Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Multiscale Study on the Intramolecular C-S Bond Formation Catalyzed by P450 Monooxygenase CxnD Involved in the Biosynthesis of Chuangxinmycin: The Critical Roles of Noncrystal Water Molecule and Conformational Change.

Cytochrome P450 monooxygenase CxnD catalyzes intramolecular C-S bond formation in the biosynthesis of chuangxinmycin, which is representative of the synthesis of sulfur-containing natural heterocyclic compounds. The intramolecular cyclization usually requires the activation of two reaction sites and a large conformational change; thus, illuminating its detailed reaction mechanism remains challengeable. Here, the reaction pathway of CxnD-catalyzed C-S bond formation was clarified by a series of calculations, including Gaussian accelerated molecular dynamics simulations and quantum mechanical-molecular mechanical calculations. Our results revealed that the C-S formation follows a diradical coupling mechanism. CxnD first employs Cpd I to abstract the hydrogen atom from the imino group of the indole ring, and then, the resulted Cpd II further extracts another hydrogen atom from the thiol group of the side chain to afford a diradical intermediate, in which a noncrystal water molecule entering into the active site after the formation of Cpd I was proved to play an indispensable role. Moreover, the diradical intermediate cannot directly perform the coupling reaction. It should first undergo a series of conformational changes leading to the proximity of two reaction sites. It is the flexibility of the active site of the enzyme and the side chain of the substrate that makes the diradical coupling to be successful.

Enzymatic Assembly of Diverse Lactone Structures: An Intramolecular C-H Functionalization Strategy.

Lactones are cyclic esters with extensive applications in materials science, medicinal chemistry, and the food and perfume industries. Nature's strategy for the synthesis of many lactones found in natural products always relies on a single type of retrosynthetic strategy, a C-O bond disconnection. Here, we describe a set of laboratory-engineered enzymes that use a new-to-nature C-C bond-forming strategy to assemble diverse lactone structures. These engineered "carbene transferases" catalyze intramolecular carbene insertions into benzylic or allylic C-H bonds, which allow for the synthesis of lactones with different ring sizes and ring scaffolds from simple starting materials. Starting from a serine-ligated cytochrome P450 variant previously engineered for other carbene-transfer activities, directed evolution generated a variant P411-LAS-5247, which exhibits a high activity for constructing a five-membered ε-lactone, lactam, and cyclic ketone products (up to 5600 total turnovers (TTN) and >99% enantiomeric excess (ee)). Further engineering led to variants P411-LAS-5249 and P411-LAS-5264, which deliver six-membered δ-lactones and seven-membered ε-lactones, respectively, overcoming the thermodynamically unfavorable ring strain associated with these products compared to the γ-lactones. This new carbene-transfer activity was further extended to the synthesis of complex lactone scaffolds based on fused, bridged, and spiro rings. The enzymatic platform developed here complements natural biosynthetic strategies for lactone assembly and expands the structural diversity of lactones accessible through C-H functionalization.

Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III.

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

Computational identification and analysis of deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) in the human POR gene: a structural and functional impact.

Cytochrome P450 oxidoreductase (POR) protein is essential for steroidogenesis, and POR gene mutations are frequently associated with P450 Oxidoreductase Deficiency (PORD), a disorder of hormone production. To our knowledge, no previous attempt has been made to identify and analyze the deleterious/pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) in the human POR gene through an extensive computational approach. Computational algorithms and tools were employed to identify, characterize, and validate the pathogenic SNPs associated with certain diseases. To begin with, all the high-confidence SNPs were collected, and their structural and functional impacts on the protein structures were explored. The results of various in silico analyses affirm that the A287P and R457H variants of POR could destabilize the interactions between the amino acids and the hydrogen bond networks, resulting in functional deviations of POR. The literature study further confirms that the pathogenic mutations (A287P and R457H) are associated with the onset of PORD. Molecular dynamics simulations (MDS) and essential dynamics (ED) studies characterized the structural consequences of prioritized deleterious mutations, representing the structural destabilization that might disrupt POR biological function. The identified deleterious mutations at the cofactor's binding domains might interfere with the essential interactions between the protein and cofactors, thus inhibiting POR catalytic activity. The consolidated insights from the computational analyses can be used to predict potential deleterious mutants and understand the disease's pathological basis and the molecular mechanism of drug metabolism for the application of personalized medication. HIGHLIGHTSNADPH cytochrome P450 oxidoreductase (POR) mutations are associated with a broad spectrum of human diseasesIdentified and analyzed the most deleterious nsSNPs of POR through the sequence and structure-based prediction toolsInvestigated the structural and functional impacts of the most significant mutations (A287P and R457H) associated with PORDMolecular dynamics and PCA-based FEL analysis were utilized to probe the mutation-induced structural alterations in PORCommunicated by Ramaswamy H. Sarma.

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification.

The vast majority of known enzymes exist as oligomers, which often gives them high catalytic performance but at the same time imposes constraints on structural conformations and environmental conditions. An example of an enzyme with a complex architecture is the P450 BM3 monooxygenase CYP102A1 from Bacillus megaterium. Only active as a dimer, it is highly sensitive to dilution or common immobilization techniques. In this study, we engineered a thermostable P450BM3 chimera consisting of the heme domain of a CYP102A1 variant and the reductase domain of the homologous CYP102A3. The dimerization of the hybrid was even weaker compared to the corresponding CYP102A1 variant. To create a stable dimer, we covalently coupled the C-termini of two monomers of the chimera via SpyTag003/SpyCatcher003 interaction. As a result, purification, thermostability, pH stability, and catalytic activity were improved. Via a bioorthogonal two-step affinity purification, we obtained high purity (94 %) of the dimer-stabilized variant being robust against heme depletion. Long-term stability was increased with a half-life of over 2 months at 20 °C and 80-90 % residual activity after 2 months at 5 °C. Most catalytic features were retained with even an enhancement of the overall activity by ~2-fold compared to the P450BM3 chimera without SpyTag003/SpyCatcher003.

Microbial P450 repertoire (P450ome) and its application feasibility in pharmaceutical industry, chemical industry, and environmental protection.

Cytochrome P450s (CYPs) are heme-thiolated enzymes that catalyze the oxidation of C-H bonds in a regio- and stereo-selective manner. CYPs are widely present in the biological world. With the completion of more biological genome sequencing, the number and types of P450 enzymes have increased rapidly. P450 in microorganisms is easy to clone and express, rich in catalytic types, and strong in substrate adaptability, which has good application potential. Although the number of P450 enzymes found in microorganisms is huge, the function of most of the microorganism P450s has not been studied, and it contains a large number of excellent biocatalysts to be developed. This review is based on the P450 groups in microorganisms. First, it reviews the distribution of P450 groups in different microbial species, and then studies the application of microbial P450 enzymes in the pharmaceutical industry, chemical industry and environmental pollutant treatment in recent years. And focused on the application fields of P450 enzymes of different families to guide the selection of suitable P450s from the huge P450 library. In view of the current shortcomings of microbial P450 in the application process, the final solution is the most likely to assist the application of P450 enzymes in large-scale, that is, whole cell transformation combined with engineering, fusion P450 combined with immobilization technology.

Enhancing ferryl accumulation in H2O2-dependent cytochrome P450s.

A facile strategy is presented to enhance the accumulation of ferryl (iron(IV)-oxo) species in H2O2 dependent cytochrome P450s (CYPs) of the CYP152 family. We report the characterization of a highly chemoselective CYP decarboxylase from Staphylococcus aureus (OleTSA) that is soluble at high concentrations. Examination of OleTSA Compound I (CpdI) accumulation with a variety of fatty acid substrates reveals a dependence on resting spin-state equilibrium. Alteration of this equilibrium through targeted mutagenesis of the proximal pocket favors the high-spin form, and as a result, enhances Cpd-I accumulation to nearly stoichiometric yields.

Understanding the Multifaceted Mechanism of Compound I Formation in Unspecific Peroxygenases through Multiscale Simulations.

Unspecific peroxygenases (UPOs) can selectively oxyfunctionalize unactivated hydrocarbons by using peroxides under mild conditions. They circumvent the oxygen dilemma faced by cytochrome P450s and exhibit greater stability than the latter. As such, they hold great potential for industrial applications. A thorough understanding of their catalysis is needed to improve their catalytic performance. However, it remains elusive how UPOs effectively convert peroxide to Compound I (CpdI), the principal oxidizing intermediate in the catalytic cycle. Previous computational studies of this process primarily focused on heme peroxidases and P450s, which have significant differences in the active site from UPOs. Additionally, the roles of peroxide unbinding in the kinetics of CpdI formation, which is essential for interpreting existing experiments, have been understudied. Moreover, there has been a lack of free energy characterizations with explicit sampling of protein and hydration dynamics, which is critical for understanding the thermodynamics of the proton transport (PT) events involved in CpdI formation. To bridge these gaps, we employed multiscale simulations to comprehensively characterize the CpdI formation in wild-type UPO from Agrocybe aegerita (AaeUPO). Extensive free energy and potential energy calculations were performed in a quantum mechanics/molecular mechanics setting. Our results indicate that substrate-binding dehydrates the active site, impeding the PT from H2O2 to a nearby catalytic base (Glu196). Furthermore, the PT is coupled with considerable hydrogen bond network rearrangements near the active site, facilitating subsequent O-O bond cleavage. Finally, large unbinding free energy barriers kinetically stabilize H2O2 at the active site. These findings reveal a delicate balance among PT, hydration dynamics, hydrogen bond rearrangement, and cosubstrate unbinding, which collectively enable efficient CpdI formation. Our simulation results are consistent with kinetic measurements and offer new insights into the CpdI formation mechanism at atomic-level details, which can potentially aid the design of next-generation biocatalysts for sustainable chemical transformations of feedstocks.

Bidirectional Charge Transfer at the Heme Iron in Reversible and Quasi-irreversible Cytochrome P450 Inhibition.

The coordination bonding between inhibitor ligands and heme iron plays a critical role in disrupting the essential catalytic functions of cytochrome P450 enzymes (P450s). Despite its intrinsic importance and consequential implications for human health, our current understanding of coordination bonding in P450 inhibition remains limited. To address this knowledge gap, we conducted a systematic theoretical analysis of the complexes between a ferric or a ferrous heme model and representative inhibitor ligands. Specifically, we evaluated the charge-transfer (CT) effect within these complexes by employing a series of theoretical methods based on density functional theory (DFT). Through a comprehensive analysis, we unveiled the relative significance of ligand-to-heme forward CT in the ferric and ferrous complexes of reversible inhibitors. In contrast, backward CT dominates over forward CT in the ferrous heme complexes of quasi-irreversible inhibitors. Further analysis using the compact frontier orbital method underscores the elevated electron-accepting abilities of quasi-irreversible inhibitors for π backdonation, which greatly amplifies their binding affinity for the ferrous heme. This study sheds light on the intricate mechanisms underlying P450 inhibition and provides valuable insights for future inhibitor design and development.

Catalytic Degradation of Triphenylmethane Dyes with an Iron Porphyrin Complex as a Cytochrome P450 Model.

The organic dyes used in printing and dyeing wastewater have complex components, diverse structures and strong chemical stability, which make them not suitable for treatment and difficult to degrade in the environment. Porphyrins are macromolecules with 18 π electrons formed by four pyrrole molecules connected with a methylene bridge that has a stable structure. Porphyrin combines with iron to form an active intermediate with a structure similar to the cytochrome P450 enzyme, so they are widely used in the biomimetic field. In the current study, 5,10,15,20-tetra (4-carboxyphenyl) porphine ferric chloride (III) (Fe(III)TCPP) was used as a catalyst and iodosobenzene was used as an oxidant to explore the catalytic degradation of triphenylmethane dyes, such as rhodamine B (RhB) and malachite green (MG). The results of UV-Vis spectral analysis have shown that the conversion rate of the rhodamine B was over 90% when the amount of Fe(III)TCPP was 0.027 mM and the amount of iodosobenzene was eight equivalents. When the catalyst was 0.00681 mM and the amount of the oxidant was five equivalents, the conversion rate of the malachite green reached over 95%. This work provides a feasible method for the degradation of triphenylmethane dyes.

A preparative small-molecule mimic of liver CYP450 enzymes in the aliphatic C-H oxidation of carbocyclic N-heterocycles.

An emerging trend in small-molecule pharmaceuticals, generally composed of nitrogen heterocycles (N-heterocycles), is the incorporation of aliphatic fragments. Derivatization of the aliphatic fragments to improve drug properties or identify metabolites often requires lengthy de novo syntheses. Cytochrome P450 (CYP450) enzymes are capable of direct site- and chemo-selective oxidation of a broad range of substrates but are not preparative. A chemoinformatic analysis underscored limited structural diversity of N-heterocyclic substrates oxidized using chemical methods relative to pharmaceutical chemical space. Here, we describe a preparative chemical method for direct aliphatic oxidation that tolerates a wide range of nitrogen functionality (chemoselective) and matches the site of oxidation (site-selective) of liver CYP450 enzymes. Commercial small-molecule catalyst Mn(CF3-PDP) selectively effects direct methylene oxidation in compounds bearing 25 distinct heterocycles including 14 out of 27 of the most frequent N-heterocycles found in U.S. Food and Drug Administration (FDA)-approved drugs. Mn(CF3-PDP) oxidations of carbocyclic bioisostere drug candidates (for example, HCV NS5B and COX-2 inhibitors including valdecoxib and celecoxib derivatives) and precursors of antipsychotic drugs blonanserin, buspirone, and tiospirone and the fungicide penconazole are demonstrated to match the major site of aliphatic metabolism obtained with liver microsomes. Oxidations are demonstrated at low Mn(CF3-PDP) loadings (2.5 to 5 mol%) on gram scales of substrate to furnish preparative amounts of oxidized products. A chemoinformatic analysis supports that Mn(CF3-PDP) significantly expands the pharmaceutical chemical space accessible to small-molecule C-H oxidation catalysis.

Structural basis of bidirectional allostery across the heme in a cytochrome P450 enzyme.

Cytochromes P450 (CYPs) are heme-containing enzymes that are present in all kingdoms of life and share a structurally homologous, globular protein fold. CYPs utilize structures distal to the heme to recognize and coordinate substrates, while the necessary interactions with redox partner proteins are mediated at the opposite, proximal surface. In the current study, we investigated the functional allostery across the heme for the bacterial enzyme CYP121A1, which utilizes a non-polar distal-to-distal dimer interface for specific binding of its dicyclotyrosine substrate. Fluorine-detected Nuclear Magnetic Resonance (19F-NMR) spectroscopy was combined with site-specific labeling of a distal surface residue (S171C of the FG-loop), one residue of the B-helix (N84C), and two proximal surface residues (T103C and T333C) with a thiol-reactive fluorine label. Adrenodoxin was used as a substitute redox protein and was found to promote a closed arrangement of the FG-loop, similar to the addition of substrate alone. Disruption of the protein-protein interface by mutagenesis of two CYP121 basic surface residues removed the allosteric effect. Moreover, 19F-NMR spectra of the proximal surface indicate that ligand-induced allostery modulates the environment at the C-helix but not the meander region of the enzyme. In light of the high degree of structural homology in this family of enzymes, we interpret the findings from this work to represent a conserved allosteric network in CYPs.

Dual factors required for cytochrome-P450-mediated hydrocarbon ring contraction in bacterial gibberellin phytohormone biosynthesis.

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction.

Structural insights into the effects of glycerol on ligand binding to cytochrome P450.

New antitubercular drugs are vital due to the spread of resistant strains. Carbethoxyhexyl imidazole (CHImi) inhibits cytochrome P450 CYP124, which is a steroid-metabolizing enzyme that is important for the survival of Mycobacterium tuberculosis in macrophages. The available crystal structure of the CYP124-CHImi complex reveals two glycerol molecules in the active site. A 1.15 Šresolution crystal structure of the glycerol-free CYP124-CHimi complex reported here shows multiple conformations of CHImi and the CYP124 active site which were previously restricted by glycerol. Complementary molecular dynamics simulations show coherence of the ligand and enzyme conformations. Spectrophotometric titration confirmed the influence of glycerol on CHImi binding: the affinity decreases more than tenfold in glycerol-containing buffer. In addition, it also showed that glycerol has a similar effect on other azole and triazole CYP124 ligands. Together, these data show that glycerol may compromise structural-functional studies and impede rational drug-design campaigns.

Biotransformation of Bisphenol by Human Cytochrome P450 2C9 Enzymes: A Density Functional Theory Study.

Bisphenol A (BPA, 2,2-bis-(4-hydroxyphenyl)propane) is used as a precursor in the synthesis of polycarbonate and epoxy plastics; however, its availability in the environment is causing toxicity as an endocrine-disrupting chemical. Metabolism of BPA and their analogues (substitutes) is generally performed by liver cytochrome P450 enzymes and often leads to a mixture of products, and some of those are toxic. To understand the product distributions of P450 activation of BPA, we have performed a computational study into the mechanisms and reactivities using large model structures of a human P450 isozyme (P450 2C9) with BPA bound. Density functional theory (DFT) calculations on mechanisms of BPA activation by a P450 compound I model were investigated, leading to a number of possible products. The substrate-binding pocket is tight, and as a consequence, aliphatic hydroxylation is not feasible as the methyl substituents of BPA cannot reach compound I well due to constraints of the substrate-binding pocket. Instead, we find low-energy pathways that are initiated with phenol hydrogen atom abstraction followed by OH rebound to the phenolic ortho- or para-position. The barriers of para-rebound are well lower in energy than those for ortho-rebound, and consequently, our P450 2C9 model predicts dominant hydroxycumyl alcohol products. The reactions proceed through two-state reactivity on competing doublet and quartet spin state surfaces. The calculations show fast and efficient substrate activation on a doublet spin state surface with a rate-determining electrophilic addition step, while the quartet spin state surface has multiple high-energy barriers that can also lead to various side products including C4-aromatic hydroxylation. This work shows that product formation is more feasible on the low spin state, while the physicochemical properties of the substrate govern barrier heights of the rate-determining step of the reaction. Finally, the importance of the second-coordination sphere is highlighted that determines the product distributions and guides the bifurcation pathways.

Tissue-preferential recruitment of electron transfer chains for cytochrome P450-catalyzed phenolic biosynthesis.

Cytochrome P450 system consists of P450 monooxygenase and redox pattern(s). While the importance of monooxygenases in plant metabolism is well documented, the metabolic roles of the related redox components have been largely overlooked. Here, we show that distinct electron transfer chains are recruited in phenylpropanoid-monolignol P450 systems to support the synthesis and distribution of different classes of phenolics in different plant tissues. While Arabidopsis cinnamate 4-hydroxylase adopts conventional NADPH-cytochrome P450 oxidoreductase (CPR) electron transfer chain for its para-hydroxylation reaction, ferulate 5-hydroxylase uses both NADPH-CPR-cytochrome b5 (CB5) and NADH-cytochrome b5 reductase-CB5 chains to support benzene ring 5-hydroxylation, in which the former route is primarily recruited in the stem for syringyl lignin synthesis, while the latter dominates in the syntheses of 5-hydroxylated phenolics in seeds and seed coat suberin. Our study unveils an additional layer of complexity and versatility of P450 system that the plants evolved for diversifying phenolic repertoires.

Efficient heterologous expression of cytochrome P450 enzymes in microorganisms for the biosynthesis of natural products.

Natural products, a chemically and structurally diverse class of molecules, possess a wide spectrum of biological activities, have been used therapeutically for millennia, and have provided many lead compounds for the development of synthetic drugs. Cytochrome P450 enzymes (P450s, CYP) are widespread in nature and are involved in the biosynthesis of many natural products. P450s are heme-containing enzymes that use molecular oxygen and the hydride donor NAD(P)H (coupled via enzymic redox partners) to catalyze the insertion of oxygen into C-H bonds in a regio- and stereo-selective manner, effecting hydroxylation and several other reactions. With the rapid development of systems biology, numerous novel P450s have been identified for the biosynthesis of natural products, but there are still several challenges to the efficient heterologous expression of active P450s. This review covers recent developments in P450 research and development, including the properties and functions of P450s, discovery and mining of novel P450s, modification and screening of P450 mutants, improved heterologous expression of P450s in microbial hosts, efficient whole-cell transformation with P450s, and current applications of P450s for the biosynthesis of natural products. This resource provides a solid foundation for the application of highly active and stable P450s in microbial cell factories to biosynthesize natural products.

Rational design of thermophilic CYP119 for progesterone hydroxylation by in silico mutagenesis and docking screening.

Steroid-based chemicals can affect the metabolism, immune functions, and development of sexual characteristics. Because of these effects, steroid derivatives are widely used in the pharmaceutical industry. Progesterone is a steroid-based hormone that mainly controls the ovulation period of women but is also a precursor molecule for the synthesis of important hormones like testosterone and cortisone. Cytochrome P450 (CYP) enzymes are important for the production of hydroxyprogesterones in the industry since they can catalyze regio- and enantioselective hydroxylation reactions. Although human CYP enzymes can catalyze hydroxyprogesterone synthesis with high selectivity, these enzymes are membrane bound, which limits their application for industrial production. CYP119 is a soluble and thermophilic enzyme from the archaea Sulfolobus acidocaldarius. Even though the native substrate of the enzyme is not known, CYP119 can catalyze styrene epoxidation, lauric acid hydroxylation, and Amplex®Red peroxidation. In this work, an in silico mutagenesis approach was used to design CYP119 mutants with high progesterone affinity. Energy scores of progesterone docking simulations were used for the design and elimination of single, double, and triple mutants of CYP119. Among designed 674 mutants, five of them match the criteria for progesterone hydroxylation. The most common mutation of these five mutants, L69G mutant was analyzed using independent molecular dynamics (MD) simulations in comparison with the wild-type (WT) enzyme. L69G CYP119, was expressed and isolated from Escherichia coli; it showed 800-fold higher affinity for progesterone compared to WT CYP119. L69G CYP119 also catalyzed progesterone hydroxylation. The novel designed enzyme L69G CYP119 is a potential versatile biocatalyst for progesterone hydroxylation that is expected to be stable under industrial production conditions.

CYP51-mediated cholesterol biosynthesis is required for the proliferation of CD4+ T cells in Sjogren's syndrome.

CYtochrome P450, family 51 (CYP51) is an important enzyme for de novo cholesterol synthesis in mammalian cells. In the present study, we found that the expression of CYP51 positively correlated with CD4+ T cell activation both in vivo and in vitro. The addition of ketoconazole, a pharmacological inhibitor of CYP51, prevented the proliferation and activation of anti-CD3/CD28-expanded mouse CD4+ T cells in a dose-dependent fashion. Liquid chromatography-tandem mass spectrometry indicated an increase in levels of lanosterol in T cells treated with ketoconazole during activation. Ketoconazole-induced blockade of the cholesterol synthesis pathway also caused Sterol regulatory element binding protein 2 (SREBP2) activation in CD4+ T cells. Additionally, ketoconazole treatment elicited an integrated stress response in T cells that up-regulated activating transcription factor 4 (ATF4) and DNA-damage inducible transcript 3 (DDIT3/CHOP) at the translational level. Furthermore, treatment with ketoconazole significantly decreased the amount of CD4+ T cells infiltrating lesions in the submandibular glands of NOD/Ltj mice. In summary, our results suggest that CYP51 plays an essential role in the proliferation and survival of CD4+ T cells, which makes ketoconazole an inhibitor of CD4+ T cell proliferation and of the SS-like autoimmune response through regulating the biosynthesis of cholesterol and inducing the integrated stress response.