Novel Sensitive High-Throughput Screening Strategy for Nitrilase-Producing Strains

Nitrilases have found wide use in the pharmaceutical industry for the production of fine chemicals, and it is important to have a method by which to screen libraries of isolated or engineered nitrilase variants (including bacteria and fungi). The conventional methods, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis, or gas chromatography, are tedious and time-consuming. Therefore, a direct and sensitive readout of the nitrilase's activity has to be considered. In this paper, we report a novel time-resolved luminescent probe: o-hydroxybenzonitrile derivatives could be applied to detect the activity of the nitrilases. By the action of nitrilases, o-hydroxybenzonitrile derivatives can be transformed to the corresponding salicylic acid derivatives, which, upon binding Tb³⁺, serve as a photon antenna and sensitize Tb³⁺ luminescence. Because of the time-resolved property of the luminescence, the background from the other proteins (especially in the fermentation system) in the assay could be reduced and, therefore, the sensitivity was increased. Moreover, because the detection was performed on a 96- or 384-well plate, the activity of the nitrilases from microorganisms could be determined quickly. Based on this strategy, the best fermentation conditions for nitrilase-producing strains were obtained.     

A luminescent affinity tag for proteins based on the terbium(III)-binding peptide

Genetically encoded tags attached to proteins of interest are widely exploited for proteome analysis. Here, we present Tb(3+)-binding peptides (TBPs) which can be used for both luminescent measurements and affinity purification of proteins. TBPs consist of acidic amino acid residues and tryptophan residues which serve as Tb(3+)-binding sites and sensitizers for Tb(3+) luminescence, respectively. The Tb(3+) complexes of TBPs fused to a target protein exhibited luminescence characteristic of Tb(3+) by excitation of the tryptophan residue, and fusion proteins fused to one of the TPBs were successfully isolated from Escherichia coli cell lysate by affinity chromatography with a Tb(3+)-immobilized solid support.     

Terbium as a luminescent probe of metal-binding sites in protein kinase C

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.     

Improved differentiation between luminescence decay components by use of time-resolved optical activity measurements and selective lifetime modulation.

The analysis of luminescence decay experiments from proteins is typically modeled as a combination of independent first-order decay functions. However, Poisson noise in the photon counting experiment limits the ability of this approach to resolve decay components from separate lumiphores with similar lifetimes. To provide further differentiation, we incorporate time-resolved circular polarization of luminescence, an additional independent observable, into the analysis. In the simplest case, for example, each lumiphore's chirality is assumed to be time independent and is determined by the position of the lumiphore with respect to the surrounding chiral environment within the protein. In this paper, we describe the analysis of simultaneously recorded time-resolved luminescence and circularly polarized luminescence data to obtain improved temporal resolution. When combined with selective dynamic luminescence quenching, in a model system comprising a mixture of Tb/transferrin and Tb/conalbumin, we demonstrate resolution between two decay components with a lifetime difference of 7% and a difference in emission anisotropy of 5 X 10(-2). Evidence for the improved discrimination is further demonstrated by the increase in curvature of the chi 2 surface that results from the additional information.     

The nitrilases of Rhodococcus rhodochrous NCIMB 11216

Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed -cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the -terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The -terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30°C and 8.0, respectively. The propionitrile-induced nitrilase had a K_m for benzonitrile of 20.7 m and a V_max of 12.4 μmol min⁻¹ mg⁻¹ protein whereas the benzonitrile-induced nitrilase had a K_m for benzonitrile of 8.83 m and a V_max of 0.57 μmol min⁻¹ mg⁻¹ protein.     

A time-resolved assay of dopamine β-hydroxylase activity utilizing high-pressure liquid chromatography

A time-resolved assay of dopamine β-hydroxylase (EC 1.14.17.1) activity utilizing high-pressure liquid chromatography is described. The conversion of tyramine to octopamine by the enzyme was used as a standard reaction. The analytical separation of the assay substrate and product employed a reversed-phase ion-pair chromatographic system, with ultraviolet absorbance detection of eluents at 280 nm. Aliquots of the assay solution were injected directly onto the high-pressure liquid chromatography column and were separated in 6 min total elapsed time, thus permitting time-resolved determination of the produet. Quantities of octopamine as small as 20 pmol could be measured. This facile method is more straightforward, convenient, and sensitive than previously published physical and spectroscopic methods of determining dopamine β-hydroxylase activity.     

Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.

The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the F?rster equation in approximating distances in these systems.     

Luminescence quenching by nitroxide spin labels in aqueous solution: studies on the mechanism of quenching.

The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water-soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the rate constant for quenching of triplet-state Tb3+ by nitroxides was about 4 orders of magnitude smaller (ca. 10(5) M-1 s-1) than those of the singlet-state probes; (II) the quenchers reduced the excited-state lifetime of both probes; (III) the rate constants for quenching of both probes were found to be apparently independent of the temperature (between 6 and 42 degrees C) and viscosity (up to 60 mPa.s) of the solutions; (IV) both singlet and triplet quenching rates were sensitive to solvent polarity; (V) there is a small but significant spectral overlap between the absorption band of weekly absorbing nitroxide radicals and the emission spectra of luminophores, the extent of which, however, does not correlate with the extent of quenching; (VI) the quenching rate declines sharply with an increasing luminophore to nitroxide distance. The distance dependence of the quenching rate showed a satisfactory fit to an exponential function. These findings indicate that the quenching reaction is dominated by an electron exchange between the excited singlet- or triplet-state luminophore and the nitroxide radical rather than controlled by diffusional properties of the reactants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distance measurements in cardiac troponin C

Intramolecular distance measurements were made in cardiac troponin C (cTnC) by fluorescence energy transfer using Eu3+ or Tb3+ as energy donors and Nd3+ or an organic chromophore as acceptors. The laser-induced luminescence of bound Eu3+ is quenched in Eu1Nd1cTnC with a lifetime of 0.328 ms, compared with 0.43 ms for Eu2cTnC. The enhanced decay corresponds to an energy transfer efficiency of 0.25, or a distance of 1.1 nm between the two high affinity sites. We have also labeled cTnC with 4-dimethylaminophenylazophenyl-4'-maleimide (DAB-Mal) at the two cysteine residues (Cys-35 and Cys-84). Energy transfer measurements were carried out between Tb3+ bound to the high affinity sites and the labels attached to the domain containing the low affinity site. Upon uv irradiation at pH 6.7, Tb1cTnCDAB emits tyrosine-sensitized Tb3+ luminescence that decays bioexponentially with lifetimes of 1.29 and 0.76 ms. The shorter lifetime is ascribed to energy transfer from Tb3+ to the DAB labels, yielding an average distance of 3.4 nm between the donor and the acceptors. At pH 5.0, however, the luminescence decays exclusively with a single lifetime of 1.31 ms, suggesting that under these conditions all Tb3+ ions are more than 5.2 nm away from the label. Thus cTnC, like skeletal TnC, undergoes a pH-dependent conformational transition which converts an elongated structure at lower pH's to a rather compact conformation in a more physiological medium.     

Lanthanide ion luminescence as a probe of DNA structure. 1. Guanine-containing oligomers and nucleotides.

Laser-induced Eu(3+) luminescence spectroscopy is used to probe the interaction of Eu(3+) ion with guanine-containing nucleotides and single-stranded oligomers. By using time-resolved and non-time-resolved Eu(3+) luminescence techniques, two classes of Eu(3+) binding site are observed in oligo(dG)10, oligo(dG)8, oligo(dG)6, oligo(dG)4, and d-GMP. One class of site binds Eu(3+) ions more strongly than the other. Since the "tight" class of bound Eu(3+) ions have two coordinated water molecules, it is inferred that six or seven atoms from the oligomers are coordinating the Eu(3+). The "weaker" class of Eu(3+) ion sites involve the coordination of six or seven water molecules and therefore, are coordinated by one or two atoms from the oligomer. The tight class of Eu(3+) binding site is attributed to an interstrand association of Eu(3+) with the oligomers forming dimeric or polymeric structures. The dissociation constants (Kd) for the 1:1 complexes Eu(d-GMP)+ and Eu(d-GTP)- have been determined as well as the Kd for the dimerization reaction of Eu(d-GMP)+. The Tb(3+) luminescence enhancement properties of these molecules are also examined in relation to their EU(3+) binding characteristics.     

Oxygen quenching of sensitized terbium luminescence in complexes of terbium with small organic ligands and proteins

Oxygen does not quench the luminescence of either free Tb or of Tb bound to dipicolinate. However, sensitized Tb luminescence in complexes of that ion with elastase, thermolysin, and alpha-amylase is quenched by oxygen at rates that far exceed that with which the intrinsic fluorescence of the proteins is quenched. We infer that this more rapid quenching of Tb luminescence indicates a major role for energy transfer from tryptophan moieties in a triplet excited state.     

Applications of thin layer chromatography,high performance liquid chromatography and mass spectrometry in the fermentation and isolation of the antibiotic nybomycin

Summary Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrometry (MS) methods have been developed for the analysis of the antibiotic nybomycin, its derivatives deoxynybomycin and nybomycin acetate, during the fermentation and isolation of nybomycin. Using a quantitative HPLC based assay, the time course of nybomycin production (nybomycin titers) in 1000 liter fermentations was determined. Desorption chemical ionization mass spectrometry (DCI/MS) of standard nybomycin samples, fermentation broth samples and purified fractions suggested the co-production of deoxynybomycin which was not reported previously from this organism. TLC and HPLC were used to confirm the presence of deoxynybomycin in the crude extracts of fermentation broths.     

Metal ion binding sites of bacteriorhodopsin. Laser-induced lanthanide luminescence study

Laser-excited luminescence lifetimes of lanthanide ions bound to bacteriorhodopsin have been measured in deionized membranes. The luminescence titration curve, as well as the binding curve of apomembrane (retinal-free) with Eu3+, has shown that the removal of the retinal does not significantly affect the affinity of Eu3+ for the two high affinity sites of bacteriorhodopsin. The D2O effects on decay rate constants indicate that Eu3+ bound to the high affinity sites of native membrane or apomembrane is coordinated by about six ligands in the first coordination sphere. Tb3+ is shown to be coordinated by four ligands. The data indicate that metal ions bind to the protein with a specific geometry. From intermetal energy transfer experiments using Eu3+-Pr3+, Tb3+-Ho3+, and Tb3+-Er3+, the distance between the two high affinity sites is estimated to be 7-8 A.     

Luminescence study of G-quadruplex formation in the presence of Tb3+ ion

The interactions of Tb3+ with the quadruplex-forming oligonucleotide bearing human telomeric repeat sequence d(G(3)T(2)AG(3)T(2)AG(3)T(2)AG(3)), (htel21), have been studied using luminescence spectroscopy and circular dichroism (CD). Enhanced luminescence of Tb3+, resulting from energy transfer from guanines, indicated encapsulation of Tb3+ ion in the central cavity of quadruplex core. The ability of lanthanide ions (Eu3+ and Tb3+) to mediate formation of quadruplex structure has been further evidenced by the fluorescence energy transfer measurements with the use of oligonucleotide probe labeled with fluorescein and rhodamine FRET partners, FAM-htel21-TAMRA. The CD spectra revealed that Tb3+/htel21 quadruplex possesses antiparallel strand orientation, similarly as sodium quadruplex. Tb3+ binding equilibria have been investigated in the absence and the presence of competing metal cations. At low Tb3+ concentration (8 microM) Tb3+/htel21 quadruplex stability is very high (5 x 10(6) M(-1)) and stoichiometry of 5-7 Tb3+ ions per one quadruplex molecule is observed. Luminescence and CD titration experiments suggested that the cavity of quadruplex accommodates two Tb3+ ions and the remaining Tb3+ ions bind probably to TTA loops of quadruplex. Higher concentration of Tb3+ (above 10 microM) results in the excessive binding of Tb3+ ions that finally destabilizes quadruplex, which undergoes transformation into differently organized assemblies. Such assemblies (probably possessing multiple positive charge) exhibit kinetic stability, which is manifested by a very slow kinetics of displacement of Tb3+ ion by competing cations (Li+, Na+, K+).     

Nitrilase and its application as a 'green' catalyst

Hydrolase-catalyzed reactions have been widely applied in organic synthesis. Nitrilases are an important class of hydrolase that converts naturally occurring, as well as xenobiotically derived, nitriles to the corresponding carboxylic acids and ammonia. Because of their inherent enantio- and regioselectivities and other benefits, nitrilases are attractive as 'green', mild, and selective catalysts for setting stereogenic centers in fine-chemical synthesis and enantiospecific synthesis of a variety of carboxylic acid derivatives. In this review, the literature has been surveyed to provide a comprehensive coverage of the application of nitrilases in organic synthesis. Literature has also been cited to describe the isolation and/or characterization of nitrilases and related enzymes.     

A catalytic site of protein disulfide isomerase probed with adenosine-5'-triphosphate analogs

Anthraniloyl adenosine-5'-triphosphate (Ant-ATP) and etheno-adenosine-5'-triphosphate (epsilon-ATP) complexed to Mg(2+) ions are substrates of protein disulfide isomerase (PDI). epsilon-ATP, coordinated to Tb(3+) ions, was used as a probe of the ATPase binding site. Sensitized luminescence arising from resonance energy transfer from epsilon-adenine to Tb(3+) is quenched by PDI. The luminescence results are discussed in reference to a model in which the distance of separation between epsilon-adenine (donor) and Tb(3+) (acceptor) is increased upon binding of PDI. The interaction of a small peptide of 14 amino acid residues with the b/b' domain of the protein does not influence the ATPase activity. The phosphorescence, fluorescence and fluorescence anisotropy of bound epsilon-ATP are not perturbed by the binding of the small molecular weight peptide to PDI. It is suggested that the peptide and ATP do not share a common binding site on the b/b' domain.     

Importance of hydrogen-bonding sites in the chiral recognition mechanism between racemic D3 terbium(III) complexes and amino acids

The perturbation of the racemic equilibrium of luminescent D3 terbium(III) complexes with chelidamic acid (CDA), a hydroxylated derivative of 2,6-pyridine-dicarboxylic acid (DPA), by added chiral biomolecules such as L-amino acids has been studied using circularly polarized luminescence and 13C NMR spectroscopy. It is shown in this work that the chiral-induced equilibrium shift of [Tb(CDA)3](6-) by L-amino acids (i.e. L-proline or L-arginine) was largely influenced by the hydrogen-bonding networks formed between the ligand interface of racemic [Tb(CDA)3](6-) and these added chiral agents. The capping of potential hydrogen-bonding sites by acetylation in L-proline led to a approximately 100-fold drop in the induced optical activity of the [Tb(CDA)3](6-):N-acetyl-L-proline system. This result suggested that the hydrogen-bonding networks serve as the basis for further noncovalent discriminatory interactions between racemic [Tb(CDA)3](6-) and added L-amino acids.     

Terbium(III) luminescence study of the spatial relationship of tryptophan residues to the two metal ion binding sites of Escherichia coli glutamine synthetase.

The luminescence of Tb(III) was used to explore the topography of the metal ion sites of Escherichia coli glutamine synthetase and the relationship between these sites and tryptophan residues of the enzyme. By irradiation of tryptophan residues at 295 nm and measurement of the resulting Tb(III) luminescence at 544 nm, a biphasic curve was obtained upon titrating apoenzyme with Tb(III) indicating sequential binding of Tb(III) ions to the two binding sites of glutamine synthetase. The luminescence intensity was greater in the second region of the titration curve which is mostly due to energy transfer from Trp-158 to the second Tb(III) binding site of the enzyme. By use of the F?rster equation for energy transfer from donor Trp to acceptor Tb(III), distances from Trp-57 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-158 was replaced by Ser, to be 16.4 and 15.7 A, respectively; distances from Trp-158 to Tb(III) at the n1 and n2 sites were calculated, by using a mutant enzyme in which Trp-57 was replaced by Leu, to be 16.8 and 9.5 A, respectively. All the distances are in reasonably good agreement with the crystal structure distances from Salmonella typhimurium glutamine synthetase except the distance from Trp-158 to the second Tb(III) binding site. The discrepancies may result from a slightly different conformation of glutamine synthetase in solution and in the crystal and/or a slightly different conformation for trivalent Ln(III) binding compared to divalent Mn(II) binding.     

The binding of terbium ions to gelsolin reveals two classes of metal ion binding sites.

Spectroscopically active terbium ions have been used to probe the Ca2+ ion-binding sites on human plasma gelsolin. The luminescence of Tb3+ ions bound to gelsolin is markedly enhanced when excited indirectly at 295 nm due to F?rster type dipole-dipole energy transfer from neighboring tryptophan residues. Titration of this luminescence with increasing concentrations of Tb3+ ions was saturable although the shape of this titration curve was complex indicating the involvement of multiple classes of sites. Luminescence lifetime measurements (obtained by indirect excitation at 295 nm) demonstrate the presence of two classes of sites characterized by a major lifetime of 1.0-1.1 ms and a minor lifetime of 0.7-0.8 ms. However, while the amplitude of the minor lifetime showed a hyperbolic dependence on the Tb3+ ion concentration, the amplitude of the major lifetime showed a strongly sigmoidal dependence. Different classes of Tb3+ ion binding sites can also be distinguished by the different Ca2+ ion concentrations needed to displace Tb3+ ions from these sites on gelsolin. It is proposed that the occupancy of one class of Tb3+ ion binding sites on gelsolin causes a conformational change in gelsolin which then allows a second class of cryptic Tb3+ ion binding sites to be expressed. The implications of these results in terms of the binding of Ca2+ ions to gelsolin and the regulation of the activities of gelsolin by calcium are discussed.     

Expanding the repertoire of nitrilases with broad substrate specificity and high substrate tolerance for biocatalytic applications

Enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. Although nitrilases from several sources have been characterized, there exists a scope for identifying broad spectrum nitrilases exhibiting higher substrate tolerance and better thermostability to develop industrially relevant biocatalytic processes. Through genome mining, we have identified nine novel nitrilase sequences from bacteria and evaluated their activity on a broad spectrum of 23 industrially relevant nitrile substrates. Nitrilases from Zobellia galactanivorans, Achromobacter insolitus and Cupriavidus necator were highly active on varying classes of nitriles and applied as whole cell biocatalysts in lab scale processes. Z. galactanivorans nitrilase could convert 4-cyanopyridine to achieve yields of 1.79 M isonicotinic acid within 3 h via fed-batch substrate addition. The nitrilase from A. insolitus could hydrolyze 630 mM iminodiacetonitrile at a fast rate, effecting 86 % conversion to iminodiacetic acid within 1 h. The arylaliphatic nitrilase from C. necator catalysed enantioselective hydrolysis of 740 mM mandelonitrile to (R)-mandelic acid in 4 h. Significantly high product yields suggest that these enzymes would be promising additions to the suite of nitrilases for upscale biocatalytic application.