependence of the pKESB for cysteine and serine, the higher pKa for K41, and the preference for the neutral species at high pH compared to the A-isozyme can be explained by titration of C280 to give the thiolate. Subtle conformational differences between O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B are detected by comparing the absorption and emission spectra of the internal aldimine in the absence and presence of the product acetate and of the external aldimine with l-serine. The two isozymes show a different equilibrium distribution of the enolimine and ketoenamine tautomers, likely as a result of a more polar active site for O-acetylserine sulfhydrylase B. The distribution of cofactor tautomers is dramatically affected by the ligation state of the enzyme. In the presence of acetate, which occupies the alpha-carboxylate subsite, the equilibrium between tautomers is shifted toward the ketoenamine tautomer, as a result of a conformational change affecting the structure of the active site. This finding, in agreement with structural data, suggests for the O-acetylserine sulfhydrylase B-isozyme a higher degree of conformational flexibility linked to catalysis.     

A rapid purification procedure and computer-assisted sulfide ion selective electrode assay for O-acetylserine sulfhydrylase from Salmonella typhimurium

An improved method for purifying O-acetylserine sulfhydrylase from Salmonella typhimurium is described as well as a new computer-controlled assay making use of the sulfide ion selective electrode. The purification method uses gradient elution from Q-Sepharose Fast Flow and phenyl-Sepharose columns to give 75 mg (50% yield) of the enzyme starting from 300 g of starting material in 3 days. The sulfide electrode assay makes use of sulfide and calomel electrodes attached to a signal buffer which serves as an impedance match. The output of the signal buffer is linked in parallel to a strip chart recorder and a Keithley Model 575 data acquisition and control system. The system 575 is interfaced to a Packard-Bell AT computer. In addition, two BASIC computer programs have been written to convert potential measured by the electrode to sulfide concentration and to convert the time course data to rates.     

Exploring O-acetylserine sulfhydrylase-B isoenzyme from Salmonella typhimurium by fluorescence spectroscopy

The pyridoxal 5′-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of cysteine in bacteria and plants. In bacteria two isoenzymes are present, OASS-A and OASS-B, with distinct structural, functional, and regulatory properties. In order to gain a deeper insight into OASS-B dynamic and functional properties, single and double mutants of the three tryptophan residues, Trp28, Trp159, and Trp212, were prepared and their fluorescence emission properties were characterized in the absence and presence of substrate and ligands by steady-state and time-resolved spectrofluorimetry. Residue Trp28 was found to be mainly responsible for Trp fluorescence emission, whereas Trp212, located in a highly flexible region near the active site, is mainly responsible for an energy-transfer to PLP leading to an emission at 500 nm. Not surprisingly, mutation of Trp212 affects OASS-B activity. Trp159 slightly contributes to both direct emission and energy transfer to PLP. Time-resolved fluorescence measurements confirmed these findings, observing a third longer tryptophan lifetime for apo-OASS-B, in addition to the two lifetimes that are present in the holo-enzyme and mutants. A comparison with the emissions previously determined for OASS-A indicates that OASS-B active site is likely to be more polar and flexible, in agreement with a broader substrate specificity and higher catalytic efficiency.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

A three-dimensional homology model of the O-acetylserine sulfhydrylase-B from Salmonella typhimurium

O-acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants. The overall pathway involves the substitution of the beta-acetoxy group of O-acetyl-L-serine with inorganic bisulfide. Two isozymes are present in S. typhimurium, the A- and B-isozymes, expressed under aerobic and anaerobic conditions, respectively. No crystal structure is presently available for the B-isozyme. Kinetic data indicate the catalytic mechanism of OASS-B is ping-pong, as found for the A-isozyme, but kinetic parameters and substrate specificity differ. In order to estimate whether structural differences may be responsible for the kinetic differences, a homology model was built using the structure of OASS-A as the template for the OASS-B model. The beta-subunit of tryptophan synthase and cystathionine beta-synthase were used for comparison. Differences between the OASS-A structure and the homology model for OASS-B are discussed.     

Heparin binding induces a conformational change in pigment epithelium-derived factor

Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J. E., and Becerra, S. P. (2003) BMC Biochem. 4, 1). We have investigated the structural basis for this observation and show that heparin induces a conformational change in the vicinity of Lys(178). This structural change was evident both when binding to intact heparin and specific heparin-derived oligosaccharides at physiological conditions or simply when exposing PEDF to low ionic strength. Binding to other glycosaminoglycans, heparin-derived oligosaccharides smaller than hexadecasaccharides (dp16), or type I collagen did not affect the structure of PEDF. The conformational change is likely to expose the epitope involved in binding to the receptor and thus regulates the interactions with cell surface receptors.     

Characterization of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1

An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5'-phosphate binding motif with both OASS and cystathionine beta-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5'-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100 degrees C. In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent K(m) values for O-acetyl-L-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s(-1). In the L-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent K(m) values for L-serine and L-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s(-1). A. pernix OASS has a high activity in the L-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-L-cysteine from L-cysteine and dithiothreitol.     

CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity

A gene encoding an O-acetyl-L-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-L-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K(M) for O-acetyl-L-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K(M) for L-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.     

Cloning of the E. coli O-acetylserine sulfhydrylase gene: ability of the clone to produce a mutagenic product from azide and O-acetylserine

The gene coding for O-acetylserine sulfhydrylase (OASS) from E. coli K12 was cloned into the vector pBR322 plasmid and expressed in a cysk mutant strain of E. coli that is deficient in O-acetylserine sulfhydrylase (OASS-). The clone containing the OASS gene was selected by using tetracycline-ammonium bismuth citrate medium. Retransformation of the hybrid plasmid into competent cysk mutant cells resulted in the recovery of a clone containing normal levels of O-acetylserine sulfhydrylase. Negative selection of retransformed cysk cells on 1,2,4-triazole plates resulted in the complete inhibition of growth indicating the presence of a functional OASS gene. The ability of the new clone to convert azide to its mutagenic metabolite was tested. Cultures of the clone cells containing significant levels of OASS activity were able to produce a mutagenic product from azide and O-acetylserine as tested on Salmonella typhimurium TA1530. This cloning method could be applied also to clone the same gene from eukaryotic sources.     

Epidermal growth factor binding induces a conformational change in the external domain of its receptor.

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha-helix and beta-sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near- and far-UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady-state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent-accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor-ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of a sulfide ion selective electrode to study O-acetylserine sulfhydrylase from germinating rapeseed

O-Acetylserine sulfhydrylase was partially purified from germinating seeds of Rape (Brassica napus L. var Target), and a sulfide ion specific electrode used to study the properties of this enzyme. It was demonstrated that the sensitivity of the electrode to sulfide ion concentration was not affected by pH, or by various concentrations of cysteine, sulfate, sulfite, and acetate.Kinetic studies showed that the K_m for O-acetyl-l-serine was 1.74 × 10^?6m, while the K_m for sulfide was 4.3 × 10^?4m. The implications of the data, together with the methods used to obtain them, are discussed.     

Tyrosine kinase inhibition: Ligand binding and conformational change in c-Kit and c-Abl

The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamily often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.     

Ligand binding to the androgen receptor induces conformational changes that regulate phosphatase interactions

We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen- and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation.     

Yeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate.

Using small-angle X-ray scattering from solutions of yeast hexokinase, we have measured the radii of gyration of the monomeric B isozyme and its complexes with sugar substrates. We find that the radius of gyration decreases by 0.95 +/- 0.24 A upon binding glucose and 1.25 +/- 0.28 A upon binding glucose 6-phosphate. This observed reduction in radius of gyration in the presence of glucose is the same as that calculated from the coordinates of the high-resolution crystal structures of native hexokinase B and a glucose complex with hexokinase A. Thus, these measurements suggest that the dramatic closing of the slit between the two lobes of hexokinase observed in the crystal structures (Bennett, W.S., & Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848--4852) occurs in solution when either glucose or glucose 6-phosphate is bound.     

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.     

Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium.

The relationship of acid adaptation to tolerance of other environmental stresses was examined in Salmonella typhimurium. S. typhimurium was adapted to acid by exposing the cells to mildly acidic conditions (pH 5.8) for one to two cell doublings. Acid-adapted cells were found to have increased tolerance towards various stresses including heat, salt, an activated lactoperoxidase system, and the surface-active agents crystal violet and polymyxin B. Acid adaptation increased cell surface hydrophobicity. Specific outer membrane proteins were induced by acid adaptation, but the lipopolysaccharide component appeared to be unaltered. These results show that acid adaptation alters cellular resistance to a variety of environmental stresses. The mechanism of acid-induced cross-protection involved changes in cell surface properties in addition to the known enhancement of intracellular pH homeostasis.     

Salmonella typhimurium induces apoptosis in human monocyte-derived macrophages

Salmonella species represent a leading cause of gastroenteritis worldwide. More recently, they have been proposed as putative vaccine delivery vehicles in humans. Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages. In this study, we investigated the fate of Salmonella typhimurium-infected human macrophages differentiated from blood monocytes by GM-CSF. Wild type S. typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30 min post-infection. Three hours later more than 80% of the macrophages in the culture were killed. In contrast, during the same period, macrophages infected with the non-invasive S. typhimurium strain BJ66 remained viable. Chromatin fragmentation is a hallmark of cells undergoing apoptosis. Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells. Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase-3, a member of the pro-apoptotic enzyme family shown to be involved in S. typhimurium-induced killing of mouse macrophages, reduced SL1344-mediated cytotoxicity by 40%. Our study provides the first evidence that invasive S. typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM-CSF, and that cell death is a caspase-dependent phenomenon.