Unique structural features define the decarboxylation activity of a CYP152 fatty acid decarboxylase from Lacicoccus alkaliphilus

Cytochrome P450 CYP152s catalyze decarboxylation of fatty acids to generate terminal alkenes, valuable compounds for various industries. Here, we identified, overexpressed, and characterized a new CYP152 enzyme from Lacicoccus alkaliphilus (OleT_LA) and compared its biophysical and biochemical properties with the well-studied OleT_JE from Jeotgalicoccus sp. 8456. Improved expression protocols gave the highest yields of CYP152 holoenzymes reported to date. OleT_LA exhibits twice the catalytic turnover number of OleT_JE when using hexadecanoic acid and H₂O₂ as substrates in 10% (v/v) ethanol (EtOH). The X-ray structure of OleT_LA in complex with icosanoic acid revealed a unique flipped heme and a substrate tunnel configuration which are different than those of other CYP152 decarboxylases. Molecular dynamics simulations revealed that in the presence of EtOH, OleT_LA displays structural dynamics which maintain structural interactions better than those of OleT_JE. As I178 in OleT_LA (equivalent to L176 in OleT_JE) shows close interactions with its substrate during simulations, I178L of OleT_LA and L176I of OleT_JE variants were constructed and investigated for their activities. While L176I in OleT_JE caused a significant loss of activity, I178L of OleT_LA had activities that were equivalent to or greater than those of the wild-type enzyme, suggesting that overall scaffold of OleT_LA is more amenable to mutation than OleT_JE. Stopped-flow investigations of OleT_LA reactions indicated that EtOH increases the rate constant of Compound I formation. We also identified a new redox partner system, ferredoxin and ferredoxin reductase that can function as effective electron donors for both in vitro and in vivo systems of CYP152s.