دورية أكاديمية
Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids.
| العنوان: | Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids. |
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| المؤلفون: | Avila-Crump S; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States., Hemshorn ML; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States., Jones CM; Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, Pennsylvania 19104, United States., Mbengi L; Department of Chemistry, Portland State University, P.O. Box 751, Portland, Oregon 97207, United States., Meyer K; Department of Chemistry, Portland State University, P.O. Box 751, Portland, Oregon 97207, United States., Griffis JA; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States., Jana S; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States., Petrina GE; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States., Pagar VV; Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104, United States., Karplus PA; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States., Petersson EJ; Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104, United States., Perona JJ; Department of Chemistry, Portland State University, P.O. Box 751, Portland, Oregon 97207, United States., Mehl RA; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States., Cooley RB; Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon 97331, United States. |
| المصدر: | ACS chemical biology [ACS Chem Biol] 2022 Dec 16; Vol. 17 (12), pp. 3458-3469. Date of Electronic Publication: 2022 Nov 16. |
| نوع المنشور: | Journal Article; Research Support, N.I.H., Extramural |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Chemical Society Country of Publication: United States NLM ID: 101282906 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1554-8937 (Electronic) Linking ISSN: 15548929 NLM ISO Abbreviation: ACS Chem Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, D.C. : American Chemical Society, c2006- |
| مواضيع طبية MeSH: | Amino Acids* , Amino Acyl-tRNA Synthetases*/metabolism, Humans ; HEK293 Cells ; Lysine/chemistry ; Methanosarcina/genetics ; Methanosarcina/metabolism ; RNA, Transfer/genetics ; Tyrosine |
| مستخلص: | Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNA Pyl pairs from Methanosarcina mazei ( Mm ) and Methanosarcina barkeri ( Mb ) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNA Pyl pair from Candidatus Methanomethylophilus alvus Mx1201 ( Ma ) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb / Mm PylRSs. An open question is how to alter Ma PylRS specificity to encode specific ncAAs with high efficiency. Prior work focused on "transplanting" ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm / Mb PylRSs in Ma PylRS. Here, we found that this strategy produced low-efficiency Ma PylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m -methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient Ma PylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 μM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All Ma PylRS variants identified through selection had at least two different active site residues when compared with their Mb PylRS counterparts. We conclude that Ma and Mm / Mb PylRSs are sufficiently different that "active site transplantation" yields suboptimal Ma GCE systems. This work establishes a paradigm for expanding the utility of the promising Ma PylRS/tRNA Pyl GCE platform. |
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| معلومات مُعتمدة: | U19 NS110456 United States NS NINDS NIH HHS; R01 NS103873 United States NS NINDS NIH HHS; R01 GM131168 United States GM NIGMS NIH HHS; T32 GM132039 United States GM NIGMS NIH HHS; RM1 GM144227 United States GM NIGMS NIH HHS; S10 OD020111 United States OD NIH HHS; T32 GM008275 United States GM NIGMS NIH HHS |
| المشرفين على المادة: | 0 (Amino Acids) K3Z4F929H6 (Lysine) EC 6.1.1.- (Amino Acyl-tRNA Synthetases) 9014-25-9 (RNA, Transfer) 42HK56048U (Tyrosine) |
| تواريخ الأحداث: | Date Created: 20221116 Date Completed: 20221219 Latest Revision: 20231217 |
| رمز التحديث: | 20231217 |
| مُعرف محوري في PubMed: | PMC9833845 |
| DOI: | 10.1021/acschembio.2c00639 |
| PMID: | 36383641 |
| قاعدة البيانات: | MEDLINE |
Find this issue from the American Chemical Society | تدمد: | 1554-8937 |
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| DOI: | 10.1021/acschembio.2c00639 |