Structure and peroxidase activity of ferric Streptomyces clavuligerus orf10-encoded protein P450CLA: UV-visible, CD, MCD and EPR spectroscopic characterization

Author(s)

Goto, Leandro S. ^[1] ; Hokka, Carlos O. ^[1] ; Lima, Jose F. ^[2] ; Prieto, Tatiana ^[2] ; Araujo, Ana Paula U. ^[2] ; Nantes, Iseli L. ^[3] ; Nascimento, Otaciro R. ^{[2, 3]}

Affiliation

^[1] Univ Fed Sao Carlos, Dept Engn Quim, Grp Engn Bioquim, BR-13565905 Sao Carlos, SP - Brazil

^[2] Univ Sao Paulo, Inst Fis Sao Carlos, Grp Biofis Mol Sergio Mascarenhas, BR-13560970 Sao Carlos, SP - Brazil

^[3] Univ Fed ABC, Ctr Ciencias Nat & Humanas, Grp Nanoestruturas Biol & Mat Avancados, BR-09210170 Santo Andre, SP - Brazil

• Document type: Journal article
• Source: Journal of the Brazilian Chemical Society; v. 23, n. 5, p. 913-920, MAY 2012.
• Web of Science Citations: 2

Abstract

The present study reports the spectroscopic characterization by UV-visible absorption spectroscopy, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) of the recombinant orf10-encoded P450-camphor like protein (P450CLA)of Streptomyces clavuligerus expressed in Escherichia coli Rosetta in the native form and associated to external ligands containing the β-lactam, oxazole and alkylamine-derived (alcohol) moieties of the clavulamic acid. Considering the diversity of potential applications for the enzyme, the reactivity with tert-butylhydroperoxide (tert-BuOOH) was also characterized. P450CLA presents a covalently bound heme group and exhibited the UV-visible, CD and MCD spectral features of P450CAM including the fingerprint Soret band at 450 nm generated by the ferrous CO-complex. P450CLA was converted to high valence species by tert-BuOOH and promoted homolytic scission of the O-O bond. The radical profile of the reaction was tert-butyloxyl as primary and methyl and butylperoxyl as secondary radicals. The secondary methyl and butylperoxyl radicals resulted respectively from the β-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen. (AU)

• FAPESP's process: 06/59693-1 - Production and purification of clavulanic acid, cephamycin C and other bioactive metabotites from Streptomyces
• Grantee: Leandro Seiji Goto
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 08/04948-0 - Structure and reactivity of hemepeptides, hemeproteins and phorphyrins in homogeneous and heterogenoeus media: basic aspects and application in nanotechnology
• Grantee: Iseli Lourenço Nantes Cardoso
• Support Opportunities: Research Projects - Thematic Grants