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.     

Crystal structure of tyrosine hydroxylase with bound cofactor analogue and iron at 2.3 A resolution: self-hydroxylation of Phe300 and the pterin-binding site

TyrOH is a non-heme iron enzyme which uses molecular oxygen to hydroxylate tyrosine to form L-dihydroxyphenylalanine (L-DOPA), and tetrahydrobiopterin to form 4a-hydroxybiopterin, in the rate-limiting step of the catecholamine biosynthetic pathway. The 2.3 A crystal structure of the catalytic and tetramerization domains of rat tyrosine hydroxylase (TyrOH) in the presence of the cofactor analogue 7,8-dihydrobiopterin and iron shows the mode of pterin binding and the proximity of its hydroxylated 4a carbon to the required iron. The pterin binds on one face of the large active-site cleft, forming an aromatic pi-stacking interaction with Phe300. This phenylalanine residue of TyrOH is found to be hydroxylated in the meta position, most likely through an autocatalytic process, and to consequently form a hydrogen bond to the main-chain carbonyl of Gln310 which anchors Phe300 in the active site. The bound pterin forms hydrogen bonds from N-8 to the main-chain carbonyl of Leu295, from O-4 to Tyr371 and Glu376, from the C-1' OH to the main-chain amides of Leu294 and Leu295, and from the C-2' hydroxyl to an iron-coordinating water. The part of the pterin closest to the iron is the O-4 carbonyl oxygen at a distance of 3.6 A. The iron is 5.6 A from the pterin 4a carbon which is hydroxylated in the enzymatic reaction. No structural changes are observed between the pterin bound and the nonliganded enzyme. On the basis of these structures, molecular oxygen could bind in a bridging position optimally between the pterin C-4a and iron atom prior to substrate hydroxylation. This structure represents the first report of close interactions between pterin and iron in an enzyme active site.     

Elevated interleukin-12 in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy

The technique of pulse radiolysis has been used to investigate the possibility of intramolecular charge transfer in the dipeptide histidyltyrosine, following one-electron oxidation of one of its amino acid residues. The radical anion, Br2.- was found to react with the dipeptide at pH 6.0 with a bimolecular rate constant of 2.3+/-0.2 x 10(7) dm3 mol(-1)s(-1) suggesting that it reacts very selectively with the histidine moiety. Spectral observations at, or close to the end of this reaction show only the presence of a tyrosinyl free radical (TyrO.), however, indicating that fast (>10(6) s(-1) intramolecular charge transfer has taken place between histidine radicals (His+.) and tyrosine (TyrOH). This finding was supported by the direct observation of the rate of formation of TyrO. in experiments with the free amino acids, histidine and tyrosine, under conditions where Br2.- reacted selectively with histidine. The bimolecular rate constant for the reaction between His+. and TyrOH was found to be 2.4+/-0.5 x 10(6) dm3 mol(-1)s(-1). Taken together, the results of the study indicate that His+. is a relatively strong oxidising agent where (E (His+./His) > 770 mV at pH 6.0.     

Mosquito-Plasmodium interactions in response to immune activation of the vector

A molecular model was built for human lecithin:cholesterol acyltransferase (LCAT) based upon the structural homology between this enzyme and lipases (Peelman et al. 1998. Prot. Sci. 7: 585-597). We proposed that LCAT belongs to the alpha/beta hydrolase fold family, and that the central domain of LCAT consists of a mixed seven-stranded beta-pleated sheet with four alpha-helices and loops linking the beta-strands. The catalytic triad of LCAT was identified as Asp345 and His377, as well as Ser181. This model is used here for the interpretation of the structural defects linked to the point mutations identified in LCAT, which cause either familial LCAT deficiency (FLD) or fish-eye disease (FED). We show that these mutations occur in separate domains of the 3D structure of the enzyme. Most mutations causing familial LCAT deficiency are either clustered in the vicinity of the catalytic triad or affect conserved structural elements in LCAT. Most mutations causing fish-eye disease are localized on the outer hydrophilic surface of the amphipathic helical segments. These mutations affect only minimally the overall structure of the enzyme, but are likely to impair the interaction of the enzyme with its co-factor and/or substrate.     

Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase from Escherichia coli

The amino acid residues involved in the metal-binding site in the iron-containing dehydrogenase family were characterized by the site-directed mutagenesis of selected candidate residues of propanediol oxidoreductase from Escherichia coli. Based on the findings that mutations H263R, H267A and H277A resulted in iron-deficient propanediol oxidoreductases without catalytic activity, we identified three conserved His residues as iron ligands, which also bind zinc. The Cys362, a residue highly conserved among these dehydrogenases, was considered another possible ligand by comparison with the sequences of the medium-chain dehydrogenases. Mutation of Cys362 to Ile, resulted in an active enzyme that was still able to bind iron, with minor changes in the Km values and decreased thermal stability. Furthermore, in an attempt to produce an enzyme specific only for the zinc ion, three mutations were designed to mimic the catalytic zinc-binding site of the medium-chain dehydrogenases: (1) V262C produced an enzyme with altered kinetic parameters which nevertheless retained a significant ability to bind both metals, (2) the double mutant V262C-M265D was inactive and too unstable to allow purification, and (3) the insertion of a cysteine at position 263 resulted in a catalytically inactive enzyme without iron-binding capacity, while retaining the ability to bind zinc. This mutation could represent a conceivable model of one of the steps in the evolution from iron to zinc-dependent dehydrogenases.     

Mutation of the distal arginine in Coprinus cinereus peroxidase--structural implications

Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron. Spectroscopic techniques, in contrast to X-ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment. Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0. This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51-->Leu, Asn, Gln, and Lys, respectively). These mutations cause changes in the coordination and spin states of the heme iron, and in the v(Fe-Im) stretching frequency. The variations are explained in terms of pH-dependent changes, charge location, size and hydrogen-bonding acceptor/donor properties of the residue at position 51. The present work indicates that the hydrogen-bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.     

Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease

Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic L-amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic L-amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic L-amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. L-DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic L-amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic L-amino acid decarboxylase produced less L-DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.     

Novel mutations of the glutaryl-CoA dehydrogenase gene in two Japanese patients with glutaric aciduria type I

We identified three different point mutations in the glutaryl-CoA dehydrogenase (GCDH) gene in two unrelated Japanese patients with glutaric aciduria type I (GA-I). One patient was a homozygote for Arg355His and the other a compound heterozygote for Ser305Leu and Met339Val. Arg355His and Met339Val are mutations hitherto undescribed, and all three mutations are predicted to alter the secondary structure of GCDH. Molecular analysis is useful for definite diagnosis and/or prenatal diagnosis of GA-I.     

Three-dimensional stereotactic posterior ischiorectal space computerized tomography guided brachytherapy of prostate cancer: a preliminary report

We reported recently that protein D2 (OprD) porin of Pseudomonas aeruginosa bears protease activity (FEBS Letters 394, 179-182, 1996). To identify the catalytic residues of OprD, we introduced the site-directed mutations replacing the putative catalytic triad His156, Asp208, and Ser296 with glutamine, asparagine, and alanine, respectively. The OprD proteins purified from the chromosomal oprD-deficient mutants harboring the plasmids encoding the site-directed mutations showed protease activity less than 0.1% of that of the wild-type OprD. These site-directed mutageneses caused undetectable changes in the pore-forming activity of OprD as measured by single-channel conductance by the planar lipid bilayer. The minimum inhibitory concentration of imipenem in mutants having the replaced catalytic triads was identical with that in the wild-type strain. On the other hand, introduction of the mutation at His367 replacing with glutamine, the site that is supposed to be unrelated to the catalytic sites, showed the unchanged protease activity. These results unequivocally demonstrate that OprD is the protease bearing porin and catalyzes the reaction at His156, Asp208, and Ser296 residues.     

Tyrosine hydroxylase and Parkinson's disease

A consistent neurochemical abnormality in Parkinson's disease (PD) is degeneration of dopaminergic neurons in substantia nigra, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation of L-DOPA, the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Similarly, some patients with hereditary L-DOPA-responsive dystonia, a neurological disorder with clinical similarities to PD, have mutations in the TH gene and decreased TH activity and/or stability. Thus, a logical and efficient treatment strategy for PD is based on correcting or bypassing the enzyme deficiency by treatment with L-DOPA, DA agonists, inhibitors of DA metabolism, or brain grafts with cells expressing TH. A direct pathogenetic role of TH has also been suggested, as the enzyme is a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury. Recently, it has been demonstrated that L-DOPA is effectively oxidized by mammalian TH in vitro, possibly contributing to the cytotoxic effects of DOPA. This enzyme may therefore be involved in the pathogenesis of PD at several different levels, in addition to being a promising candidate for developing new treatments of this disease.     

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.     

Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis-- X-ray structure analysis of site-directed mutants

We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 A of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 A and 2.5 A, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.     

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter

To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.     

Mutational analyses support a model for the HRV2 2A proteinase

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity. A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase. The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular ("cis") and intermolecular ("trans") activities. The HRV2 proteinase 2A is proposed to have a two domain structure, with the catalytic site and substrate binding region on one face of the molecule and a Zn-binding motif on the opposite face. Residues Gly 123, Gly 124, Thr 121, and Cys 101 are proposed to be involved in the architecture of the substrate binding pocket and to provide the correct environment for the catalytic triad of His 18, Asp 35, and Cys 106. Residues Tyr 85 and Tyr 86 are thought to participate in substrate recognition. The presence of an extensive C-terminal helix, in which Asp 132, Arg 134, Phe 130, and Phe 136 play important roles, explains why mutations in this region are generally detrimental to proteinase activity. The proposed Zn-binding motif comprises Cys 52, Cys 54, Cys 112, and His 114. Exchange of these residues inactivates the enzyme. Furthermore, as measured by atom emission spectroscopy, Zn was absent from purified preparations of proteinase 2A in which His 114 had been replaced by Asn. The absence of disulphide bridges was confirmed by subjecting highly purified HRV2 proteinase 2A to one- and two-step alkylation procedures.     

Domain flexibility in retroviral proteases: structural implications for drug resistant mutations

Rigid body rotation of five domains and movements within their interfacial joints provide a rational context for understanding why HIV protease mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites. Domain motions associated with substrate binding in the retroviral HIV-1 and SIV proteases are identified and characterized. These motions are in addition to closure of the flaps and result from rotations of approximately 6-7 degrees at primarily hydrophobic interfaces. A crystal structure of unliganded SIV protease (incorporating the point mutation Ser 4 His to stabilize the protease against autolysis) was determined to 2.0 A resolution in a new space group, P3221. The structure is in the most "open" conformation of any retroviral protease so far examined, with six residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by difference distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompassing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes, mutations at interdomain interfaces that favor the unliganded form of the target active site will increase the off-rate of the inhibitor, allowing the substrate greater access for catalysis. This offers a mechanism of resistance to competitive inhibitors, especially when the forward enzymatic reaction rate exceeds the rate of substrate dissociation.     

Contribution to activity of histidine-aromatic, amide-aromatic, and aromatic-aromatic interactions in the extended catalytic site of cysteine proteinases

Within the papain family of cysteine proteinases few other residues in addition to the catalytic triad, Cys25-His159-Asn175 (papain numbering) are completely conserved [Berti & Storer (1995) J. Mol. Biol. 246, 273-283]. One such residue is tryptophan 177 which participates in a Trp-His-type interaction with the catalytic His159. In all enzymes of this class for which a three-dimensional structure has been reported, an additional highly conserved tryptophan, Trp181, also interacts with Trp177 via an aromatic-aromatic interaction in which the planes of the indole rings are essentially perpendicular. Also, both indole rings participate as pseudo-hydrogen bond acceptors in interactions with the two side chain amide protons of Asn175. Clearly, the proximity of Trp177 and Trp181 to the catalytic triad residues His159 and Asn175 and their network of interactions points to potential contributions of these aromatic residues to catalysis. In this paper, using cathepsin S, a naturally occurring variant that has a phenylalanine residue at position 181, we report the kinetic characterization of mutants of residues 175, 177, and 181. The results are interpreted in terms of the side chain contributions to catalytic activity and thiolate-imidazolium ion-pair stability. For example, the side chain of Asn175 has a major influence on the ion-pair stability presumably through its hydrogen bond to His159. The magnitude of this effect is modulated by Trp177, which shields the His159-Asn175 hydrogen bond from solvent. The His159-Trp177 interaction also contributes significantly to ion-pair stability; however, Trp181 and its interactions with Asn175 and Trp177 do not influence ion-pair stability to a significant degree. The observation that certain mutations at positions 177 and 181 result in a reduction of kcat/Km but do not appear to influence ion-pair stability probably reflects the contributions of these residues to substrate binding.     

Comparative study of the catalytic domain of phosphorylating glyceraldehyde-3-phosphate dehydrogenases from bacteria and archaea via essential cysteine probes and site-directed mutagenesis

Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to form 1.3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. Two essential aminoacids, Cys149 and His176 are involved in the chemical mechanism of bacterial and eukaryotic GraP-DHs. Roles have been assigned to the His176 as (a) a chemical activator for enhancing the reactivity of Cys149, (b) a stabilizator of the tetrahedral transition states, and (c) a base catalyst facilitating hydride transfer towards NAD. In a previous study carried out on Escherichia coli GraP-DH [Soukri, A., Mougin, A., Corbier, C., Wonacott, A. J., Branlant, C. & Branlant, G. (1989) Biochemistry, 28, 2586-2592], the role of His176 as an activator of the reactivity of Cys149 was studied. Here, we further investigated the role of the His residue in the chemical mechanism of phosphorylating GraP-DH from E. coli and Bacillus stearothermophilus. The chemical reactivity of Cys149 in the His176Asn mutant was reinvestigated. At neutral pH, its reactivity was shown to be at least as high as that observed in the Cys-/His+ ion pair present in the wild type. No pre-steady state burst of NADH was found with the His176Asn mutant in contrast to what is observed for the wild type, and a primary isotope effect was observed when D-[1-2H]glyceraldehyde-3-phosphate was used as the substrate. Therefore, the major role of the His176 in the catalytic mechanism under physiological conditions is not to activate the nucleophilicity of Cys149 but first to facilitate the hydride transfer. These results hypothesized that a phosphorylating GraP-DH possessing a different protein environment competent to increase the nucleophilic character of the essential Cys residue and to favor the hydride transfer in place of His, could be enzymically efficient. This is most likely the case for archaeal Methanothermus fervidus GraP-DH which shares less than 15% amino-acid identity with the bacterial or eukaryotic counterparts. No Cys-/His+ ion pair was detectable. Only one thiolate entity was observed with an apparent pKa of 6.2. This result was confirmed by the fact that none of the mutations of the five invariant His changed the catalytic efficiency.     

Neurochemical characterization of the alterations in the noradrenergic afferents to the cerebellum of adult rats exposed to X-irradiation at birth

A single dose of x-irradiation was applied on the cephalic end of newborn rats, and the alterations in the noradrenergic afferents to the cerebellum were studied 180 days later. A net increase in the noradrenaline content of cerebellum was found (122% of nonirradiated controls). The response of noradrenaline content to reserpine injection (0.9 mg/kg, i.p.) was similar in exposed and control rats. Likewise, the 3H release induced by Ro 4-1284 from cerebellar cortex slices labeled with [3H]noradrenaline was unmodified by x-rays, although a mild increase in the spontaneous efflux of 3H was found. The retention of 3H by the slices was reduced in exposed animals (58% of controls). Both the in vitro activity of tyrosine hydroxylase and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) were not significantly different between x-treated rats and controls. In contrast, monoamine oxidase activity was markedly reduced in x-irradiated cerebellum (38% of controls). The x-ray-induced decrease in cerebellar weight (-60%) resulted in marked increases in noradrenaline concentration (223%), tyrosine hydroxylase activity per milligram of protein (206%), and 3H retention (50%). The accumulation of L-DOPA per gram of tissue was also increased at every time considered. These data indicate that x-irradiation at birth produces a cerebellar loss not completely shared by the noradrenergic afferents, and a permanent imbalance between the noradrenergic afferent input and its target cells might eventually result.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease

To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.     

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.