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      Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids

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          Abstract

          Unnatural amino acids (UAAs) are chiral amines with high application potential in drug discovery and synthesis of other valuable chemicals. Biocatalysis offers the possibility to synthesise novel optically pure UAAs with different physical and chemical properties. While the biocatalytic potential of transaminases in the synthesis of UAAs has been demonstrated, there is still a need to improve the activity with non-native substrates and to understand which amino acids residues are important for activity with these UAAs. Using a rational design approach, six variants of Chromobacterium violaceum DSM30191 transaminase (CV_TA) carrying a single and one variant carrying two substitutions were generated. Among the variants with a single substitution, CV_Y168F showed a 2 to 2.6-fold increased affinity for 2-oxooctanoic acid (2-OOA) and 3-oxobutyric acid (3-OBA) methyl ester used to synthesise an α- and β-UAA. Analysis of the first half of the transaminase reaction showed no change in the activity with the donor ( S)-1-phenylethylamine. The combination of W60C and Y168F substitutions improved the CV_TA affinity for 2-OOA 10-fold compared to the wild type. Other substitutions showed no change, or reduced activity with the tested substrates. Our findings provide structural information on CV_TA and demonstrate the potential of rational design for biosynthesis of UAAs.

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          How Taxol/paclitaxel kills cancer cells

          Taxol (generic name paclitaxel) is a microtubule-stabilizing drug that is approved by the Food and Drug Administration for the treatment of ovarian, breast, and lung cancer, as well as Kaposi's sarcoma. It is used off-label to treat gastroesophageal, endometrial, cervical, prostate, and head and neck cancers, in addition to sarcoma, lymphoma, and leukemia. Paclitaxel has long been recognized to induce mitotic arrest, which leads to cell death in a subset of the arrested population. However, recent evidence demonstrates that intratumoral concentrations of paclitaxel are too low to cause mitotic arrest and result in multipolar divisions instead. It is hoped that this insight can now be used to develop a biomarker to identify the ∼50% of patients that will benefit from paclitaxel therapy. Here I discuss the history of paclitaxel and our recently evolved understanding of its mechanism of action.
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            Unusual Amino Acids in Medicinal Chemistry.

            Unusual amino acids are fundamental building blocks of modern medicinal chemistry. The combination of readily functionalized amine and carboxyl groups attached to a chiral central core along with one or two potentially diverse side chains provides a unique three-dimensional structure with a high degree of functionality. This makes them invaluable as starting materials for syntheses of complex molecules, highly diverse elements for SAR campaigns, integral components of peptidomimetic drugs, and potential drugs on their own. This Perspective highlights the diversity of unnatural amino acid structures found in hit-to-lead and lead optimization campaigns and clinical stage and approved drugs, reflecting their increasingly important role in medicinal chemistry.
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              Biocatalytic Approaches to the Synthesis of Enantiomerically Pure Chiral Amines

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                Author and article information

                Contributors
                tanja.narancic@ucd.ie
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                26 November 2018
                26 November 2018
                2018
                : 8
                : 17397
                Affiliations
                [1 ]ISNI 0000 0001 0768 2743, GRID grid.7886.1, UCD Earth Institute and School of Biomolecular and Biomedical Science, , University College Dublin, ; Belfield, Dublin 4 Ireland
                [2 ]ISNI 0000 0001 0768 2743, GRID grid.7886.1, BEACON - Bioeconomy Research Centre, Ireland, , University College Dublin, ; Belfield, Dublin 4 Ireland
                [3 ]ISNI 0000 0004 1936 9705, GRID grid.8217.c, Molecular Design Group, School of Biochemistry and Immunology, , Trinity Biomedical Sciences Institute, Trinity College Dublin, ; Dublin 2, Ireland
                Author information
                http://orcid.org/0000-0003-3269-2200
                Article
                35688
                10.1038/s41598-018-35688-7
                6255834
                30478262
                fd194940-1f02-43e7-83d5-a19c8b4f1874
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 31 July 2018
                : 16 October 2018
                Funding
                Funded by: Ministry of education of Kingdom of Saudi Arabia, King Abdullah scholarship programme
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