Structure Based Discovery of Inhibitors of CYP125 and CYP142 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.

An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme.

The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the α subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes.

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism.

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3's high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production.

Catalytic Mechanism of Aromatic Nitration by Cytochrome P450 TxtE: Involvement of a Ferric-Peroxynitrite Intermediate.

The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso-hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.

Cytochrome P450 1A1 opens up to new substrates.

The cytochromes P450 (CYPs) oxidatively transform a huge number of substrates in both prokaryotic and eukaryotic organisms, but the mechanisms by which they accommodate these diverse molecules remain unclear. A new study by Bart and Scott reports two co-crystal structures of CYP1A1 that reveal structural rearrangements and flexible interaction networks that explain how the active site cavity shapes itself around new ligands. These data open the door to an increased understanding of fundamental enzyme behavior and improved searches for anti-cancer compounds.

Synthesis and biological evaluation of novel cYY analogues targeting Mycobacterium tuberculosis CYP121A1.

The rise in multidrug resistant (MDR) cases of tuberculosis (TB) has led to the need for the development of TB drugs with different mechanisms of action. The genome sequence of Mycobacterium tuberculosis (Mtb) revealed twenty different genes coding for cytochrome P450s. CYP121A1 catalyzes a CC crosslinking reaction of dicyclotyrosine (cYY) producing mycocyclosin and current research suggests that either mycocyclosin is essential or the overproduction of cYY is toxic to Mtb. A series of 1,4-dibenzyl-2-imidazol-1-yl-methylpiperazine derivatives were designed and synthesised as cYY mimics. The derivatives substituted in the 4-position of the phenyl rings with halides or alkyl group showed promising antimycobacterial activity (MIC 6.25 µg/mL), with the more lipophilic branched alkyl derivatives displaying optimal binding affinity with CYP121A1 (iPr KD = 1.6 µM; tBu KD = 1.2 µM). Computational studies revealed two possible binding modes within the CYP121A1 active site both of which would effectively block cYY from binding.

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1.

The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands "moonlight" as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE.

The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleTJE (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 OleTJE 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 OleTJE active site residues Phe79, His85, and Arg245 to interrogate their roles in substrate binding and catalytic activity. His85 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 OleTJE 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 OleTJE, indicating the key role of His85 in this process. Phe79 interacts with His85, and Phe79 mutants showed diminished affinity for shorter chain (C10-C16) fatty acids and weak substrate-induced high spin conversion. F79A OleTJE is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg245 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 OleTJE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

Structure and function of the cytochrome P450 peroxygenase enzymes.

The cytochromes P450 (P450s or CYPs) constitute a large heme enzyme superfamily, members of which catalyze the oxidative transformation of a wide range of organic substrates, and whose functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The P450 peroxygenases are a subgroup of the P450s that have evolved in microbes to catalyze the oxidative metabolism of fatty acids, using hydrogen peroxide as an oxidant rather than NAD(P)H-driven redox partner systems typical of the vast majority of other characterized P450 enzymes. Early members of the peroxygenase (CYP152) family were shown to catalyze hydroxylation at the α and ß carbons of medium-to-long-chain fatty acids. However, more recent studies on other CYP152 family P450s revealed the ability to oxidatively decarboxylate fatty acids, generating terminal alkenes with potential applications as drop-in biofuels. Other research has revealed their capacity to decarboxylate and to desaturate hydroxylated fatty acids to form novel products. Structural data have revealed a common active site motif for the binding of the substrate carboxylate group in the peroxygenases, and mechanistic and transient kinetic analyses have demonstrated the formation of reactive iron-oxo species (compounds I and II) that are ultimately responsible for hydroxylation and decarboxylation of fatty acids, respectively. This short review will focus on the biochemical properties of the P450 peroxygenases and on their biotechnological applications with respect to production of volatile alkenes as biofuels, as well as other fine chemicals.

Design, synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics.

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH2-imidazole or CH2-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC = 12.5 µg/mL, 17a 50 µg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2 Å. A model generated from a 1.5 Šcrystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.

The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the ß-lactam-negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6-epi-pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450(Prava) bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450(Prava) fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.

Rapid Rule-out of Acute Myocardial Infarction With a Single High-Sensitivity Cardiac Troponin T Measurement Below the Limit of Detection: A Collaborative Meta-analysis.

Background: High-sensitivity assays for cardiac troponin T (hs-cTnT) are sometimes used to rapidly rule out acute myocardial infarction (AMI). Purpose: To estimate the ability of a single hs-cTnT concentration below the limit of detection (<0.005 µg/L) and a nonischemic electrocardiogram (ECG) to rule out AMI in adults presenting to the emergency department (ED) with chest pain. Data Sources: EMBASE and MEDLINE without language restrictions (1 January 2008 to 14 December 2016). Study Selection: Cohort studies involving adults presenting to the ED with possible acute coronary syndrome in whom an ECG and hs-cTnT measurements were obtained and AMI outcomes adjudicated during initial hospitalization. Data Extraction: Investigators of studies provided data on the number of low-risk patients (no new ischemia on ECG and hs-cTnT measurements <0.005 µg/L) and the number who had AMI during hospitalization (primary outcome) or a major adverse cardiac event (MACE) or death within 30 days (secondary outcomes), by risk classification (low or not low risk). Two independent epidemiologists rated risk of bias of studies. Data Synthesis: Of 9241 patients in 11 cohort studies, 2825 (30.6%) were classified as low risk. Fourteen (0.5%) low-risk patients had AMI. Sensitivity of the risk classification for AMI ranged from 87.5% to 100% in individual studies. Pooled estimated sensitivity was 98.7% (95% CI, 96.6% to 99.5%). Sensitivity for 30-day MACEs ranged from 87.9% to 100%; pooled sensitivity was 98.0% (CI, 94.7% to 99.3%). No low-risk patients died. Limitation: Few studies, variation in timing and methods of reference standard troponin tests, and heterogeneity of risk and prevalence of AMI across studies. Conclusion: A single hs-cTnT concentration below the limit of detection in combination with a nonischemic ECG may successfully rule out AMI in patients presenting to EDs with possible emergency acute coronary syndrome. Primary Funding Source: Emergency Care Foundation.

What makes a P450 tick?

The cytochromes P450 (P450s) are probably nature's most versatile enzymes in terms of both their vast substrate range and the diverse types of molecular transformations performed across the P450 enzyme superfamily. The P450s exquisitely perform highly specific oxidative chemistry, utilizing a sophisticated catalytic reaction mechanism. Recent studies have provided the first definitive characterization of the transient reaction cycle intermediate (compound I) responsible for the majority of P450 oxidative reactions. This major advance comes at a time when P450 engineering has facilitated the elucidation of several mammalian P450 structures and generated P450 variants with novel substrate specificity and reactivity. This review describes recent advances in P450 research and the ramifications for biotechnological and biomedical exploitation of these enzymes.

Fragment Profiling Approach to Inhibitors of the Orphan M. tuberculosis P450 CYP144A1.

Similarity between the ligand binding profiles of enzymes may aid functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach for characterization of the ligand binding profile of an enzyme and has been applied here to study the family of cytochrome P450 enzymes (P450s) expressed by Mycobacterium tuberculosis (Mtb). The Mtb P450s have important roles in bacterial virulence, survival, and pathogenicity. Comparing the fragment profiles of seven of these enzymes revealed that P450s which share a similar biological function have significantly similar fragment profiles, whereas functionally unrelated or orphan P450s exhibit distinct ligand binding properties, despite overall high structural homology. Chemical structures that exhibit promiscuous binding between enzymes have been identified, as have selective fragments that could provide leads for inhibitor development. The similarity between the fragment binding profiles of the orphan enzyme CYP144A1 and CYP121A1, a characterized enzyme that is important for Mtb viability, provides a case study illustrating the subsequent identification of novel CYP144A1 ligands. The different binding modes of these compounds to CYP144A1 provide insight into structural and dynamic aspects of the enzyme, possible biological function, and provide the opportunity to develop inhibitors. Expanding this fragment profiling approach to include a greater number of functionally characterized and orphan proteins may provide a valuable resource for understanding enzyme-ligand interactions.

Effect of DMSO on Protein Structure and Interactions Assessed by Collision-Induced Dissociation and Unfolding.

Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization-ion mobility-mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.

Nodular Colloid Milium of the Conjunctiva and Anterior Orbit.

Colloid milium is a rare deposition disorder with accumulation of a characteristic amorphous substance in both protected and sun-exposed areas depending upon the disease subtype. The patient was a 59-year-old woman who was referred for evaluation of a gelatinous-appearing mass in the inferior fornix of the right eye, extending from the medial to the lateral canthal region, and extending up onto the bulbar conjunctival surface and anterior lacrimal gland. Hematoxylin and eosin stain of an incisional biopsy showed deposits of an amorphous brightly eosinophilic material within the conjunctiva with scattered plasma cells at the periphery. There was no evidence of monoclonality, and a Congo red stain was negative. Based on histopathologic findings, a diagnosis of nodular orbital colloid milium was made. To our knowledge, the adult and nodular forms of colloid milium have not been previously described as occurring in the conjunctiva or anterior orbit.

Analysis of Heme Iron Coordination in DGCR8: The Heme-Binding Component of the Microprocessor Complex.

DGCR8 is the RNA-binding partner of the nuclease Drosha. Their complex (the "Microprocessor") is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Binding of heme to DGCR8 is essential for pri-miRNA processing. On the basis of the split Soret ultraviolet-visible (UV-vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys(-)) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman, and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys(-)/Cys(-)) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8's optical spectrum, consistent with the axial ligand environment being separated from the substrate-binding site. UV-vis absorption spectra of the Fe(II) and Fe(II)-CO forms indicate discrete species exhibiting peaks with absorption coefficients substantially larger than those for ferric DGCR8 and that previously reported for a ferrous form of DGCR8. Electron-nuclear double resonance spectroscopy data exclude histidine or water as axial ligands for ferric DGCR8 and favor bis-thiolate coordination in this form. UV-vis MCD and near-infrared MCD provide data consistent with this conclusion. UV-vis MCD data for ferrous DGCR8 reveal features consistent with bis-thiol heme iron coordination, and resonance Raman data for the ferrous-CO form are consistent with a thiol ligand trans to the CO. These studies support retention of DGCR8 cysteine coordination upon reduction, a conclusion distinct from those of previous studies of a different ferrous DGCR8 isoform.

Limits of Detection and Quantification of Electrochemical Quartz-Crystal Nanobalance in Platinum Electrochemistry and Electrocatalysis Research.

The electrochemical quartz-crystal nanobalance has been used in electrochemistry research for over three decades. It provides an atomic/molecular level insight into the nature of interfacial electrochemical phenomena by measuring in situ mass changes on the nanogram scale. The sensitivity of this technique remains unknown because there have been no attempts to determine its limits of detection (LOD) or quantification (LOQ). We propose an experimental approach for determining the values of LOD and LOQ for Pt electrodes in aqueous H2SO4 solutions that employs cyclic voltammetry and frequency variation measurements. However, this methodology is also appropriate to other electrode materials and electrolytes. The LOD and LOQ values depend on the electrolyte concentration and decrease (i.e., the sensitivity increases) as the concentration decreases. Knowledge of the LOD and LOQ values determines the applicability of this technique in research on the oxidation and degradation of Pt catalysts employed in fuel cells.

Structure and biochemical properties of the alkene producing cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 bacterium.

The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical characterization and crystal structures of a cytochrome P450 fatty acid peroxygenase: the terminal alkene forming OleTJE (CYP152L1) from Jeotgalicoccus sp. 8456. OleTJE is stabilized at high ionic strength, but aggregation and precipitation of OleTJE in low salt buffer can be turned to advantage for purification, because resolubilized OleTJE is fully active and extensively dissociated from lipids. OleTJE binds avidly to a range of long chain fatty acids, and structures of both ligand-free and arachidic acid-bound OleTJE reveal that the P450 active site is preformed for fatty acid binding. OleTJE heme iron has an unusually positive redox potential (-103 mV versus normal hydrogen electrode), which is not significantly affected by substrate binding, despite extensive conversion of the heme iron to a high spin ferric state. Terminal alkenes are produced from a range of saturated fatty acids (C12-C20), and stopped-flow spectroscopy indicates a rapid reaction between peroxide and fatty acid-bound OleTJE (167 s(-1) at 200 µm H2O2). Surprisingly, the active site is highly similar in structure to the related P450BSß, which catalyzes hydroxylation of fatty acids as opposed to decarboxylation. Our data provide new insights into structural and mechanistic properties of a robust P450 with potential industrial applications.