9KGP

The structure of natural P450BM3-H derived from Bacillus megaterium for catalyzing the steroid DHEA

Summary for 9KGP

• Entry DOI: 10.2210/pdb9kgp/pdb
• Descriptor: Bifunctional cytochrome P450/NADPH--P450 reductase, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• Functional Keywords: oxidoreductase, cytochrome p450bm3, heme-binding domain, steroid compound, dehydroepiandrosterone dhea
• Biological source: Priestia megaterium NBRC 15308 = ATCC 14581 (Bacillus megaterium)
• Total number of polymer chains: 2
• Total formula weight: 105389.56

Authors

Deng, Q.B.,Zhang, L.J.,Li, H. (deposition date: 2024-11-08, release date: 2025-05-07, Last modification date: 2025-05-21)

Primary citation

Deng, Q.,Lu, Z.M.,Yuan, Z.,Feng, Y.,Zhang, L.,Shi, J.,Xu, Z.,Kofas, M.A.G.,Li, H.
Rationally designing P450BM3-H to excavate a novel channel for product exit and enhance overall performance.
Int.J.Biol.Macromol., 307:142162-142162, 2025

PubMed Abstract: P450 dihydroxylase plays a crucial role in steroid drug synthesis by efficiently catalyzing two-step selective hydroxylation reactions. However, natural P450 dihydroxylases are scarce, with poor catalytic performance and intermediate accumulation limiting production. Here, we report a P450 dihydroxylase BM3-H that synthesizes 7α,15α-diOH-DHEA with 76 % selectivity. To enhance 7α,15α-diOH-DHEA synthesis, we engineered a novel exit channel for the intermediate by modifying key residues in the solvent channel. The triple mutant D182K/E143D/V178A exhibited significant improvements in product concentration (10.08-fold), enzymatic activity (2.16-fold), catalytic efficiency (k/K, 42.32-fold), electron transfer rate (k, 27.14-fold), and coupling efficiency (CE, 3.93-fold). Molecular dynamics simulations revealed that D182K/E143D/V178A created a novel exit channel for 7α-OH-DHEA, with channel length, polarity, and steric hindrance influencing enzyme performance. Our approach enhances the overall catalytic performance of P450BM3-H by excavating new intermediate product exit channels, providing theoretical guidance for the design of other enzyme molecules.

PubMed: 40107536
DOI: 10.1016/j.ijbiomac.2025.142162

Experimental method

X-RAY DIFFRACTION (1.52 Å)