Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production

Abstract

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleT_JE. Herein, we describe OleTP_RN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTP_RN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTP_RN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleT_JE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTP_RN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

Keywords: CYP152 peroxygenase; alkene production; decarboxylation activity; molecular mechanism; renewable hydrocarbons.

Fig. 1.

(A) Phylogenetic tree of putative CYP-decarboxylases obtained from HMM analysis. Node highlighted in blue represents OleTP_RN, and nodes highlighted in salmon display species with OleT already characterized. Red dots highlight bacterium genera with alkene production already reported. Red star highlights OleT_JE. (B) Product yield of turnover reactions of OleTP_RN with different FA substrates (experimental conditions: 2 µM enzyme, 500 µM substrate, 600 µM H₂O₂, 35 °C and 1 h). (C) Substrate preference assay of OleTP_RN, performed with a mixture of even chain-length FAs ranging from C12 to C20. (D) Time course of myristic (blue) and oleic acid (green) simultaneous consumption (dashed line) and alkene production (straight line) through stepped H₂O₂ addition (red pointed line). (E) PDA spectra of the single turnover reaction between 10 µM C14D and 10 mM H₂O₂. Arrows indicate the direction of decreasing and increasing absorbances during the reactions. (F) Difference spectra generated by subtracting 1 s PDA trace from all other PDA traces collected during C14D single turnover. The blue spectrum (1 ms) was obtained by subtracting the 1 s PDA trace and shows the features of OleTP_RN Compound I, which has a major Soret absorbance at 373 nm and a secondary absorption at 694 nm due to the π-cation radical. Wine red spectrum (30 ms) represents the split Soret absorbance at 370 and 445 nm attributed to Compound II. Two summed-exponential fits of 370 nm (G) and 440 nm time courses (H) PMT traces were collected during the single turnover reaction of C14D bound OleTP_RN with 10 mM H₂O₂. The PMT data is shown in dark gray, with two-summed exponential fits in red, and the residuals below blue. Numbers in the figures represent the RRT constants associated with Compound I decay and Compound II formation and decay.

Fig. 2.

(A) Cartoon representation of superimposed OleTP_RN, OleT_JE, and P450_Bsβ structure, showing substrates and helices names. (B) View of important amino acids for substrate positioning in OleTP_RN pocket. (C) Catalytic site showing amino acid interactions, hydrogen bounds are displayed in gray and van der Waals forces in yellow. (D) Product yield of turnover reactions with OleTP_RN H90Q (Experimental conditions: 2 µM of enzyme, 500 µM of substrate, 600 µM H₂O₂, 35 °C and 1 h of reaction).

Fig. 3.

(A) Representation of longer F-G loop present in OleT_JE in contrast to the shorter loop of OleTP_RN and P450_Bsβ. (B) Phe17 present in OleTP_RN in substitution of the longer F-G loop and A’-B loop interactions of OleT_JE. (C) Comparison of alkene yield from turnover reactions with OleTP_RN WT and mutants (Experimental conditions: 2 µM of enzyme, 500 µM of substrate, 600 µM H₂O₂, 35 °C and 1 h of reaction).

Fig. 4.

(A) Free energy profile along the Arg246-FA distance, measured between the Arg246 Cζ atom and the FA carboxylate C atom. Snapshots corresponding to the first minimum (bound state) and second minimum (pseudo-bound state) are shown for C10. The free energy profiles show the existence of bound-pseudo-bound equilibrium which shifts to the bound state with an increase in the chain-length. Error bars correspond to the SD associated with four free energy profiles, computed for blocks of 10 ns of sampling per window. (B) Alkene yield from turnover reactions with OleTP_RN mutants of the hydrophobic cradle amino acid (Experimental conditions: 2 µM of enzyme, 500 µM of substrate, 600 µM H₂O₂, 35 °C and 1 h of reaction).

Fig. 5.

(A) Cartoon representation of OleTP_RN dimerization, highlighting helices involved in the dimer interface. One protomer is shown in yellow and the other in salmon. (B) Highlight of important amino acids involved in the dimer interface contact. Hydrogen bonds are shown in gray. (C) Ideal placement of Phe24 established by the dimerization, anchoring Phe17 and Phe328, via Phe44, which are both important for substrate positioning. Van der Waals forces are displayed in yellow. (D) Tyr22 interactions that hold important amino acids for substrate positioning in the binding pocket. (E) Product yield of turnover reactions of myristic acid with OleTP_RN mutants that destabilize either protein dimerization or substrate positioning.