9LN5

Crystal structure of P450revI A241L mutant in complex with reveromycin T

Summary for 9LN5

• Entry DOI: 10.2210/pdb9ln5/pdb
• Descriptor: Putative monooxygenase, PROTOPORPHYRIN IX CONTAINING FE, (2E,4S,5S,6E,8E)-10-{(2R,3S,6S,8R,9S)-9-butyl-8-[(1E,3E)-4-carboxy-3-methylbuta-1,3-dien-1-yl]-3-methyl-1,7-dioxaspiro[5.5]undec-2-yl}-5-hydroxy-4,8-dimethyldeca-2,6,8-trienoic acid, ... (5 entities in total)
• Functional Keywords: cytochrome p450, heme, reveromycin, biosynthesis, oxidoreductase
• Biological source: Streptomyces sp. SN-593
• Total number of polymer chains: 1
• Total formula weight: 46026.19

Authors

Fujiyama, K.,Yong, Y.F.,Takahashi, S. (deposition date: 2025-01-20, release date: 2025-07-02, Last modification date: 2025-07-30)

Primary citation

Yong, Y.F.,Liu, S.,Sakai, K.,Fujiyama, K.,Takagi, H.,Futamura, Y.,Shimizu, T.,Osada, H.,Ong, E.B.B.,Takahashi, S.
Biosynthesis of reveromycin derivatives by altering the regioselectivity of cytochrome P450revI.
Chem Sci, 16:13106-13114, 2025

PubMed Abstract: Reveromycin A (RM-A) (1) has a 6,6-spiroacetal core structure that is important for its biological activity. However, 1 undergoes a spiroacetal rearrangement to form RM-B (2) with a 5,6-spiroacetal core, which exhibits reduced bioactivity. This undesired rearrangement is partly due to the hemisuccinate moiety at the C18 position of 1. In 1 biosynthesis, P450revI catalyses the C18-hydroxylation of RM-T (3), which is essential for its subsequent hemisuccinylation to generate 1. In this study, we aimed to alter the P450revI regioselectivity to improve the stability of the 6,6-spiroacetal core and expand the structural diversity of RMs. Candidate amino acid residues for mutagenesis studies were selected by comparing the co-crystal structure of P450revI with the docking models of the P450revI mutant-3 complexes. Notably, the P450revI-A241L mutant selectively produced novel RM derivatives. Nuclear magnetic resonance analysis revealed that P450revI-A241L catalysed the C17-hydroxylation of 3 to produce 17-hydroxy-RM-T (6). Co-crystal structure analysis of the P450revI-A241L-3 complex revealed that the pro- hydrogen at the C17 position faces toward the haem iron. Introduction of the P450revI-A241L mutant gene into the SN-593-Δ strain led to the production of 17-hemisuccinyloxy-RM-T (7). After the successful bioproduction of RM derivatives, we evaluated their structural stabilities and biological activities. Compounds 6 and 7 exhibited better stabilities than 18-hydroxylated-3 (RM-T1; 4) and 1, respectively. Biological activity analysis revealed that 6 and 7 exhibited anti-malarial and anti-multiple myeloma activities, respectively, comparable to those of 1 and 3, while showing low cytotoxicity against human cell lines. Overall, this study highlights the potential of RM derivatives as pharmaceuticals.

PubMed: 40556721
DOI: 10.1039/d5sc01355k

Experimental method

X-RAY DIFFRACTION (2.13 Å)