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Expression of manga...
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Cristea, MirelaUppsala universitet,Institutionen för farmaceutisk biovetenskap
(author)
Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative manganese ligands
- Article/chapterEnglish2005
Publisher, publication year, extent ...
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Elsevier BV,2005
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printrdacarrier
Numbers
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LIBRIS-ID:oai:DiVA.org:uu-94110
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https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-94110URI
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https://doi.org/10.1016/j.abb.2004.10.026DOI
Supplementary language notes
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Language:English
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Summary in:English
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
Notes
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Manganese lipoxygenase is secreted by the fungus Gaeumannomyces graminis. We expressed the enzyme in Pichia pastoris, which secreted approximately 30 mg Mn-lipoxygenase/L culture medium in fermentor. The recombinant lipoxygenase was N- and O-glycosylated (80-100 kDa), contained approximately 1 mol Mn/mol protein, and had similar kinetic properties (K(m) approximately 7.1 microM alpha-linolenic acid and V(max) 18 nmol/min/microg) as the native Mn-lipoxygenase. Mn-lipoxygenase could be quantitatively converted, presumably by secreted Pichia proteases, to a smaller protein (approximately 67 kDa) with retention of lipoxygenase activity (K(m) approximately 6.4 microM alpha-linolenic acid and V(max) approximately 12 nmol/min/microg). 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 alpha-linolenic acid to 11- and 13-hydroperoxylinolenic acids. We conclude that His-274, His-278, His-462, and Val-602 likely coordinate manganese.
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Engström, ÅkeUppsala universitet,Institutionen för medicinsk biokemi och mikrobiologi(Swepub:uu)akeengs
(author)
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Su, ChaoUppsala universitet,Institutionen för farmaceutisk biovetenskap
(author)
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Hörnsten, Lena
(author)
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Oliw, Ernst H.Uppsala universitet,Institutionen för farmaceutisk biovetenskap
(author)
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Uppsala universitetInstitutionen för farmaceutisk biovetenskap
(creator_code:org_t)
Related titles
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In:Archives of Biochemistry and Biophysics: Elsevier BV434:1, s. 201-2110003-98611096-0384
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