Biomedical subjects

Yuan-Wei Zhang

Publications and source records attributed to Yuan-Wei Zhang.

10 recordsLinked to original sources

PubMed2006

Direct observation of substrate-enzyme complexation by surface forces measurement.

The substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM) and a quartz crystal microbalance (QCM) in order to obtain new insights into the molecular mechanism of the enzyme reaction. This enzyme is composed of two dissociable subunits that exhibit a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). The QCM measurement revealed that FPP was preferentially bound to subunit II in the presence of Mg2+, while the AFM measurement showed that the adhesive force between the subunits was observed only in the presence of both Mg2+ and FPP. This is the first direct demonstration of the specific interaction involved in the enzyme reaction. The dependence of the Mg2+ concentration on the specific interaction between subunits I and II well agreed with that on the enzyme activity of heptaprenyl diphosphate synthase. This indicated that the observed adhesive forces were indeed involved in the catalytic reaction of this enzyme. On the basis of these results, we discussed the processes involved in the substrate-enzyme complexation. The first, the substrate FPP bound to subunit II using Mg2+, followed by the formation of the subunit I-FPP-Mg2+-subunit II complex. Our study showed a very useful methodology for examining the elemental processes of biological reactions such as an enzyme reaction.

Alkyl and Aryl Transferases↗

PubMed2006

The cytoplasmic substrate permeation pathway of serotonin transporter.

Serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin (5-HT) and is a target for antidepressant drugs and psychostimulants. It is a member of a large family of neurotransmitter and amino acid transporters. A recent study using site-directed cysteine modification identified a helical region of the transporter with high accessibility to the cytoplasm. Subsequently, the high resolution structure of LeuT, a prokaryotic homologue, showed that the residues corresponding to this helical region are part of the fifth transmembrane domain. The accessibility of these positions is now shown to depend on conformational changes corresponding to interconversion of SERT between two forms that face the extracellular medium and the cytoplasm, respectively. Binding of the extracellular inhibitor cocaine decreased accessibility at these positions, whereas 5-HT, the transported substrate, increased it. The effect of 5-HT required the simultaneous presence of Na+ and Cl-, which are transported into the cell together (symported) with 5-HT. In light of the LeuT structure, these results begin to define the pathway through which 5-HT diffuses between its binding site and the cytoplasm. They also confirm a prediction of the alternating access model for transport, namely, that all symported substrates must bind together before translocation.

Animals↗

PubMed2005

Serotonin transporter mutations associated with obsessive-compulsive disorder and phosphorylation alter binding affinity for inhibitors.

Mutants of serotonin transporter that are altered in their regulation by cGMP were tested for the ability of cocaine and the antidepressant drugs imipramine, sertraline, citalopram and fluoxetine to inhibit serotonin transport. Mutation at Ile-425 to valine, found in some patients with obsessive-compulsive disorder, altered the response of SERT to cGMP (Kilic, F., Murphy, D.L., Rudnick, G., 2003. A human serotonin transporter mutation causes constitutive activation of transport activity. Mol. Pharmacol. 64, 440-446). This mutation selectively decreased the potency of sertraline for inhibiting serotonin transport. The potencies of imipramine, citalopram, fluoxetine and cocaine for inhibiting transport were not affected by this mutation. In binding measurements with the cocaine analog 2beta-carbomethoxy-3beta-(4-[(125)I]-iodophenyl)-tropane (beta-CIT), sertraline potency was reduced by the I425V mutation but citalopram potency was unchanged. Mutation at the site of cGMP-dependent phosphorylation, Thr-276, decreased the potency of each of the drugs tested. This effect was also observed in studies with beta-CIT where both citalopram and sertraline were less potent at displacing this high-affinity ligand. These results support an influence of Thr-276 on the conformation of inhibitor binding sites of serotonin transporter, and also suggest that the sertraline binding site contains unique determinants that are not shared with the other tested inhibitors.

Binding, Competitive↗

PubMed2005

Cysteine-scanning mutagenesis of serotonin transporter intracellular loop 2 suggests an alpha-helical conformation.

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.

Amino Acid Sequence↗

PubMed2004

Analysis of transmembrane domain 2 of rat serotonin transporter by cysteine scanning mutagenesis.

The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an alpha-helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)-methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na+ and Cl-, and was likely to result from conformational changes during the transport process.

Animals↗

PubMed2003

Mutational analysis of allylic substrate binding site of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

Undecaprenyl diphosphate (UPP) synthase catalyzes the sequential cis-condensation of isopentenyl diphosphate (IPP) onto (E,E)-farnesyl diphosphate (FPP). In our previous reports on the Micrococcus luteus B-P 26 UPP synthase, we have shown that the conserved residues in the disordered region from Ser-74 to Val-85 is crucial for the binding of FPP and the catalytic function [Fujikura, K., et al. (2000) J. Biochem. (Tokyo) 128, 917-922] and the existence of a structural P-loop motif for the FPP binding site [Fujihashi, M., et al. (2001) Proc. Natl. Acad. Sci. U.S.A., 98, 4337-4342]. To elucidate the allylic substrate binding site in more detail, we prepared eight mutant enzymes and examined their kinetic behavior. The mutant with respect to the two complementarily conserved Arg residues among the structural P-loop motif, G32R-R42G, retained the activity and showed product distribution pattern exactly similar to that of the wild-type, indicating that the complementarily conserved Arg is important for maintaining the catalytic function. Substitutions of Asp-29, Arg-33, or Arg-80 with Ala resulted in a large loss of enzyme activity, suggesting that these residues are essential for catalytic function. However, the K(m) values of these mutant enzymes for Z-GGPP, which is the first intermediate during the enzymatic cis-condensations of IPP onto FPP, were only moderately different or little changed from those of the wild type. These results suggest that the binding site for the intermediate Z-GGPP having a cis double bond is different to that for the intrinsic allylic substrate, FPP, whose diphosphate moiety is recognized by the structural P-loop.

Alkyl and Aryl Transferases↗

PubMed2003

Identification of human dehydrodolichyl diphosphate synthase gene.

We isolated a cDNA encoding human dehydrodolichyl diphosphate (Dedol-PP) synthase and expressed the gene in a yeast mutant strain SNH23-7D, which is deficient in Dedol-PP synthase activity. The identity of the cloned enzyme was confirmed by functional complementation of SNH23-7D strain together with in vitro Dedol-PP synthase activity assay. Northern blot analysis indicated that testis and kidney expressed Dedol-PP synthase mRNA at high levels.

Alkyl and Aryl Transferases↗

PubMed2003

Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis.

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.

Alkyl and Aryl Transferases↗

PubMed2003

Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis.

Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration. The enzyme responsible for the cis-1,4-polymerization of isoprene units has been idengified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis. Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase. Northern blot analysis indicated that the transcript(s) of the Hevea cis-prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined. In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 x 103-1 x 104 Da. Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased. These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber.

Amino Acid Sequence↗

PubMed2003

Significance of highly conserved aromatic residues in Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al. (2001) J. Biol. Chem. 276, 28459-28464]. In order to know the IPP binding mode in more detail, we selected six highly conserved residues in Regions III, IV, and V among nine conserved aromatic residues in Micrococcus luteus B-P 26 UPP synthase for substitution by site-directed mutagenesis. The mutant enzymes were expressed and purified to homogeneity, and then their effects on substrate binding and the catalytic function were examined. All of the mutant enzymes showed moderately similar far-UV CD spectra to that of the wild-type, indicating that none of the replacement of conserved aromatic residues affected the secondary structure of the enzyme. Kinetic analysis showed that the replacement of Tyr-71 with Ser in Region III, Tyr-148 with Phe in Region IV, and Trp-210 with Ala in Region V brought about 10-1,600-fold decreases in the kcat/Km values compared to that of the wild-type but the Km values for both substrates IPP and FPP resulted in only moderate changes. Substitution of Phe-207 with Ser in Region V resulted in a 13-fold increase in the Km value for IPP and a 1,000-2,000-fold lower kcat/Km value than those of the wild-type, although the Km values for FPP showed about no significant changes. In addition, the W224A mutant as to Region V showed 6-fold and 14-fold increased Km values for IPP and FPP, respectively, and 100-250-fold decreased kcat/Km values as compared to those of the wild-type. These results suggested that these conserved aromatic residues play important roles in the binding with both substrates, IPP and FPP, as well as the catalytic function of undecaprenyl diphosphate synthase.

Alkyl and Aryl Transferases↗