Application of ω-Transaminases in the Pharmaceutical Industry.

Chiral amines are valuable building blocks for the pharmaceutical industry. ω-TAms have emerged as an exciting option for their synthesis, offering a potential "green alternative" to overcome the drawbacks associated with conventional chemical methods. In this review, we explore the application of ω-TAms for pharmaceutical production. We discuss the diverse array of reactions available involving ω-TAms and process considerations of their use in both kinetic resolution and asymmetric synthesis. With the aid of specific drug intermediates and APIs, we chart the development of ω-TAms using protein engineering and their contribution to elegant one-pot cascades with other enzymes, including carbonyl reductases (CREDs), hydrolases and monoamine oxidases (MAOs), providing a comprehensive overview of their uses, beginning with initial applications through to the present day.

Polymorphisms of RDH16 and VEGFR1 influence M. trapezius steatosis in Japanese Black carcass.

The exact cause of steatosis, one of defects in Japanese beef carcasses, has not been elucidated to date, because it is very difficult to diagnose cyclopedically with certain reproducibility due to the bias in the outbreak. Therefore, the objective of this study was to assess the influence of polymorphisms in retinol dehydrogenase 16 (RDH16), myoferlin (MYOF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR1, VEGFR2) on carcass-graded Musculus trapezius steatosis. For logistic regression analysis, 646 carcasses shipped from 29 farms in Miyazaki, Japan, were used. The GG genotype in RDH16 showed significant odds ratios against AA and AG. In VEGFR1, CT had a significant odds ratio against CC. After evaluating for interaction, highly significant odds ratios were observed in the combinations that included the GG risk genotype in RDH16. It is noteworthy that there was no steatosis in the combination GG (RDH16) and CC (VEGFR1). It may be concluded that there is a possibility that steatosis can be suppressed by the CC genotype in VEGFR1. The current study revealed the influence of genetic polymorphisms on M. trapezius steatosis that had not been reported until now, and may help elucidate the cause of steatosis.

Apparent Epigenetic Meiotic Double-Strand-Break Disparity in Saccharomyces cerevisiae: A Meta-Analysis.

Previously published, and some unpublished, tetrad data from budding yeast (Saccharomyces cerevisiae) are analyzed for disparity in gene conversion, in which one allele is more often favored than the other (conversion disparity). One such disparity, characteristic of a bias in the frequencies of meiotic double-strand DNA breaks at the hotspot near the His4 locus, is found in diploids that undergo meiosis soon after their formation, but not in diploids that have been cloned and frozen. Altered meiotic DNA breakability associated with altered metabolism-related chromatin states has been previously reported. However, the above observations imply that such differing parental chromatin states can persist through at least one chromosome replication, and probably more, in a common environment. This conclusion may have implications for interpreting changes in allele frequencies in populations.

MR spectroscopy for in vivo assessment of the oncometabolite 2-hydroxyglutarate and its effects on cellular metabolism in human brain gliomas at 9.4T.

PURPOSE: To examine in vivo metabolic alterations in the isocitrate dehydrogenase (IDH) mutated gliomas using magnetic resonance spectroscopy (MRS) at magnetic field 9.4T. MATERIALS AND METHODS: Spectra were acquired with a 9.4T whole-body scanner with the use of a custom-built head coil (16 channel transmit and 31 channel receive). A modified stimulated echo acquisition mode (STEAM) sequence was used for localization. Eighteen patients with brain tumors of probable glial origin participated in this study. The study was performed in accordance with the guidelines of the local Ethics Committee. RESULTS: The increased spectral resolution allowed us to directly address metabolic alterations caused by the specific pathophysiology of IDH mutations including the presence of the oncometabolite 2-hydroxglutarate (2HG) and a significant decrease of the pooled glutamate and glutamine (20%, P = 0.024), which probably reflects an attempt to replenish α-ketoglutarate lost by conversion to 2HG. We also observed significantly reduced glutathione (GSH) levels (39%, P = 0.019), which could be similarly caused by depletion of dihydronicotinamide-adenine dinucleotide phosphate (NADPH) during this conversion in IDH mutant gliomas. CONCLUSION: We demonstrate that MRS at 9.4T provides a noninvasive measure of 2HG in vivo, which may be used for therapy planning and prognostication, and may provide insights into related pathophysiologic metabolic alterations associated with IDH mutations. J. MAGN. RESON. IMAGING 2016;44:823-833.

Non-motor symptoms in genetically defined dystonia: Homogenous groups require systematic assessment.

INTRODUCTION: Dystonia is a movement disorder involving sustained or intermittent muscle contractions resulting in abnormal movements and postures. Identification of disease causing genes has allowed examination of genetically homogenous groups. Unlike the motor symptoms, non-motor characteristics are less clearly defined, despite their impact on a patient's quality of life. This review aims to examine the evidence for non-motor symptoms, addressing cohort size and methods of assessment in each study. METHODS: A systematic and standardised search strategy was used to identify the published literature relating to psychiatric symptoms, cognition, sleep disorders, sensory abnormalities and pain in each of the genetically determined dystonias. Studies were divided according to cohort size, method of assessment and whether comparison was made to an appropriate control group. RESULTS: Ninety-five articles were identified including reported clinical histories (n = 42), case reports and smaller case series (n = 12), larger case series (n = 23) and case-control cohorts (n = 18). Psychiatric symptoms were the most frequently investigated with anxiety, depression and Obsessive-Compulsive disorder being most common. Cognitive impairment involved either global deficits or isolated difficulties in specific domains. Disturbances to sleep were most common in the dopa-responsive dystonias. Sensory testing in DYT1 cases identified an intermediate subclinical phenotype. CONCLUSION: Non-motor symptoms form an integral component of the dystonia phenotype. However, future studies should involve a complete assessment of all symptom subtypes in order to understand the frequency and gene-specificity of these symptoms. This will enable early symptom identification, appropriate clinical management, and provide additional outcome measures in future clinical trials.

Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates.

The halophile Halomonas TD01 and its derivatives have been successfully developed as a low-cost platform for the unsterile and continuous production of chemicals. Therefore, to increase the genetic engineering stability of this platform, the DNA restriction/methylation system of Halomonas TD01 was partially inhibited. In addition, a stable and conjugative plasmid pSEVA341 with a high-copy number was constructed to contain a LacI(q)-Ptrc system for the inducible expression of multiple pathway genes. The Halomonas TD01 platform, was further engineered with its 2-methylcitrate synthase and three PHA depolymerases deleted within the chromosome, resulting in the production of the Halomonas TD08 strain. The overexpression of the threonine synthesis pathway and threonine dehydrogenase made the recombinant Halomonas TD08 able to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV consisting of 4-6 mol% 3-hydroxyvalerate or 3 HV, from various carbohydrates as the sole carbon source. The overexpression of the cell division inhibitor MinCD during the cell growth stationary phase in Halomonas TD08 elongated its shape to become at least 1.4-fold longer than its original size, resulting in enhanced PHB accumulation from 69 wt% to 82 wt% in the elongated cells, further promoting gravity-induced cell precipitations that simplify the downstream processing of the biomass. The resulted Halomonas strains contributed to further reducing the PHA production cost.

A multi-year assessment of the environmental impact of transgenic Eucalyptus trees harboring a bacterial choline oxidase gene on biomass, precinct vegetation and the microbial community.

A 4-year field trial for the salt tolerant Eucalyptus globulus Labill. harboring the choline oxidase (codA) gene derived from the halobacterium Arthrobacter globiformis was conducted to assess the impact of transgenic versus non-transgenic trees on biomass production, the adjacent soil microbial communities and vegetation by monitoring growth parameters, seasonal changes in soil microbes and the allelopathic activity of leaves. Three independently-derived lines of transgenic E. globulus were compared with three independent non-transgenic lines including two elite clones. No significant differences in biomass production were detected between transgenic lines and non-transgenic controls derived from same seed bulk, while differences were seen compared to two elite clones. Significant differences in the number of soil microbes present were also detected at different sampling times but not between transgenic and non-transgenic lines. The allelopathic activity of leaves from both transgenic and non-transgenic lines also varied significantly with sampling time, but the allelopathic activity of leaves from transgenic lines did not differ significantly from those from non-transgenic lines. These results indicate that, for the observed variables, the impact on the environment of codA-transgenic E. globulus did not differ significantly from that of the non-transformed controls on this field trial.

Assessment of a putative proton relay in Arabidopsis cinnamyl alcohol dehydrogenase catalysis.

Extended proton relay systems have been proposed for various alcohol dehydrogenases, including the Arabidopsis thaliana cinnamyl alcohol dehydrogenases (AtCADs). Following a previous structural biology investigation of AtCAD5, the potential roles of three amino acid residues in a putative proton relay system, namely Thr49, His52 and Asp57, in AtCAD5, were investigated herein. Using site-directed mutagenesis, kinetic and isothermal titration calorimetry (ITC) analyses, it was established that the Thr49 residue was essential for overall catalytic conversion, whereas His52 and Asp57 residues were not. Mutation of the Thr49 residue to Ala resulted in near abolition of catalysis, with thermodynamic data indicating a negative enthalpic change (ΔH), as well as a significant decrease in binding affinity with NADPH, in contrast to wild type AtCAD5. Mutation of His52 and Asp57 residues by Ala did not significantly change either catalytic efficiency or thermodynamic parameters. Therefore, only the Thr49 residue is demonstrably essential for catalytic function. ITC analyses also suggested that for AtCAD5 catalysis, NADPH was bound first followed by p-coumaryl aldehyde.

Bioinformatic and biochemical characterization of DCXR and DHRS2/4 from Caenorhabditis elegans.

Several reductases belonging to the large enzyme superfamily of the short-chain dehydrogenases/reductases (SDR) are involved in the reductive metabolism of carbonyl containing xenobiotics. In order to characterize the human enzymes dicarbonyl/l-xylulose reductase (DCXR), and dehydrogenase/reductase members 2 and 4 (DHRS2, DHRS4) in terms of metabolism of xenobiotics, orthologues from the model organism Caenorhabditis elegans (C. elegans) were identified by using hidden Markov models that were developed in the present study. Accordingly, we describe the characterization of proteins from C. elegans as orthologous to the human enzymes DCXR and DHRS2/4 using a combined approach of bioinformatic and biochemical methods. With the hidden Markov model based system we identified the C. elegans proteins SDR20C18, SDR25C21 and SDR25C22 as being homologous to the human enzymes DCXR, and DHRS2 or DHRS4, respectively. After cloning and overexpression of these three C. elegans genes in Escherichia coli we could purify SDR20C18 and SDR25C22 as soluble proteins by Ni-affinity chromatography, whereas recombinant SDR25C21 was only found in inclusion bodies. Both SDR20C18 (UniProtAcc: Q21929) and SDR25C22 (UniProtAcc: Q93790) were tested with a variety of xenobotic carbonyl compounds as substrates. A comparison of the catalytic activities of SDR20C18 and SDR25C22 with well-known substrates of the human forms revealed that SDR20C18 is the DCXR-orthologue enzyme to the human enzyme and that SDR25C22 might be a DHRS2/4 homologue. Due to their high sequence identity, it was so far not possible to distinguish between SDR25C22 and the human DHRS2/4 proteins by means of sequence analysis alone. However, the study of homologue genes in the model organism C. elegans can provide valuable information on the putative physiological role of the corresponding human form.

Highly selective L-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of L-threonine.

l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD(+)-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and dl-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD(+)-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.

Safety assessment of bacterial choline oxidase protein introduced in transgenic crops for tolerance against abiotic stress.

Genetically modified crops have resistance to abiotic stress by introduction of choline oxidase protein. In the present study, the safety of choline oxidase protein derived from Arthrobacter globiformis was assessed for toxicity and allergenicity. The protein was stable at 90 degrees C for 1 h. Toxicity studies of choline oxidase in mice showed no significant difference (p > 0.05) from control in terms of growth, body weight, food consumption, and blood biochemical indices. Histology of gut tissue of mice fed protein showed normal gastric mucosal lining and villi in jejunum and ileum sections. Specific IgE in serum and IL-4 release in splenic culture supernatant were low in choline oxidase treated mice, comparable to control. Intravenous challenge with choline oxidase did not induce any adverse reaction, unlike ovalbumin group mice. Histology of lung tissues from choline oxidase sensitized mice showed normal airways, whereas ovalbumin-sensitized mice showed inflamed airways with eosinophilic infiltration and bronchoconstriction. ELISA carried out with food allergic patients' sera revealed no significant IgE affinity with choline oxidase. Also, choline oxidase did not show any symptoms of toxicity and allergenicity in mice.

Allergenicity assessment of transgenic mustard (Brassica juncea) expressing bacterial codA gene.

BACKGROUND: Assessing the allergenicity and toxicity of genetically modified (GM) crops is essential before they become a regular part of our food supply. The present study aimed to assess the allergenicity of Brassica juncea (mustard) expressing choline oxidase (codA) gene from Arthrobacter globiformis that provides resistance against abiotic stresses. METHODS: SDAP, Farrp, and Swiss-Prot databases were used to study allergenicity of choline oxidase. Digestibility of choline oxidase was assessed in simulated gastric fluid (SGF). Specific immunoglobulin E (IgE) reactivity of native and GM mustard was compared by using enzyme-linked immunosorbent assay (ELISA) and skin tests in respiratory-allergic patients. Allergenicity of GM and native mustard proteins was compared in Balb/c mice. RESULTS: Choline oxidase showed no significant homology with allergenic proteins in SDAP and Farrp databases. Cross-reactive epitope search showed a stretch similar to Hev b 6 having some antigenic properties. Purified choline oxidase showed complete degradation with SGF. Skin prick test of native and GM mustard extract on respiratory allergic patients showed significant correlation (P < 0.05). ELISA with 96 patients' sera showed comparable IgE reactivity. Balb/c mice immunized with native and GM mustard proteins showed low IgE response. Presensitized mice on intravenous challenge with Brassica extract showed no anaphylactic symptoms unlike ovalbumin (OVA) sensitization that showed anaphylactic reaction in mice. Lung histology of OVA-sensitized mice showed narrowing of airway and large eosinophilic infiltration, whereas native and GM Brassica extract showed normal airway. CONCLUSION: Genetically modified mustard with the codA gene possessed allergenicity similar to that of native mustard and no enhancement of IgE binding was observed due to genetic manipulation.

Assessment of the essentiality of ERG genes late in ergosterol biosynthesis in Saccharomyces cerevisiae.

Isogenic strains of yeast were constructed, differing only in insertionally inactivated genes for ergosterol biosynthesis. These and their allelic wild-types were grown in competition to ascertain growth differences and any selective advantage for organisms producing sterols with or without specific features of ergosterol. In every instance tested, the wild-type allele afforded a competitive advantage over the isogenic pair producing modified sterol structures instead of ergosterol. A general trend was seen in which the earlier in the biosynthetic pathway that a mutation occurred, the less able the strain producing the defective sterols could compete with the ergosterol-producing strains.

Assessment of variability within electromorphs of alcohol dehydrogenase in Drosophila melanogaster.

Ninety-six isochromosomal lines of Drosophila melanogaster from a natural population were screened electrophoretically for unusual mobility variants at the alcohol dehydrogenase locus, using a total of eight conditions of acrylamide electrophoresis. No additional mobility variation was found among the 50 "slow" and 46 "fast" mobility lines beyond that detected by standard methods of electrophoresis. However, two thermostability variants recovered by R. MILKMAN from a natural population, whose electrophoretic mobilities were previously thought to be distinguishable from those of "standard" alleles, are distinguishable from the standard electromorphs by these procedures. These results suggest that the Adh locus, although polymorphic, does not harbor substantial amounts of "hidden" allelic variability. This study also reports the appearance of substantial mobility variation among isogenic lines that can be induced under specific conditions of sample preparation involving the pretreatment of samples with NAD and acetone. However, genetic analysis demonstrates that this variability cannot be attributed to allelic differences at the structural locus, but instead appears to be dependent upon the concentration of the enzyme in a sample. These results are discussed in relation to the distribution of allelic variation at other enzyme loci.