Crystallization and preliminary X-ray analysis of phenol hydroxylase from Trichosporon cutaneum

OBJECTIVE: To determine the relation between dysplasia at cervical cone margins and the presence or absence of residual dysplasia in post-cone hysterectomy specimens. METHODS: We performed a 6-year retrospective, multicenter study and reviewed 250 cases in which the patient had a cold-knife cervical cone biopsy followed by a hysterectomy within 6 months. Pathology reports from 23 institutions described the margins in conization specimens and the subsequent status of residual dysplasia in the hysterectomy specimens. RESULTS: There was a statistically significant difference in the prevalence of residual dysplasia in hysterectomy specimens between patients with positive margins on cone biopsy (47%) and those with negative margins (23%) (P < .01). The positive predictive value for residual dysplasia given positive cone margins was 47%, and the negative predictive value was 77%. The grade of post-cone residual dysplasia increased commensurately with the grade of dysplasia in the conization specimen. CONCLUSIONS: The presence of dysplasia at the cervical cone margin relates significantly with the presence of residual dysplasia in the post-cone hysterectomy specimen. The grade of residual dysplasia in the post-cone hysterectomy specimen increased as the grade of dysplasia in the conization specimen increased. Free margins on a cone biopsy specimen with dysplasia offer reassurance that invasive cancer is not present in the remaining uterus.     

Crystallization and preliminary X-ray analysis of salicylate hydroxylase from Pseudomonas putida S-1

Apo-salicylate hydroxylase from Pseudomonas putida S-1 has been crystallized by the dialysis method, using ammonium sulfate as the precipitant. The crystals belong to hexagonal space group P6(2) or P6(4) with unit cell dimensions of a = b = 142.8 A and c = 63.8 A, and diffract X-rays at higher than 3.5 A resolution. A heavy-atom derivative has been prepared by soaking a crystal in an ammonium sulfate solution containing p-chloromercuriphenylsulfonate.     

Crystallization and preliminary X-ray analysis of human antithrombin III

Human antithrombin III has been crystallized from 18 to 21% (w/v) polyethylene glycol 4000 at pH 7.15. The spacegroup is P2(1) with cell parameters a = 89.8 A, b = 100.8 A, c = 70.0 A and beta = 106 degrees. The diffraction limit is 3.2 A. The asymmetric unit contains two protein molecules. Analysis of dissolved crystals for biological activity and by gel electrophoresis suggests that one protein molecule in the asymmetric unit is intact, while the other is cleaved.     

Crystallization and preliminary X-ray analysis of a truncated family A alkaline endoglucanase isolated from Bacillus sp. KSM-635

The catalytic domain of an alkaline endo-1,4-beta-glucanase (family A) isolated from Bacillus sp. KSM-635 (Mr = 40.2 kDa) was crystallized using the hanging drop vapor diffusion method. Two different crystal forms were obtained. Form 2 crystals (trigonal space group R3 with cell dimensions of a = b = 111.9 and c = 207.1 angstroms in a hexagonal lattice) were found to be more stable than form 1 ones upon X-ray irradiation. A full data set for form 2 crystals has been collected up to 3.3 angstroms resolution.     

Crystallization and preliminary X-ray diffraction studies on a soluble Escherichia coli quinone oxidoreductase

Single crystals suitable for X-ray diffraction studies have been obtained from a soluble Escherichia coli NAD(P)H-dependent quinone oxidoreductase (QOR) of molecular mass 35 kDa. Crystals were grown by the vapour diffusion method in the presence of NADPH and diffract strongly to beyond 2.0 A. The space group was determined to be P2(1)2(1)2(1) with unit cell dimensions of a = 107.44 A, b = 104.06 A, c = 77.45 A. There is one dimer in the asymmetric unit.     

Crystallization and preliminary X-ray analysis of human tissue factor extracellular domain

The extracellular domain (residues 1 to 220) of human tissue factor has been cloned and expressed in Escherichia coli and purified to isoelectric homogeneity. Single crystals suitable for X-ray analysis have been obtained by vapour diffusion. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 45.2 A, c = 231.5 A, contain one molecule per asymmetric unit and diffract to 2.6 A resolution. Native and derivative data sets have been collected to 3.6 and 3.9 A, respectively.     

Crystallization and preliminary X-ray analysis of a trimeric form of human mannose binding protein

A trimeric form of the carbohydrate recognition domain of human mannose binding protein has been crystallized in two different forms. The first form crystallizes with symmetry consistent with space group P2(1)2(1)2(1) and a = 61 A; b = 144 A; c = 107 A with presumably two trimers in the asymmetric unit. The second form crystallizes with symmetry consistent with space group P321 and a = b = 77 A; c = 58 A and one monomer per asymmetric unit. The molecular and crystallographic 3-folds must be coincident in this crystal form.     

Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties

In earlier studies we found that treatment with interferon-gamma (IFN-gamma) produced an 8- to 11-fold increase in choline acetyltransferase (ChAT) in cultured cells taken from Embryonic Day 16 (E16) septal nuclei with adjacent basal forebrain (SN/BF). Since younger cultures responded even more profoundly to IFN treatment, we have tested the possibility that the action of IFN (or its intermediate; see below) is to prompt the cholinergic differentiation of neuronal precursors. SN/BF cultures of various ages were labeled with a retrovirus engineered to express beta-galactosidase (Lac-Z), and ChAT-positive descendants of the retrovirally labeled precursors were counted. IFN-gamma treatment of cultures caused as much as an 8.8-fold increase in the proportion of ChAT-positive cells present in Lac-Z-positive clones, suggesting that IFN promoted cholinergic differentiation in precursor populations. By contrast, bFGF increased clone size but did not change the proportion of ChAT-positive cells. NGF affected neither. Only ameboid microglia present in the cultures responded to IFN with characteristic nuclear translocation of the signal transducing molecule p91, suggesting that a microglial-derived molecule may mediate the action of IFN. Consistent with this hypothesis, conditioned media from cultures of enriched, activated microglia also increased ChAT activity in a dose-dependent fashion. Conditioned media from an unstimulated macrophage/monocyte cell line (RAW 264.7) also proved extremely efficacious in raising ChAT activity. In addition, conditioned media from both activated microglia and RAW 264.7 cells increased the proportion of ChAT-positive cells in retrovirally labeled clones to the same extent as IFN itself, suggesting the possibility that they contain the molecule(s) that mediates the action of IFN. Preliminary characterization of this molecule suggests that it is a very stable and large protein. Together these data suggest that a molecule promoting cholinergic differentiation is produced by activated microglia and other macrophage-like cells. The identity of this molecule and its precise role in normal development await its further purification.     

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.     

The role of phenylalanine in structure-function relationships of phenylalanine hydroxylase revealed by radiation target analysis

The activity of rat liver phenylalanine hydroxylase (PAH; phenylalanine 4-monooxygenase, EC 1.14.16.1) is regulated by interaction with its substrate, phenylalanine, and its coenzyme, BH4 [tetrahydrobiopterin (6R-dihydroxypropyl-L-erythro-5,6,7,8-tetrahydropterin)]. The structural changes accompanying these interactions have been studied by radiation target analysis. PAH purified from rat liver was incubated with 2 mM phenylalanine to achieve complete activation of the enzyme. Frozen samples were irradiated with various doses of high energy electrons; samples were subsequently thawed, and several surviving properties of the enzyme were determined. Each parameter decreased as a single exponential function of radiation dose. Radiation target analysis of enzymatic activity yielded a dimeric target size. Similar radiation effects on subunit monomers and on tetrameric structure were observed. Together with results from unactivated enzyme, these data show that phenylalanine increases the interactions between the subunits in a dimer and weakens the interactions between dimers in a tetramer. These alterations prevent the natural cofactor, a tetrahydrobiopterin, from exerting a negative effect on activity.     

Crystallization and preliminary X-ray diffraction analysis of gingipain R2 from Porphyromonas gingivalis in complex with H-D-Phe-Phe-Arg-chloromethylketone

Gingipain R2 is a 50 kDa proteinase from the oral pathogenic bacterium Porphyromonas gingivalis. This proteinase, which displays no significant sequence homology to any protein previously analyzed by X-ray crystallography, has been crystallized using the vapor diffusion method. Two different crystal forms were obtained from a solution containing polyethylene glycol (MW 8,000) (space group P2(1)2(1)2(1)) or magnesium sulfate (space group R3) as precipitating agent. Complete diffraction data sets have been collected up to 2.0 and 2.9 A resolution, respectively. Cell dimensions are a = 51.9 A, b = 79.9 A, and c = 99.6 A (P2(1)2(1)2(1)), and a = b = 176.6 A, and c = 143.4 A (R3). Considerations of the possible values of Vm accounts for the presence of one monomer per asymmetric unit in the case of the orthorhombic crystal form, whereas the rhombohedral crystal form, together with the analysis of the self-rotation function, could accommodate a tetramer in the asymmetric unit.     

Crystallization and atomic resolution X-ray diffraction analysis of antichymotrypsin variants

Crystals of two recombinant antichymotrypsin (rACT) variants have been prepared: variant rACT-T345R crystallizes in space group P2(1) (a = 109.2 A, b = 79.4 A, c = 111.9 A, beta = 116.3 degrees, with 2 molecules in the asymmetric unit), and variant ACT' crystallizes in space group P2(1)22(1) (a = 69.7 A, b = 77.2 A, c = 83.8 A, with one molecule in the asymmetric unit). The latter variant is an engineered dimer having the P3-P3' hexapeptide sequence of the related serpin, alpha 1-proteinase inhibitor, substituted for the corresponding wild-type sequence. Crystals of each variant diffract to a limiting resolution of 2.5 A, which represents the best diffraction yet achieved for a crystalline, inhibitory serpin. The exceptional quality of ACT' crystals probably arises from favorable protein-protein interactions as well as a stabilizing disulfide crosslink engineered between the monomers.     

Large-scale purification and preliminary x-ray diffraction studies of human aspartylglucosaminidase

Selective vestibular neurectomy is an effective treatment for intractable vertigo of peripheral vestibular origin when preservation of hearing is a goal. The retrolabyrinthine and retrosigmoid-intracanalicular approaches have been used predominantly at our institutions over the last 10 years. The results and complications of these two techniques were compared. No significant differences were found between hearing results in these two patient groups. The retrosigmoid-internal auditory can approach yielded better control of recurrent episodic vertigo, as well as superior ablation of postoperative ice-water caloric responses (p < 0.05). Surgical complications, including the incidence of cerebrospinal fluid leakage (greater in retrolabyrinthine approach) and postoperative headache (more prevalent in retrosigmoid approach), were also analyzed. To further evaluate the results of this study, data were reanalyzed and compared with previously published reports of selective vestibular nerve section.     

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.     

Crystallization and preliminary X-ray diffraction studies of the soluble 14 kDa beta-galactoside-binding lectin from bovine heart

The soluble 14 kDa beta-galactoside-binding lectin from bovine heart, a member of the S-type lectin family, has been crystallized in a form suitable for X-ray diffraction analysis. The crystals, in the absence of a saccharide ligand, diffract beyond 2.5 A resolution. They are obtained from polyethylene glycol 6000 at pH 6.0. Crystals grow as monoclinic plates, space group P2(1), with cell dimensions: a = 35.47 A, b = 64.33 A, c = 58.78 A and beta = 91.7 degrees. The asymmetric unit contains two molecules related by a 2-fold non-crystallographic axis. Two lectin monomers in the asymmetric unit give a Vm of 2.4 A3/Da, i.e. a solvent content of approximately 50%. The complex of lectin with the saccharide ligand, N-acetyllactosamine, crystallizes in the space group P2(1)2(1)2 with cell dimensions: a = 63.55 A, b = 82.13 A and c = 62.39 A. Crystals of this complex diffract beyond 2.0 A resolution. Two complexes in the asymmetric unit lead to a Vm value of 2.8 A3/Da (57% solvent).     

Crystallization and preliminary X-ray investigation of human erythrocytic purine nucleoside phosphorylase

Crystals of human erythrocytic purine nucleoside phosphorylase have been grown from solutions of ammonium sulfate. The crystals are trigonal, space group R32; the hexagonal axes are a = 143.8(2) and c = 165.1(2) A. The crystals are moderately stable to x-rays and diffract beyond 3.0 A resolution. The experimental density of the crystals indicates that the molecular weight of the protein is 94,000. The three subunits are not related by crystallographic symmetry.     

Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine

The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 A, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the KM value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.     

Characterization of mutations at the mouse phenylalanine hydroxylase locus

By using a three-dimensional computed tomography (CT) scanner, we compared the anatomic features of the pelvis of three fetuses of same gestational age, one with a normal pelvis representing the reference model, one with classic bladder exstrophy, and one with cloacal exstrophy. The tomography slices were selected at the same levels for each case. Three angles expressing external opening of the pelvis were defined. Comparing normal and abnormal pelvises allowed definition of three criteria for the correction of the malformation: (a) the sum of the differential angles gives the amplitude of the correction needed; (b) a supraacetabular osteotomy appears to allow best closure of the pelvic ring; (c) only three slices of a CT scan are needed, which cannot be harmful, especially for neonates. Therefore, we believe that a CT scan of the pelvis should be performed whenever an osteotomy is planned in the surgical reconstruction of bladder and cloacal exstrophy.     

Extensive restriction site polymorphism at the human phenylalanine hydroxylase locus and application in prenatal diagnosis of phenylketonuria

A total of 10 restriction site polymorphisms have been identified at the human phenylalanine hydroxylase locus using a full-length human phenylalanine hydroxylase cDNA clone as a hybridization probe to analyze human genomic DNA. These polymorphic patterns segregate in a Mendelian fashion and concordantly with the disease state in various PKU kindreds. The frequencies of the restriction site polymorphisms at the human phenylalanine hydroxylase locus among Caucasians are such that the observed heterozygosity in the population is 87.5%. Thus, most families with a history of classical phenylketonuria can take advantage of the genetic analysis for prenatal diagnosis and carrier detection of the hereditary disorder.     

Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA

8 patients with paludism diagnosis due to Plasmodium vivax and deficiency of glucose-6-phosphate dehydrogenase that should receive antipaludism radical treatment with primaquine were studied. It was determined that 87.5% of the patients presented hemolysis but its relation with the enzymatic activity was not significant (p > 0.05). 50% of the patients could not finish their treatment because of the appearance of important hemolysis. It is concluded that primaquine should not be used indiscriminately among those patients with deficit of glucose 6 phosphate dehydrogenase.