5M0N

Crystal structure of cytochrome P450 OleT in complex with formate

Summary for 5M0N

• Entry DOI: 10.2210/pdb5m0n/pdb
• Descriptor: Terminal olefin-forming fatty acid decarboxylase, PROTOPORPHYRIN IX CONTAINING FE, SULFATE ION, ... (5 entities in total)
• Functional Keywords: cytochrome p450, decarboxylase, oxidase, peroxide, oxidoreductase
• Biological source: Jeotgalicoccus sp. ATCC 8456
• Total number of polymer chains: 1
• Total formula weight: 50016.36

Authors

Tee, K.L.,Munro, A.,Matthews, S.,Leys, D.,Levy, C. (deposition date: 2016-10-05, release date: 2017-01-11, Last modification date: 2024-01-17)

Primary citation

Matthews, S.,Belcher, J.D.,Tee, K.L.,Girvan, H.M.,McLean, K.J.,Rigby, S.E.,Levy, C.W.,Leys, D.,Parker, D.A.,Blankley, R.T.,Munro, A.W.
Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE.
J. Biol. Chem., 292:5128-5143, 2017

PubMed Abstract: The sp. peroxygenase cytochrome P450 OleT (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleT could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleT active site residues Phe, His, and Arg to interrogate their roles in substrate binding and catalytic activity. His is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several peroxygenases that favor fatty acid hydroxylation. H85Q OleT still favors alkene production, suggesting alternative protonation mechanisms. However, the mutant undergoes only minor substrate binding-induced heme iron spin state shift toward high spin by comparison with WT OleT, indicating the key role of His in this process. Phe interacts with His, and Phe mutants showed diminished affinity for shorter chain (C10-C16) fatty acids and weak substrate-induced high spin conversion. F79A OleT is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg is crucial for binding the substrate carboxylate, and R245E/L mutations severely compromise activity and heme content, although alkene products are formed from some substrates, including stearic acid (C18:0). The results identify crucial roles for the active site amino acid trio in determining OleT catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

PubMed: 28053093
DOI: 10.1074/jbc.M116.762336

Experimental method

X-RAY DIFFRACTION (1.44 Å)