دورية أكاديمية
Expression and kinetic characterization of PYCR3.
| العنوان: | Expression and kinetic characterization of PYCR3. |
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| المؤلفون: | Meeks KR; Department of Biochemistry, University of Missouri, Columbia, MO, 65211, United States., Tanner JJ; Department of Biochemistry, University of Missouri, Columbia, MO, 65211, United States; Department of Chemistry, University of Missouri, Columbia, MO, 65211, United States. Electronic address: tannerjj@missouri.edu. |
| المصدر: | Archives of biochemistry and biophysics [Arch Biochem Biophys] 2023 Jan 01; Vol. 733, pp. 109468. Date of Electronic Publication: 2022 Nov 19. |
| نوع المنشور: | Journal Article; Research Support, N.I.H., Extramural |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Country of Publication: United States NLM ID: 0372430 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-0384 (Electronic) Linking ISSN: 00039861 NLM ISO Abbreviation: Arch Biochem Biophys Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: <2000- > : San Diego, CA : Elsevier Original Publication: New York, NY : Academic Press |
| مواضيع طبية MeSH: | Pyrroline Carboxylate Reductases*/genetics , Pyrroline Carboxylate Reductases*/chemistry , NAD*/metabolism, Humans ; Kinetics ; Proline/chemistry ; NADP/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism |
| مستخلص: | PYCRs are proline biosynthetic enzymes that catalyze the NAD(P)H-dependent reduction of Δ 1 -pyrroline-5-carboxylate (P5C) to proline in humans. PYCRs - especially PYCR1 - are upregulated in many types of cancers and have been implicated in the altered metabolism of cancer cells. Of the three isoforms of PYCR, PYCR3 remains the least studied due in part to the lack of a robust recombinant expression. Herein, we describe a procedure for the expression of soluble SUMO-PYCR3 in Escherichia coli, purification of the fusion protein, and removal of the SUMO tag. PYCR3 is active with either NADPH or NADH as the coenzyme. Bi-substrate kinetic measurements obtained by varying the concentrations of both L-P5C and NADH, along with product inhibition data for l-proline, suggest a random ordered bi bi mechanism. A panel of 19 proline analogs was screened for inhibition, and the kinetics of competitive inhibition (with L-P5C) were measured for five of the compounds screened, including N-formyl-l-proline, a validated inhibitor of PYCR1. N-formyl-l-proline was found to be ten times more selective for PYCR1 over PYCR3. The SUMO-PYCR3 expression system should be useful for testing the isoform specificity of PYCR1 inhibitors. Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2022 Elsevier Inc. All rights reserved.) |
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| معلومات مُعتمدة: | R01 GM132640 United States GM NIGMS NIH HHS |
| فهرسة مساهمة: | Keywords: Enzyme inhibition; PYCR3; PYCRL; Proline biosynthesis; SUMO; Δ(1)-pyrroline-5-carboxylate reductase |
| المشرفين على المادة: | EC 1.5.1.- (Pyrroline Carboxylate Reductases) 0U46U6E8UK (NAD) 9DLQ4CIU6V (Proline) 53-59-8 (NADP) |
| تواريخ الأحداث: | Date Created: 20221122 Date Completed: 20221216 Latest Revision: 20240102 |
| رمز التحديث: | 20240102 |
| مُعرف محوري في PubMed: | PMC9772221 |
| DOI: | 10.1016/j.abb.2022.109468 |
| PMID: | 36414121 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1096-0384 |
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| DOI: | 10.1016/j.abb.2022.109468 |