Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3).

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.

A highly active single-mutation variant of P450BM3 (CYP102A1).

The power of proline: Bold amino acid substitutions in sensitive protein regions are frequently unproductive, while more subtle mutations can be sufficient to bring about dramatic changes. But introducing proline at the residue next to the sulfur ligand in P450(BM3) (CYP102A1) has the unexpected and desirable effect of enhancing the activity of this fatty acid hydroxylase with a broad range of non-natural substrates, as illustrated by the figure.