Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling.

PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3). Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE-Trisacryl. The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor. 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (K(i) approximately 38 --> 3 nM) against the pterin substrate and both NADP(+) and folate are inhibitors although somewhat weaker. Dihydropteridines are poor substrates compared to the fully oxidized pteridine. Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP(+) allows the formation of ternary nonproductive complexes with folate. The kinetic results are consistent with a sequential ordered bi-bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme. Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)(3)K motif. In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity. Two Y(Xaa)(3)K regions are present in wild-type PTR1 at 152 and 194. Only Y194F gives protein with zero activity. This observation coupled with affinity labeling of PTR1 by oNADP(+) (2', 3'-dialdehyde derivative of NADP(+)) followed by NaBH(4) reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194. The mutation K198Q eliminates inactivation by oNADP(+) supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family.

Formation of a novel four-helix bundle and molecular recognition sites by dimerization of a response regulator phosphotransferase.

A basis for understanding specificity of molecular recognition between phosphorelay proteins has been deduced from the 2.6 A structure of the Spo0B phosphotransferase of the phosphorelay regulating sporulation initiation. Spo0B consists of two domains: an N-terminal alpha-helical hairpin domain and a C-terminal alpha/beta domain. Two subunits of Spo0B dimerize by a parallel association of helical hairpins to form a novel four-helix bundle from which the active histidine protrudes. Docking studies show that both the monomers interact with a Spo0F molecule at the region surrounding the active site aspartate to position it for phosphotransfer. It is apparent that different surfaces of response regulators may be involved in recognition of the protein partners to which they are paired.

Antibacterial agents that inhibit two-component signal transduction systems.

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA::Spo0F two-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase::response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.

A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site.

Two-component signaling systems are used by bacteria, plants, and lower eukaryotes to adapt to environmental changes. The first component, a protein kinase, responds to a signal by phosphorylating the second component; a response regulator protein that often acts by inducing the expression of specific genes. Response regulators also have an autophosphatase activity that ensures that the proteins are not permanently activated by phosphorylation. The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecular features responsible for this varied autophosphatase activity have not been clearly defined. Using wild-type and mutant derivatives of the sporulation response regulator Spo0F, it has been demonstrated that a key residue in determining the magnitude of this activity is that at position 56 of Spo0F approximately P; this residue is adjacent to the site of phosphorylation, Asp 54. For example, Spo0F approximately P K56N has a 23-fold greater autophosphatase activity (t1/2 = 8 min) than wild-type Spo0F approximately P (t1/2 = 180 min). It is suggested that, by analogy to the GTPase activity of p21(ras) and by examining the crystallographic structure of Spo0F, that the carboxyamide of the mutant Asn 56 may favorably position a catalytic water near the protein acyl phosphate to promote Spo0F approximately P K56N hydrolysis. It is also deduced that Lys 56 in the wild-type protein is critical for the efficient interaction and phosphoryl transfer between Spo0F and it's cognate protein kinase, KinA. Comparison of the known response regulators shows that inefficient autophosphatases (t1/2 on the order of hours) typically contain an amino acid residue with a long side chain at the position equivalent to 56 in Spo0F, whereas efficient autophosphatases (t1/2 on the order of minutes) frequently contain a residue with a carboxyamide or carboxylate side chain at this position. It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match the diverse biological roles played by these proteins.

Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis.

The four individual phosphotransfer steps in the multicomponent phosphorelay system controlling sporulation in Bacillus subtilis have been characterized kinetically using highly purified samples of the individual protein components in vitro. The autophosphorylation of KinA is the initial occurrence, and a divalent metal ion is required. KinA-mediated phosphotransfer, which displays a 57,000-fold preference (kcat/Km) for catalysis of Spo0F-P formation relative to Spo0A-P formation, is shown to proceed via a hybrid ping-pong/sequential mechanism with pronounced (> or = 40-fold) substrate synergism by Spo0F of KinA autophosphorylation. In addition, evidence is presented for formation of an abortive KinA.Spo0F complex. Kinetic parameters derived for Spo0F-P and Spo0A as substrates for Spo0B, the second phosphotransferase in the phosphorelay chain, indicate that Spo0B-mediated production of Spo0A-P is 1.1-million-fold more efficient (kcat/KSpo0A) than the direct KinA-mediated process. A rationale is presented for a four component cascade as the means for controlling sporulation, which focuses on the utility of synergistic interactions among the phosphorelay components that may be modulated by environmental stimuli.

A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: crystal structure of Spo0F from Bacillus subtilis.

Spo0F is a secondary messenger in the "two-component" system controlling the sporulation of Bacillus subtilis. Spo0F, like the chemotaxis protein CheY, is a single-domain protein homologous to the N-terminal activator domain of the response regulators. We recently reported the crystal structure of a phosphatase-resistant mutant Y13S of Spo0F with Ca2+ bound in the active site. The crystal structure of wild-type Spo0F in the absence of a metal ion is presented here. A comparison of the two structures reveals that the cation induces significant changes in the active site. In the present wild-type structure, the carboxylate of Asp11 points away from the center of the active site, whereas when coordinated to the Ca2+, as in the earlier structure, it points toward the active site. In addition, Asp54, the site of phosphorylation, is blocked by a salt bridge interaction of an Arg side chain from a neighboring molecule. From fluorescence quenching studies with Spo0F Y13W, we found that only the amino acid Arg binds to Spo0F in a saturable manner (Kd = 15 mM). This observation suggests that a small molecule with a shape complementary to the active site and having a guanidinium group might inhibit phosphotransfer between response regulators and their cognate histidine kinases.

High-resolution NMR structure and backbone dynamics of the Bacillus subtilis response regulator, Spo0F: implications for phosphorylation and molecular recognition.

NMR has been employed for structural and dynamic studies of the bacterial response regulator, Spo0F. This 124-residue protein is an essential component of the sporulation phosphorelay signal transduction pathway in Bacillus subtilis. Three-dimensional 1H, 15N, and 13C experiments have been used to obtain full side chain assignments and the 1511 distance, 121 dihedral angle, and 80 hydrogen bonding restraints required for generating a family of structures (14 restraints per residue). The structures give a well-defined (alpha/beta)5 fold for residues 4-120 with average rms deviations of 0.59 A for backbone heavy atoms and 1.02 A for all heavy atoms. Analyses of backbone 15N relaxation measurements demonstrate relative rigidity in most regions of regular secondary structure with a generalized order parameter (S2) of 0.9 +/- 0.05 and a rotational correlation time (taum) of 7.0 +/- 0.5 ns. Loop regions near the site of phosphorylation have higher than average rms deviation values and T1/T2 ratios suggesting significant internal motion or chemical exchange at these sites. Additionally, multiple conformers are observed for the beta4-alpha4 loop and beta-strand 5 region. These conformers may be related to structural changes associated with phosphorylation and also indicative of the propensity this recognition surface has for differential protein interactions. Comparison of Spo0F structural features to those of other response regulators reveals subtle differences in the orientations of secondary structure in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation. These may be important in providing specificity for protein-protein interactions and for determining the lifetimes of the phosphorylated state.

The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases.

Kinetic parameters and primary deuterium isotope effects have been determined for wild-type dihydropteridine reductase (EC 1.6.99.7) and the Ala133Ser, Lys150Gln, Tyr146His, Tyr146Phe single, and Tyr146Phe/Ala133Ser and Tyr146Phe/Lys150Gln double mutant enzyme forms using the natural substrate, quinonoid (6R)-l-erythro-dihydrobiopterin (qBH2) and an alternate substrate, quinonoid 6,7-dimethyldihydropteridine (q-6,7-diMePtH2). Mutation at either Tyr146 or Lys150 resulted in pronounced changes in kinetic parameters and isotope effects for both pterin substrates, confirming a critical role for these residues in enzyme-mediated hydride transfer. By contrast, the Ala133Ser mutant was practically indistinguishable from wild-type enzyme. The changes observed, however, were quite different for the two pterin substrates. Thus, kcat for q-6,7-diMePtH2 decreased across the series of mutants from a value of 150 s-1 for wild-type enzyme to essentially zero activity for the Tyr146Phe/Lys150Gln double mutant. Conversely, kcat for qBH2 increased 3-11-fold across the same series of mutants from the wild-type value of 23 s-1. For both pterin substrates, the Km (KPt) increased several orders of magnitude upon mutation of Tyr146 or Lys150, with the greater relative increase using qBH2. Significant primary deuterium isotope effects on kcat (Dkcat) and kcat/KPt (D(kcat/KPt)) observed for the Tyr146 and Lys150 mutants varied depending on the pterin substrate used and ranged up to a maximum value of 5.5-6. For qBH2, where Dkcat < Dkcat/KPt was consistently observed, the rate determining step is ascribed to release of the tetrahydropterin product. For q-6,7-diMePtH2, where in all cases Dkcat = Dkcat/KPt, catalysis is probably limited by an isomerization step occurring prior to hydride transfer. Modeling studies in which qBH2 was docked into the binary E:NADH complex provide a structural rationale for the observed differences between the two pterin substrates. The natural substrate, qBH2, displays a higher affinity for the enzyme active site, presumably due to interaction of the dihydroxypropyl side chain of the substrate with a polar loop of residues containing Asn186, Ser189, and Met190. The location of this loop within the three-dimensional structure is consistent with putative substrate binding loops for other members of the short chain dehydrogenase/reductase (SDR) family, which includes dihydropteridine reductase.

Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1.

Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in the protozoan parasite Leishmania. Genetic analysis has previously implicated PTR1 in pterin and folate metabolism. PTR1 was purified from a fusion protein expressed in Escherichia coli. Purified PTR1 exhibits NADPH-dependent biopterin, dihydrobiopterin, folate, and dihydrofolate reductase activities. The highest activity was found with the most oxidized pterins. The active protein was found to be a tetramer as demonstrated by gel-filtration chromatography. Kinetic constants (K(m)), as determined by double-reciprocal plots, were calculated for NADPH and for several of PTR1's substrates. The PTR1 of Leishmania tarentolae had a K(m) of 16.9 microM for the cofactor NADPH and K(m) values ranging from 3.5 to 85 microM for the various substrates. The dissociation constant (KD), as determined by fluorescence titration, for NADPH was estimated to be 130 microM. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism of Leishmania should be useful for structure-function analysis and for developing specific inhibitors against this putative important chemotherapeutic target.

Purification and preliminary crystallographic studies on the sporulation response regulatory phosphotransferase protein, Spo0B, from Bacillus subtilis.

The phosphotransferase protein Spo0B, a component of the sporulation signal transduction system in Bacillus subtilis was expressed from the Escherichia coli strain BL21DE3. It was purified, crystallized, and 2.25 A data measured using the synchrotron source at the Stanford Linear Accelerator Center. The search for heavy atom derivatives is in progress.

Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

Crystallization and preliminary X-ray analysis of a Y13S mutant of Spo0F from Bacillus subtilis.

Spo0F, a member of a superfamily of bacterial response regulatory proteins, is crucial to the regulation of sporulation in Bacillus subtilis. As there were difficulties in reproducing crystals of wild-type Spo0F, we report here the crystallization and preliminary studies of a mutant, Y13S protein, which gave well diffracting reproducible crystals. The crystals of the mutant obtained by the hanging-drop method belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2) a = b = 105.1, c = 85.9 A. Diffraction data were collected at 2.8 A at the laboratory source and subsequently 2.05. A data were collected upon flash freezing the crystal at the Stanford Synchrotron Radiation Laboratory. This mutant participates in the phosphorelay in a similar manner to the wild-type protein. The presence of divalent cations are essential for wild-type phosphorylation and the present mutant crystal form is obtained in the presence of calcium.

A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates.

Transient phosphorylation at an aspartate residue on the Spo0F protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli. The response regulator Spo0F-P is stable to hydrolysis (t1/2 > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated forms of Spo0F. Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate Spo0F, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors. Rapid hydrolysis of Spo0F-P generated with phosphoramidate by proteins downstream in the phosphorelay (Spo0B and Spo0A) is consistent with phosphorylation at the active site of Spo0F. The initial rate of Spo0F-P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as Spo0F, function as phosphoryl transferase enzymes (McCleary et al., 1993). The results establish that Spo0F functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation.

Altered structural and mechanistic properties of mutant dihydropteridine reductases.

Nine single genetic mutants of rat dihydropteridine reductase (EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position.

1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.

Crystal structure of a monoclinic form of dihydropteridine reductase from rat liver.

A binary complex of dihydropteridine reductase and NADH crystallizes in the space group C2, with a = 222.2, b = 46.5, c = 95.3 A and beta = 101.1 degrees. There are two dimers in the asymmetric unit. The structure was solved by molecular-replacement techniques and refined with 2.6 A data to a crystallographic R factor of 16.8%. Each dimer has twofold non-crystallographic symmetry and the four individual monomers in the asymmetric unit have the same overall molecular conformation.

Structural and mechanistic implications of incorporating naturally occurring aberrant mutations of human dihydropteridine reductase into a rat model.

Phenylketonuria (PKU) is a debilitating hereditary disorder related to an individual's inability to convert phenylalanine to its usual tyrosine product. The genetic errors occur in three regions: in the cooperative enzymes phenylalanine hydroxylase (PAH) and dihydropteridine reductase (DHPR), and in the biosynthetic pathway from GTP to the hydroxylation cofactor, tetrahydrobiopterin (BH4). Many instances of naturally occurring defects in DHPR metabolism have been identified, and in most cases the error has been equated with an altered enzyme gene sequence. Using computer graphics, this report analyses the altered structural characteristics of eight of the enzymes encoded by mutant gene sequence and provides logical explanations for their diminished enzyme activities. In one instance, that of a threonine insertion, a mutant construct of the rat analog has been expressed in Escherichia coli and the DHPR isolated and characterised, confirming the marked changes this insert can create.

Structural and mechanistic characteristics of dihydropteridine reductase: a member of the Tyr-(Xaa)3-Lys-containing family of reductases and dehydrogenases.

Dihydropteridine reductase (EC 1.6.99.7) is a member of the recently identified family of proteins known as short-chain dehydrogenases. When the x-ray structure of dihydropteridine reductase is correlated with conserved amino acid sequences characteristic of this enzyme class, two important common structural regions can be identified. One is close to the protein N terminus and serves as the cofactor binding site, while a second conserved feature makes up the inner surface of an alpha-helix in which a tyrosine side chain is positioned in close proximity to a lysine residue four residues downstream in the sequence. The main function of this Tyr-Lys couple may be to facilitate tyrosine hydroxyl group participation in proton transfer. Thus, it appears that there is a distinctive common mechanism for this group of short-chain or pyridine dinucleotide-dependent oxidoreductases that is different from their higher molecular weight counterparts.

Subunit composition and domain structure of the Spo0A sporulation transcription factor of Bacillus subtilis.

The Spo0A transcription factor is responsible for the initiation of sporulation and is active in transcription only after phosphorylation by a specific signal transduction pathway, the phosphorelay. The effect of phosphorylation on the physical properties of Spo0A was determined. Spo0A and Spo0A approximately P both behaved as monomers during Sephacryl chromatography and gel electrophoresis, suggesting that phosphorylation did not modify the oligomerization state of the protein. Trypsin digested Spo0A at a single cleavage site between residues 142 and 143 within a hinge connecting two tightly folded domains. The amino domain retains ability to be phosphorylated by the phosphorelay. The carboxyl domain is active as a DNA-binding protein and retains the sequence specificity of the intact molecule for 0A boxes on the abrB promoter as revealed by footprinting studies. The carboxyl domain stimulated in vitro transcription from the spoIIG promoter 5-fold greater than an equal amount of Spo0A and about half as well as equivalent amounts of Spo0A approximately P. Thus, the unphosphorylated amino domain inhibits the transcription stimulation activity of the carboxyl domain. We suggest that phosphorylation activates transcription regulation functions of Spo0A by modifying the spatial relationships of the amino and carboxyl domains.