کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843427 | 1069258 | 2008 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Hydrolysis of the 5â²-p-nitrophenyl ester of TMP by oligoribonucleases (ORN) from Escherichia coli, Mycobacterium smegmatis, and human
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3â²-5â² exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from the β and γ subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E. coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5â²-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (kcat = 100-650 minâ1, KM = 0.4-2.0 mM, at pH 8.00 and 25 °C, with 1 mM Mn2+). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn2+ being preferred over Mg2+ as cofactor, and was inhibited by Ni2+. The concentration dependency of Mn2+ was examined, giving KMn values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 57, Issue 2, February 2008, Pages 180-187
Journal: Protein Expression and Purification - Volume 57, Issue 2, February 2008, Pages 180-187
نویسندگان
Ah Young Park, Christopher M. Elvin, Samir M. Hamdan, Robert J. Wood, Nancy E. Liyou, Tamarind E. Hamwood, Phil A. Jennings, Nicholas E. Dixon,