Kuakarun Krusong

Structure and molecular mechanism of a nucleobase-cation- symport-1 family transporter

The nucleobase–cation–symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuTAa and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport.

Histidine Decarboxylase Deficiency Causes Tourette Syndrome: Parallel Findings in Humans and Mice

Tourette syndrome (TS) is characterized by tics, sensorimotor gating deficiencies, and abnormalities of cortico-basal ganglia circuits. A mutation in histidine decarboxylase (Hdc), the key enzyme for the biosynthesis of histamine (HA), has been implicated as a rare genetic cause. Hdc knockout mice exhibited potentiated tic-like stereotypies, recapitulating core phenomenology of TS; these were mitigated by the dopamine (DA) D2 antagonist haloperidol, a proven pharmacotherapy, and by HA infusion into the brain. Prepulse inhibition was impaired in both mice and humans carrying Hdc mutations. HA infusion reduced striatal DA levels; in Hdc knockout mice, striatal DA was increased and the DA-regulated immediate early gene Fos was upregulated. DA D2/D3 receptor binding was altered both in mice and in humans carrying the Hdc mutation. These data confirm histidine decarboxylase deficiency as a rare cause of TS and identify HA-DA interactions in the basal ganglia as an important locus of pathology.

A Comparative Study of Uracil-DNA Glycosylases from Human and Herpes Simplex Virus Type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (kcat/Km) compared with the viral enzyme due to a lower Km. The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2′-deoxypseudouridine and 2′-α-fluoro-2′-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U·A and U·G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.

A comparative study of antimicrobial properties of crustinPm1 and crustinPm7 from the black tiger shrimp Penaeus monodon

Several isoforms of crustin have been identified in the black tiger shrimp Penaeus monodon. These cationic cysteine-rich antimicrobial peptides contain a single whey acidic protein (WAP) domain at the C-terminus and exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigate the binding properties and antimicrobial actions of crustinPm1 and crustinPm7, the two most abundant crustin isoforms found in the haemocyte of P. monodon. Previously, crustinPm1 showed strong inhibition against Gram-positive bacteria, whilst crustinPm7 acted against both Gram-positive and Gram-negative bacteria. A binding study showed that both crustins can bind to Gram-positive and Gram-negative bacterial cells. Enzyme-linked immunosorbent (ELISA) assay suggested that crustins bind to the cell wall components, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) with positive cooperativity of Hill slope (H)>2. This indicates that at least two molecules of crustins interact with one LTA or LPS molecule. In addition, both crustins can induce bacterial agglutination and cause inner membrane permeabilization in Escherichia coli. Scanning Electron Microscopy (SEM) revealed the remarkable change on the cell surface of Staphylococcus aureus, Vibrio harveyi and E. coli after the bacteria were treated with the recombinant crustinPm7. Meanwhile, crustinPm1 can cause a visible change on the cell surface of S. aureus and E. coli only. This is in agreement with the fact that crustinPm1 has shown no antimicrobial activity against V. harveyi. It is likely that the antimicrobial activity of crustins mainly relies on their ability to agglutinate bacterial cells and to disrupt the physiochemical properties of bacterial surface.

High levels of histidine decarboxylase in the striatum of mice and rats.

The neurotransmitter histamine is produced in the tuberomamillary nucleus of the posterior hypothalamus; these neurons project broadly throughout central nervous system. Histidine decarboxylase (HDC) synthesizes histamine from histidine; in the brain, its mRNA is expressed exclusively in the posterior hypothalamus. Histamine receptors are expressed throughout the forebrain, including in cortex, hippocampus, and basal ganglia, suggesting functional innervation of these structures. We investigated the distribution of HDC protein in dissected tissue from mouse and rat, anticipating that it would reflect the density of hypothalamic histaminergic axonal projections and thus qualitatively parallel the known distribution of histamine receptors. HDC protein was found at high levels in hypothalamus, as anticipated. Surprisingly, it was found at comparably high levels in mouse striatum. HDC protein was 10-fold lower in cortex, hippocampus, and cerebellum. Specificity of HDC detection by Western blot was confirmed using HDC knockout mice. Similar high levels of HDC protein were found in dissected striatum from rat. Striatum does not, however, contain comparably elevated of histamine, relative to other forebrain structures; we confirmed this fact using HPLC. This discrepancy between HDC protein and histamine levels in the striatum suggests that histamine metabolism and neurotransmission in basal ganglia may have unique characteristics, the details of which remain to be elucidated.

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

ABSTRACT Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The k cat/Km of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM.

Insights into structure and function of the active site of SoxAX cytochromes.

SoxAX cytochromes catalyze the formation of heterodisulfide bonds between inorganic sulfur compounds and a carrier protein, SoxYZ. They contain unusual His/Cys-ligated heme groups with complex spectroscopic signatures. The heme-ligating cysteine has been implicated in SoxAX catalysis, but neither the SoxAX spectroscopic properties nor its catalysis are fully understood at present. We have solved the first crystal structure for a group 2 SoxAX protein (SnSoxAX), where an N-terminal extension of SoxX forms a novel structure that supports dimer formation. Crystal structures of SoxAX with a heme ligand substitution (C236M) uncovered an inherent flexibility of this SoxA heme site, with both bonding distances and relative ligand orientation differing between asymmetric units and the new residue, Met236, representing an unusual rotamer of methionine. The flexibility of the SnSoxAXC236M SoxA heme environment is probably the cause of the four distinct, new EPR signals, including a high spin ferric heme form, that were observed for the enzyme. Despite the removal of the catalytically active cysteine heme ligand and drastic changes in the redox potential of the SoxA heme (WT, −479 mV; C236M, +85 mV), the substituted enzyme was catalytically active in glutathione-based assays although with reduced turnover numbers (WT, 3.7 s−1; C236M, 2.0 s−1). SnSoxAXC236M was also active in assays using SoxYZ and thiosulfate as the sulfur substrate, suggesting that Cys236 aids catalysis but is not crucial for it. The SoxYZ-based SoxAX assay is the first assay for an isolated component of the Sox multienzyme system.

Synthesis of rotenoid derivatives with cytotoxic and topoisomerase II inhibitory activities

6-Deoxyclitoriacetal (1) and a series of 11 further derivatives of it (2-12) were synthesized and evaluated for their cytotoxic and topoisomerase IIα inhibitory activities. Compounds bearing epoxide (2), morpholine (6) and benzylamine (10) moieties showed promising in vitro cytotoxic activities against four cancer cell lines, with IC(50) values ranging from 0.38 to 0.73 μM. These three compounds also strongly inhibited topoisomerase II activity at 68.3-93.5% and showed a moderately high DNA intercalating property.

The essential role of clathrin-mediated endocytosis in yellow head virus propagation in the black tiger shrimp Penaeus monodon

Yellow head virus (YHV) is one of the most widespread viruses seriously affecting black tiger shrimp (Penaeus monodon) cultivation. A previous microarray study demonstrated that clathrin coat assembly protein 17 (AP17) was significantly up-regulated after YHV infection (Pongsomboon et al., 2011). Clathrin coat AP17 is a part of the assembly protein σ2 (AP-2) complex which is involved in clathrin-mediated endocytosis. Quantitative RT-PCR (qRT-PCR) revealed that the clathrin coat AP17 gene was up-regulated 3-fold at 12 h post YHV infection. In addition, immunofluorescence microscopy showed that clathrin coat AP17 was highly expressed in the cytoplasm of the YHV-infected hemocytes. Knockdown of the clathrin coat AP17 gene dramatically reduced YHV replicativity by 32-fold. Interestingly, shrimp pre-treated with chlorpromazine, a commercial drug that inhibits clathrin-dependent endocytosis, exhibited significantly low levels of YHV infection. Taken together, these results suggest that clathrin-mediated endocytosis is involved in YHV propagation in P. monodon.

Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum.

This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50 °C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40 °C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme.