کد مقاله کد نشریه سال انتشار مقاله انگلیسی ترجمه فارسی نسخه تمام متن
8305396 1538429 2014 11 صفحه PDF سفارش دهید دانلود رایگان
عنوان انگلیسی مقاله ISI
Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor
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موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor
چکیده انگلیسی
Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimie - Volume 101, June 2014, Pages 39-49
نویسندگان
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