کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8333252 | 1540260 | 2013 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala
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کلمات کلیدی
N-ethylmaleimideWoodward's reagent KPMSFDEPCWRKNBSPHMBNAIN-bromosuccinimideChemical modification - اصلاح شیمیاییsubstrate protection - حفاظت بسترdiethylpyrocarbonate - دیاتیلپیر کربناتSite directed mutagenesis - سایت موتاگنسیس را هدایت می کندDocking simulations - شبیه سازی dockingphenylmethylsulfonyl fluoride - فنیل متیل سولفونیل فلورایدPhenylglyoxal - فنیل گلیکسالHomology modeling - مدل سازی همگراNEM - نه
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: International Journal of Biological Macromolecules - Volume 60, September 2013, Pages 33-38
Journal: International Journal of Biological Macromolecules - Volume 60, September 2013, Pages 33-38
نویسندگان
Prashant Sonawane, Krunal Patel, Rishi Kishore Vishwakarma, Sameer Srivastava, Somesh Singh, Sushama Gaikwad, Bashir M. Khan,