Structural Basis for the Properties of Two Single‐Site Proline Mutants of CYP102A1 (P450_BM3)

Abstract

The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single-site proline variants of CYP102A1 (P450(BM3)) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C-H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non-natural substrates, displays a number of structural similarities to substrate-bound forms of the wild-type enzyme, notably an off-axial water ligand, a drop in the proximal loop, and the positioning of two I-helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second-generation I401P variants gave high in vitro oxidation rates with non-natural substrates as varied as fluorene and propane, towards which the wild-type enzyme is essentially inactive. The substrate-free I401P haem domain had a reduction potential slightly more oxidising than the palmitate-bound wild-type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate-free wild-type enzyme.

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

“…k f was (1.5 ± 0.3) s −1 in 50 mmol/L Tris or 100 mmol/L phosphate in the absence of substrate (Whitehouse et al, 2010), but significantly higher for the fatty acid-bound forms. In 50 mmol/L Tris, k f was (58 ± 7.6) s −1 for laurate, (102 ± 7.0) s −1 for myristate, and (227 ± 15) s −1 for palmitate ( Fig.…”

Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias

“…For both these mutants, large increases in catalytic activity with non-natural substrates such as toluene, propylbenzene, and 3-methylpentane were observed (18,49).…”

“…Although alterations that influence local structure in the BM3 active site cavity clearly have potential to alter the binding position of fatty acids and to enable docking of novel substrates, other BM3 mutations were shown to have more profound effects on the P450's conformational landscape (18,19). One of the more common activity-altering BM3 mutants (A82F) shows greatly enhanced affinity for fatty acid substrates.…”

Monooxygenation of Nonnative Substrates Catalyzed by Bacterial Cytochrome P450s Facilitated by Decoy Molecules

“…A variety of mutants were also constructed for the hydroxylation of substituted benzenes, such as toluene (R3/I401P mutant with 4 amino acid replacements, R47S, Y51W, A330P, I401P), 25 oxylene (RLYF/A330P mutant with 3 amino acid replacements, R47S, Y51W, A330P), 26 p-xylene, and halogenated benzenes Figure 5. The overall structure (a) and the active site structure (b) of P450BM3 (PDB code: 1FAG).…”