کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9882368 | 1536555 | 2005 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands
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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
Manganese lipoxygenase is secreted by the fungus Gaeumannomyces graminis. We expressed the enzyme in Pichia pastoris, which secreted â¼30 mg Mn-lipoxygenase/L culture medium in fermentor. The recombinant lipoxygenase was N- and O-glycosylated (80-100 kDa), contained â¼1 mol Mn/mol protein, and had similar kinetic properties (Km â¼7.1 μM α-linolenic acid and Vmax 18 nmol/min/μg) as the native Mn-lipoxygenase. Mn-lipoxygenase could be quantitatively converted, presumably by secreted Pichia proteases, to a smaller protein (â¼67 kDa) with retention of lipoxygenase activity (Km â¼6.4 μM α-linolenic acid and Vmax â¼12 nmol/min/μg). Putative manganese ligands were investigated by site-directed mutagenesis. The iron ligands of soybean lipoxygenase-1 are two His residues in the sequence HWLNTH, one His residue and a distant Asn residue in the sequence HAAVNFGQ, and the C-terminal Ile residue. The homologous sequences of Mn-lipoxygenase are H274VLFH278 and H462HVMN466QGS, respectively, and the C-terminal amino acid is Val-602. The His274Gln, His278Glu, His462Glu, and the Val-602 deletion mutants of Mn-lipoxygenase were inactive, and had lost >95% of the manganese content. His-463, Asn-466, and Gln-467 did not appear to be critical for Mn-lipoxygenase activity, as His463Gln, Asn466Gln, Asn466Leu, and Gln467Asn mutants metabolized α-linolenic acid to 11- and 13-hydroperoxylinolenic acids. We conclude that His-274, His-278, His-462, and Val-602 likely coordinate manganese.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 434, Issue 1, 1 February 2005, Pages 201-211
Journal: Archives of Biochemistry and Biophysics - Volume 434, Issue 1, 1 February 2005, Pages 201-211
نویسندگان
Mirela Cristea, Ã
ke Engström, Chao Su, Lena Hörnsten, Ernst H. Oliw,