Yong Kee Choi

Purification and characterization of UDP-glucose:tetrahydrobiopterin glucosyltransferase from Synechococcus sp. PCC 7942

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. PCC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferase producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cytosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on SDS-PAGE. The native enzyme exists as a monomer having a molecular mass of 39.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6.5 to 10.5 but most active at pH 10.0. The enzyme required Mn(2+) for maximal activity. Optimum temperature was 42 degrees C. Apparent K(m) values for BH4 and UDP-glucose were determined as 4.3 microM and 188 microM, respectively, and V(max) values were 16.1 and 15.1 pmol min(-1) mg(-1), respectively. The N-terminal amino acid sequence was Thr-Ala-His-Arg-Phe-Lys-Phe-Val-Ser-Thr-Pro-Val-Gly-, sharing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. PCC 6803, which is known to produce a pteridine glycoside cyanopterin.

Asenapine exerts distinctive regional effects on ionotropic glutamate receptor subtypes in rat brain.

Asenapine, a new pyschopharmacologic agent being developed for the treatment of schizophrenia and bipolar disorder, has a unique human receptor binding signature with strong affinity for dopaminergic, α‐adrenergic, and, in particular, serotonergic receptors raising the possibility of interactions with glutamatergic receptors. Changes in ionotropic glutamate (Glu) N‐methyl‐D‐aspartic acid (NMDA) receptors and 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl)propionic acid (AMPA) receptors in rat forebrain regions were quantified after repeated administration of multiple doses of asenapine (0.03, 0.1, or 0.3 mg/kg, subcutaneous, twice/day) or vehicle for 4 weeks. Brain sections were collected from the medial prefrontal cortex (mPFC), dorsolateral frontal cortex, caudate putamen (CPu), nucleus accumbens (NAc), and hippocampus (HIP), and processed for in vitro receptor autoradiography. Four weeks of treatment with 0.03, 0.1, or 0.3 mg/kg of asenapine significantly (P < 0.01) decreased binding of [3H]MK‐801 to NMDA/MK‐801 modulatory sites in NAc (by 27%, 29%, and 26%, respectively), medial CPu (by 25%, 28%, and 24%), and lateral CPu (by 24%, 31%, and 26%). In contrast, the same doses of asenapine did not alter binding of [3H]glycine to NMDA/glycine modulatory sites in any of the brain regions examined. [3H]AMPA binding to AMPA receptors was selectively and significantly (P < 0.001) elevated in hippocampal CA1 (41%) and CA3 (40%) regions but only at the highest dose tested. These results indicate that chronic treatment with asenapine has region‐specific and dose‐dependent effects on ionotropic Glu‐receptor subtypes in rat forebrain, which might contribute to the unique psychopharmacologic properties of asenapine. Synapse 63:413–420, 2009.

Effects of repeated risperidone exposure on serotonin receptor subtypes in developing rats

Risperidone is an atypical antipsychotic drug that is widely prescribed to young patients with different psychotic disorders. The long-term effects of this antipsychotic agent on neuronal receptors in developing brain remain unclear and require further investigation. In this study, we examined the effects of long-term treatment of risperidone on two serotonin receptor subtypes in brain regions of juvenile rat. Levels of 5-HT(1A) and 5-HT(2A) receptors in forebrain regions of juvenile rats were quantified after 3 weeks of treatment with three different doses of risperidone (0.3, 1.0 and 3.0mg/kg). Findings were compared to previously reported changes in 5-HT receptors after risperidone treatment (3.0mg/kg) in adult rat brain. The three doses of risperidone selectively and dose-dependently increased levels of 5-HT(1A) receptors in medial-prefrontal and dorsolateral-frontal cortices of juvenile animals. The higher doses (1.0 and 3.0mg/kg) of risperidone also increased 5-HT(1A) receptor binding in hippocampal CA(1) region of juvenile but not adult rats. In contrast, the three doses of risperidone significantly reduced 5-HT(2A) labeling in medial-prefrontal and dorsolateral-frontal cortices in juvenile as well as in adult animals in an equipotent fashion. 5-HT(1A) and 5-HT(2A) receptors in other forebrain regions were not altered by repeated risperidone treatment. These findings indicate that there are differential effects of risperidone on 5-HT(1A) and 5-HT(2A) receptors in juvenile animals, and that the 5-HT system in developing animals is more sensitive than adults to the long-term effects of risperidone.

Repeated effects of asenapine on adrenergic and cholinergic muscarinic receptors.

Adrenergic (alpha1 and alpha2) and cholinergic muscarinic (M1-M5) receptor binding in rat forebrain was quantified after 4 wk of twice-daily subcutaneous administration of asenapine or vehicle. Asenapine (0.03, 0.1, and 0.3 mg/kg) produced increases in [3H]prazosin binding to alpha1-adrenergic receptors in the medial prefrontal cortex (mPFC: 30%, 39%, 57%) and dorsolateral frontal cortex (DFC: 27%, 37%, 53%) and increased [3H]RX821002 binding to alpha2-adrenergic receptors in mPFC (36%, 43%, 50%) and DFC (41%, 44%, 52%). Despite showing no appreciable affinity for muscarinic receptors, asenapine produced regionally selective increases in binding of [3H]QNB to M1-M5 receptors in mPFC (26%, 31%, 43%), DFC (27%, 34%, 41%), and hippocampal CA1 (40%, 44%, 42%) and CA3 (25%, 52%, 48%) regions. These regionally selective effects of asenapine on adrenergic and cholinergic muscarinic receptor subtypes may contribute to its beneficial clinical effects in the treatment of schizophrenia and bipolar disorder.

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

The putative gene (ygcM) of Escherichia coli was verified in vitro to encode the ortholog of 6‐pyruvoyltetrahydropterin synthase (PTPS). Unexpectedly, the enzyme was found to convert sepiapterin to 7,8‐dihydropterin without any cofactors. The enzymatic product 7,8‐dihydropterin was identified by HPLC and mass spectrometry analyses, suggesting a novel activity of the enzyme to cleave the C6 side chain of sepiapterin. The optimal activity occurred at pH 6.5–7.0. The reaction rate increased up to 3.2‐fold at 60–80°C, reflecting the thermal stability of the enzyme. The reaction required no metal ion and was activated slightly by low concentrations (1–5 mM) of EDTA. The apparent K m value for sepiapterin was determined as 0.92 mM and the V max value was 151.3 nmol/min/mg. The new catalytic function of E. coli PTPS does not imply any physiological role, because sepiapterin is not an endogenous substrate of the organism. The same activity, however, was also detected in a PTPS ortholog of Synechocystis sp. PCC 6803 but not significant in Drosophila and human enzymes, suggesting that the activity may be prevalent in bacterial PTPS orthologs.

Molecular cloning and disruption of a novel gene encoding UDP‐glucose:tetrahydrobiopterin α‐glucosyltransferase in the cyanobacterium Synechococcus sp. PCC 7942

The gene encoding UDP‐glucose:tetrahydrobiopterin α‐glucosyltransferase (BGluT) was cloned from the genomic DNA of Synechococcus sp. PCC 7942. The encoded protein consisting of 359 amino acid residues was verified in vitro and in vivo to be responsible for the synthesis of tetrahydrobiopterin (BH4)‐glucoside produced in the organism. The BGluT gene is the first cloned in pteridine glycosyltransferases and also a novel one cloned so far in UDP‐glycosyltransferases. The mutant cells disrupted in the BGluT gene produced only aglycosidic BH4 at a level of 8.3% of the BH4‐glucoside in wild type cells and exhibited half of the wild type growth in normal photoautotrophic conditions. These results suggest that the glucosylation of BH4 is required for the maintenance of the high cellular concentration of the compound, thereby supporting the normal growth of Synechococcus sp. PCC 7942.

Pharmacological characterization of the norepinephrine and dopamine reuptake inhibitor EB-1020: Implications for treatment of attention-deficit hyperactivity disorder

We report on the pharmacological, behavioral, and neurochemical characterization of a novel dual norepinephrine (NE)/dopamine (DA) transporter inhibitor EB‐1020 (1R,5S)‐1‐(naphthalen‐2‐yl)‐3‐azabicyclo[3.1.0]hexane HCl). EB‐1020 preferentially inhibited monoamine reuptake in cloned cell lines transfected with human transporters with IC50 values of 6 and 38, respectively, for NE and DA transporters. In microdialysis studies, EB‐1020 markedly increased NE, and DA concentrations levels in rat prefrontal cortex in vivo with peak increases of 375 and 300%, respectively with the greatest effects on NE, and also increased DA extracellular concentrations in the striatum to 400% of baseline concentrations. Behavioral studies demonstrated that EB‐1020 dose‐dependently decreased immobility in the mouse tail suspension test of depression to 13% of control levels, and did not stimulate locomotor activity in adult rats in the optimal dose range. EB‐1020 dose‐dependently inhibited locomotor hyperactivity in juvenile rats lesioned with the neurotoxin 6‐hydroxydopamine (100 μg intracisternally) as neonates; a well‐established animal model for attention‐deficit hyperactivity disorder (ADHD). These data suggest that EB‐1020 mediates its actions by stimulating NE and DA neurotransmission, which are typically impaired in ADHD. Synapse, 2012.

Production of Sepiapterin in Escherichia coli by Coexpression of Cyanobacterial GTP Cyclohydrolase I and Human 6-Pyruvoyltetrahydropterin Synthase

ABSTRACT Synechocystis sp. strain PCC 6803 GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin synthase were coexpressed in Escherichia coli. The E. coli transformant produced sepiapterin, which was identified by high-performance liquid chromatography and enzymatically converted to dihydrobiopterin by sepiapterin reductase. Aldose reductase, another indispensable enzyme for sepiapterin production, may be endogenous in E. coli.

Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH4) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH4 product, l-threo-BH4 (6R-(1′S,2′S)-5,6,7,8-BH4), whereas animal enzymes produce l-erythro-BH4 (6R-(1′R,2′S)-5,6,7,8-BH4) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 Å resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180° around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.

Functional investigation of a gene encoding pteridine glycosyltransferase for cyanopterin synthesis in Synechocystis sp. PCC 6803.

A gene (slr1166) putatively encoding pteridine glycosyltransferase was disrupted with a kanamycin resistance cassette in Synechocystis sp. PCC 6803, which produces cyanopterin. The deduced polypeptide from slr1166 consisted of 354 amino acid residues sharing 45% sequence identity with UDP-glucose:tetrahydrobiopterin alpha-glucosyltransferase (BGluT) isolated previously from Synechococcus sp. PCC 7942. The knockout mutant was unable to produce cyanopterin but only 6-hydroxymethylpterin-beta-galactoside, verifying that slr1166 encodes a pteridine glycosyltransferase, which is responsible for transfer of the second sugar glucuronic acid in cyanopterin synthesis. The mutant was affected in its intracellular pteridine content and growth rate, which were 74% and 80%, respectively, of wild type, demonstrating that the second sugar residue is still required for quantitative maintenance of cyanopterin. This supports the previous suggestion that glycosylation may contribute to high cellular concentration of pteridine compounds.