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.
Highlights
Based on the specific sequence homology, the orf[10] gene of Streptomyces clavuligerus has been assignedStructure and Peroxidase Activity of Ferric Streptomyces clavuligerus orf10-encoded Protein P450CLA J
The sequence of the S. clavuligerus orf10-encoded protein is consistent with a P450 enzyme and yielded a structured protein with covalently bound heme
As described in the Supplementary Information (SI) section, two oligonucleotides were synthesized as PCR primers to produce the engineered P450CLA-encoding DNA for expression experiments
Summary
Based on the specific sequence homology, the orf[10] gene of Streptomyces clavuligerus has been assignedStructure and Peroxidase Activity of Ferric Streptomyces clavuligerus orf10-encoded Protein P450CLA J. The presence of a partially filled set of d-orbitals results in ligand to metal and metal to ligand transitions that are responsible for the charge transfer bands (LMCT, MLCT) observed in both UV‐visible and MCD spectra and, in addition, for the π → π* bands.[20,21] The room temperature MCD spectrum for the resting P450CLA heme closely matched that reported for P450CAM of P. putida.[19] The MCD spectrum of P450CLA (Figure 1B) obtained after the subtraction of CD signals presents a pair of oppositely signed MCD bands of nearly equal amplitude that peak at 427 and 409 nm with the zero crossing at the absorption maximum at 418 nm.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
