9m47
Substrate Promiscuous Cytochrome P450 RufO
Structural highlights
FunctionRUFO_STRAR Cytochrome P450 involved in the biosynthesis of rufomycins, a class of cyclic heptapeptides with anti-mycobacterial activity (PubMed:28774961). Catalyzes the 3-nitration of tyrosine, leading to the formation of 3-nitrotyrosine (3NTyr) (PubMed:28774961, PubMed:40568218, Ref.2). It was initially postulated that RufO acts on free L-tyrosine (PubMed:28774961). However, further characterization demonstrates that free L-tyrosine is not the native substrate of RufO, although it could serve as a poor alternative under certain circumstances (PubMed:37451485, PubMed:37555759). It was subsequently demonstrated that RufO nitrates a biarylitide-type ribosomal pentapeptide precursor (Met-Arg-Tyr-Leu-His or MRYLH), which is encoded by bytA in the rufomycin gene cluster, producing the ribosomally synthesized and post-translationally modified peptide MR(NO(2)-Tyr)LH (PubMed:40568218, Ref.2). Following the RufO-catalyzed nitration, the nitrated tyrosine (3-NO2-Tyr), key for antibiotic activity, is cleaved out by a dedicated aminopeptidase, before being activated and incorporated into the non-ribosomal peptide chain by the rufomycin synthetase assembly line (Ref.2). RufO can also nitrate the synthetic pentapeptide Nle-RYLH, where norleucine (Nle) is used to avoid non-enzyme catalyzed Met oxidation (Ref.2). It cannot use phenylalanine, tryptophan, 4-hydroxyphenylpyruvate or 4-aminophenylalanine (PubMed:28774961).[1] [2] [3] [4] [5] Publication Abstract from PubMedRufO is a Cytochrome P450 enzyme involved in synthesising Rufomycin, a circular peptide with antibacterial activity. Herein, we present structural and biophysical analyses to resolve the ambiguity of RufO's substrate specificity. The structure of unliganded RufO, alongside a series of computational and biophysical studies investigating its substrate specificity in the presence of ferredoxin, which is known to serve as an effector of the redox activities of several P450 enzymes. Contrary to reports on RufO's catalytic activity, monomeric L-tyrosine was not recognized by RufO in our isothermal titration calorimetry (ITC) experiments. Instead, RufO recognizes a range of putative substrates, particularly those containing methyl and nitro groups, suggesting a broader substrate scope. Additionally, we see that RufO binds to its redox partner CamB with micromolar affinity, and its interaction significantly enhances the putative substrate binding by approximately 10-fold. Our crystal structure of RufO reveals similarities and differences in putative substrates and ferredoxin binding regions compared to other CYP450 enzymes. Our findings establish RufO might be a substrate-promiscuous enzyme with potential applications in the biocatalytic nitration of industrially relevant compounds. RufO, a cytochrome P450 (CYP) enzyme, recognition to putative substrates and a redox partner: Binding and structural insights.,Saniya D, Shivani P, Abhishek S, Abithaa V, Bajaj P, Rajakumara E Biophys Chem. 2026 Feb;329:107546. doi: 10.1016/j.bpc.2025.107546. Epub 2025 Oct , 26. PMID:41167129[6] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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