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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
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- Elsevier BV,2005
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- LIBRIS-ID:oai:DiVA.org:uu-94110
- https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-94110URI
- https://doi.org/10.1016/j.abb.2004.10.026DOI
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- Language:English
- Summary in:English
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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)
- Su, ChaoUppsala universitet,Institutionen för farmaceutisk biovetenskap (author)
- Hörnsten, Lena (author)
- Oliw, Ernst H.Uppsala universitet,Institutionen för farmaceutisk biovetenskap (author)
- Uppsala universitetInstitutionen för farmaceutisk biovetenskap (creator_code:org_t)
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- In:Archives of Biochemistry and Biophysics: Elsevier BV434:1, s. 201-2110003-98611096-0384
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