کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9882253 | 1536550 | 2005 | 12 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
The unexpected structural role of glutamate synthase [4Fe-4S]+1, +2 clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme β subunit
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کلمات کلیدی
AmidotransferaseSite-directed mutagenesis - mutagenesis مواجه با سایتAmmonia assimilation - آمونیاک جذبIron–sulfur clusters - خوشه های آهن گوگردdihydropyrimidine dehydrogenase - دی هیدروپیریمیدین دهیدروژنازFlavoprotein - فلاوپروتئینProtein engineering - مهندسی پروتئینglutamate synthase - گلوتامات سنتاز
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active αβ protomer (α subunit, 162 kDa; β subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S]0, +1, and two [4Fe-4S]+1, +2 clusters. The structure of the α subunit has been determined providing information on the mechanism of ammonia transfer from l-glutamine to 2-oxoglutarate through a 30 Ã
-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS β subunit with alanyl residues. The engineered genes encoding the β subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type α subunit gene. In all cases the C/A substitutions prevented α and β subunits association to yield the GltS αβ protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S]+1, +2 clusters within the N-terminal region of the β subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in αβ heterodimer formation by structuring an N-terminal [Fe-S] β subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal β subunit-like domain.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 436, Issue 2, 15 April 2005, Pages 355-366
Journal: Archives of Biochemistry and Biophysics - Volume 436, Issue 2, 15 April 2005, Pages 355-366
نویسندگان
Paola Agnelli, Laura Dossena, Paolo Colombi, Sara Mulazzi, Paola Morandi, Gabriella Tedeschi, Armando Negri, Bruno Curti, Maria A. Vanoni,