Industrial production of antibiotics, such as semisynthetic penicillins and cephalosporins, requires optically pure D-p-hydroxylphenylglycine and its derivatives as important side-chain precursors. To produce optically pure D-amino acids, microbial D-hydantoinase (E.C. 3.5.2.2) is used for stereospecific hydrolysis of chemically synthesized cyclic hydantoins. We report the apo-crystal structure of D-hydantoinase from B. stearothermophilus SD1 at 3.0 A resolution. The structure has a classic TIM barrel fold. Despite an undetectable similarity in sequence, D-hydantoinase shares a striking structural similarity with the recently solved structure of dihydroorotase. A structural comparison of hydantoinase with dihydroorotase revealed that the catalytic chemistry is conserved, while the substrate recognition is not. This structure provides insight into the stereochemistry of enantioselectivity in hydrolysis and illustrates how the enzyme recognizes stereospecific exocyclic substituents and hydrolyzes hydantoins. It should also provide a rationale for further directed evolution of this enzyme for hydrolysis of new hydantoins with novel exocyclic substituents.

No abstract

We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of D-hydantoinase, a (beta/alpha)(8)-barrel enzyme, might be major structural determinants of the substrate specificity [Cheon, Y. H., et al. (2002) Biochemistry 41, 9410-9417]. To construct a mutant D-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities. In the SGLs of D-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities. In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by approximately 200-fold compared with that of the wild-type enzyme. Saturation mutagenesis at position 159 revealed that k(cat) for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains. When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited approximately 10-fold higher k(cat) values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover. These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of D-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs.

There is compelling evidence that alcohol-induced neurotoxicity is related to glutamate excitotoxicity. It was hypothesized that the low-affinity NMDA receptor antagonist memantine would improve the cognitive function of patients with alcoholic dementia. The aim of this study was to test this hypothesis and to evaluate the effect of memantine on the cognitive improvement of patients with alcohol-related dementia (ARD). The study was designed as a 12-wk open-label study investigating the efficacy of 20 mg memantine, a low-affinity NMDA receptor antagonist, as a treatment for cognitive and behavioural problems in 19 patients with probable ARD according to the criteria for ARD proposed by Oslin and colleagues. The CERAD-K (Consortium to Establish a Registry for Alzheimer's Disease - Korean version) and several clinical assessment scales were completed before and after the 12-wk memantine treatment period. Significant improvements in the mean scores from baseline to final assessment were observed in the Global Deterioration Scale (p<0.05), Brief Psychiatric Rating Scale (p<0.01), Geriatric Quality of Life - Dementia scale (p<0.01) and Neuropsychiatric Inventory (p<0.01) at the end of week 12. The CERAD-K subscales of word list recall (p<0.05), word list recognition (p<0.05), time orientation (p<0.01), drawing an interlocking pentagon (p<0.05), and the total MMSE-K (Mini Mental State Examination - Korean version) scores (p<0.01) of the patients all showed significant improvement following the memantine trial. In this open-label study, patients with ARD treated with 20 mg/d memantine for 12 wk showed improvement on global cognition, quality of life and behavioural symptoms. The result of this study suggests the possible usefulness of memantine for the treatment of ARD. As this was an open-label study, the possibility that participants improved cognitively on their own due to protracted abstinence from alcohol cannot be discounted.

The purpose of this study was to compare differences of the psychological burdens between young male transsexuals and age-gender matched non-transsexuals with standardized psychiatric rating scales in Korea. A total of 43 biologically unrelated young male transsexuals and 49 age-gender matched non-transsexuals participated in the study. All subjects completed Beck's Depression Inventory (BDI), Social Avoidance and Distress Scale (SADS), Self-Esteem Scale (SES) and Family Adaptability and Cohesion Evaluation Scale (FACES-III). The transsexuals showed significantly higher scores on the BDI (P < 0.0001) and SADS (P = 0.002) and lower scores on the SES (P < 0.0001) and Adaptability and Cohesion subscales (P = 0.016 and P < 0.0001, respectively) of the FACES-III than those of the non-transsexuals. The present study found young male transsexuals may be potentially vulnerable to develop psychiatric and familial problems in comparison with non-transsexuals, at least in Korea, although methodological limitations exist. Further welldesigned researches should be launched to confirm this preliminary study.

No abstract

The primary aim of this study was to investigate the genetic predisposition of Internet gaming disorder (IGD), and the secondary aim was to compare the results to those of alcohol dependence (AD). Two independent case-control studies were conducted. A total of 30 male participants with IGD, diagnosed according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria, and 30 sex-matched controls participated in study 1. We designed targeted exome sequencing (TES) to test for 72 candidate genes that have been implicated in the pathogenesis of addiction. The genes included seven neurotransmitter (dopamine, serotonin, glutamate, r-aminobutyric acid (GABA), norepinephrine, acetylcholine, and opioid) system genes. A total of 31 male in-patients with AD and 29 normal male controls (NC) were enrolled in study 2. The same 72 genes included in study 1 and ten additional genes related to alcohol-metabolic enzyme were selected as the target genes, and we identified the genetic variants using the same method (TES). The IGD group had a lower frequency of the allele of rs1044396 in the nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4), and this variant represents a protective allele against IGD. However, we did not find a significant difference in the polymorphisms of the 72 genes that encode neurotransmitter systems between the AD and NC groups. This study demonstrated that rs1044396 of CHRNA4 was significantly associated with IGD.

Bifunctional enzymes find a wide application as a monitoring facility and a potential biocatalyst in molecular biology and biotechnology. Recombination of natural enzymes to a bifunctional fusion offers valuable tools, but the functional and structural instability of artificial fusion enzymes remains to be solved. Based on structural traits of microbial D-hydantoinase, we attempted to construct a bifunctional N-carbamylase/Dhydantoinase fusion enzyme that would be useful for the synthesis of nonnatural D-amino acids in a concerted fashion. The bifunctional ability of D-hydantoinase, as a fusion partner, was noticeable, but the resulting fusion enzyme was subjected to serious proteolysis in vivo, as generally encountered in the expression of large the multidomain polypeptide in E. coli. In an effort to improve the structural instability imposed by artificial linear fusion, directed evolution of the fusion enzyme was performed using DNA shuffling with a consensus primer to maintain a crucial domain for the enzyme activity. The evolved fusion enzyme, F11, was selected after repeated rounds, and this enzyme was found to show sixfold increased performance in the production of D-amino acid compared with the parent fusion enzyme, which was mainly due to the enhanced structural stability of the evolved fusion enzyme. This result is an example showing that directed evolution of the linearly fused polypeptide may broaden the opportunity to generate a fusion enzyme with greater potential.