The Cytochrome P450 OxyA from the Kistamicin Biosynthesis Cyclization Cascade is Highly Sensitive to Oxidative Damage

Abstract

Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilize a cysteine thiolate–ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of the active intermediate formed within P450s during their active cycle, it is remarkable that these enzymes can avoid auto-oxidation and retain the axial cysteine ligand in the deprotonated—and thus highly acidic—thiolate form. While little is known about the process of heme incorporation during P450 folding, there is an overwhelming preference for one heme orientation within the P450 active site. Indeed, very few structures to date contain an alternate heme orientation, of which two are OxyA homologs from glycopeptide antibiotic (GPA) biosynthesis. Given the apparent preference for the unusual heme orientation shown by OxyA enzymes, we investigated the OxyA homolog from kistamicin biosynthesis (OxyAkis), which is an atypical GPA. We determined that OxyAkis is highly sensitive to oxidative damage by peroxide, with both UV and EPR measurements showing rapid bleaching of the heme signal. We determined the structure of OxyAkis and found a mixed population of heme orientations present in this enzyme. Our analysis further revealed the possible modification of the heme moiety, which was only present in samples where the alternate heme orientation was present in the protein. These results suggest that the typical heme orientation in cytochrome P450s can help prevent potential damage to the heme—and hence deactivation of the enzyme—during P450 catalysis. It also suggests that some P450 enzymes involved in GPA biosynthesis may be especially prone to oxidative damage due to the heme orientation found in their active sites.

Exploring the Flexibility of the Glycopeptide Antibiotic Crosslinking Cascade for Extended Peptide Backbones

“…The acceptance of such amino acids in GPA biosynthesis could be beneficial in selective modification with further payloads [24] . However, modifying the C terminus of the linear GPA precursor peptide has been associated with a loss P450 activity, particularly with the second enzyme (OxyA) in the P450 cyclisation cascade that forms the D‐ O ‐E ring, [22] which has also been shown to be less stable than OxyB in certain cases [25] . In contrast to modification of the C terminus, modifications at the peptide N terminus appear to be more widely tolerated since it has been shown that the incorporation of aromatic residues such as those found in teicoplanin [26] or positively charged residues can be tolerated [18] .…”

“…[24] However, modifying the C terminus of the linear GPA precursor peptide has been associated with a loss P450 activity, particularly with the second enzyme (OxyA) in the P450 cyclisation cascade that forms the D-O-E ring, [22] which has also been shown to be less stable than OxyB in certain cases. [25] In contrast to modification of the C terminus, modifications at the peptide N terminus appear to be more widely tolerated since it has been shown that the incorporation of aromatic residues such as those found in teicoplanin [26] or positively charged residues can be tolerated. [18] In addition to l-and d-Arg, we also explored the incorporation of l-and d-4hydroxyphenylglycine (Hpg) at position "0" of the peptide to provide substrates for the Oxy cyclisation cascade that are structurally related to newly identified examples of type V GPAs.…”

Newest perspectives of glycopeptide antibiotics: biosynthetic cascades, novel derivatives, and new appealing antimicrobial applications

“…The related research had verified that the P450s enzymes performed the reactions as the scanning model depending on the experimental evidence of the continuous association/dissociation of P450s with the peptide substrates (Peschke et al 2016 ). In addition, the P450 proteins have many Tyr residues to maintain the typical heme orientation and avoid potential oxidative damage based on crystallographic experiments (Greule et al 2022 ).…”

Efficient biocatalytic C–H bond oxidation: an engineered heme-thiolate peroxygenase from a thermostable cytochrome P450

“…S4–S7, ESI†) and demonstrates that the new biocatalyst is resistant to heme destruction by H 2 O 2 in the presence of a suitable substrate. 18,19…”