The Role of Serine-246 in Cytochrome P450eryF-Catalyzed Hydroxylation of 6-Deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronol ide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed participate in the proton shuttling pathway, and also support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction. Copyright 2000 Academic Press.     

The role of serine-246 in cytochrome P450eryF-catalyzed hydroxylation of 6-deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of the crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed indirectly participate in the proton shuttling pathway, and also strongly support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.     

Structure of genes encoding steroidogenic enzymes

Synthesis of adrenal steroid hormones from cholesterol entails the actions of only five enzymes, four of which are specific forms of cytochrome P450. These cytochrome P450 enzymes have all been isolated and their activities reconstituted in vitro, showing that each enzyme catalyses multiple steroidal conversions. Genes or complementary DNAs have been cloned for human P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20 lyase) and P450c21 (21-hydroxylase). The sequences for microsomal P450c17 and P450c21 are much more closely related to one another than either is to the sequence for mitochondrial P450scc. Each of these P450 enzymes is encoded by a single human gene; the gene for P450scc lies on chromosome 15, that for P450c17 lies on chromosome 10, and that for P450c21 lies on chromosome 6. The human, mouse and bovine genomes each have two P450c21 genes. While only one of these is active in mouse and man, both genes may be active in cattle. A wide variety of lesions in the human P450c21(B) gene causes congenital adrenal hyperplasia, a common genetic disorder.     

Regulation of expression of the genes encoding steroidogenic enzymes

In recent years it has become apparent that tropic hormones involved in steroidogenesis act to regulate the expression of the enzymes involved in the various steroidogenic pathways. This is particularly evident in the ovary where the episodic secretion of steroids throughout the ovarian cycle is regulated largely by changes in the levels of the particular enzymes involved in each step of the steroid biosynthetic pathways. Recently, the genes for the various cytochrome P450 species involved in ovarian steroidogenesis, namely cholesterol side-chain cleavage P450 (P450SCC), 17 alpha-hydroxylase P450 (P450(17 alpha], and aromatase cytochrome P450 (P450AROM) have been isolated and characterized, making it possible to study the regulation of expression at the molecular level. To this end, a series of chimeric constructs have been prepared in which fragments of the 5'-untranslated region of bovine P450(17 alpha) and P450SCC have been inserted upstream of the chloramphenicol acetyl transferase (CAT) and beta-globin reporter genes. These constructs have been used to transfect primary cultures of bovine luteal and thecal cells. The results indicate that cAMP responsiveness lies within defined regions of genes which do not contain a classical CRE, similar to previous results utilizing adrenal cells in culture. Furthermore, although constructs containing both the P450(17 alpha) and P450SCC 5'-upstream regions are expressed in both luteal and thecal cell cultures, only those containing the P450SCC sequences are expressed in luteal cells. Studies on the expression of P450AROM indicate that the promoter which is responsible for its expression in human placenta is not operative in the corpus luteum. Thus estrogen biosynthesis may be regulated by the differential use of tissue specific promoters, thus accounting for the complexity and multifactorial nature of the expression of this activity.     

Progress towards the easier use of P450 enzymes

The cytochrome P450 enzymes (P450s or CYPs) form a large family of heme proteins involved in drug metabolism and in the biosynthesis of steroids, lipids, vitamins and natural products. Their remarkable ability to catalyze the insertion of oxygen into non-activated C-H bonds has attracted the interest of chemists for several decades. Very few chemical methods exist that directly hydroxylate aliphatic or aromatic C-H bonds, and most of them are not selective or of limited scope. Biocatalysts such as P450s represent a promising alternative: however, their applications have been limited by substrate specificity, low activity, poor stability and the need for cofactors. This review covers the attempts to overcome these limitations using approaches such as mutagenesis, chemical modifications, conditions engineering and immobilization.     

细胞色素P450酶与微生物药物创制北大核心CSCD

细胞色素P450酶广泛存在于动植物和微生物体内,具有底物结构多样性和催化反应类型多样性,在天然产物生物合成中扮演重要作用。P450酶可在温和条件下高选择性地催化结构复杂有机化合物中惰性C-H键的氧化反应,具备化学催化剂难以比拟的优势,因此在微生物制药领域具有广阔的应用空间。本文综述了参与天然产物生物合成的P450酶近年来的研究进展;P450酶的酶工程改造、生物转化实践及其在微生物药物创制方面的应用现状;探讨了P450酶的工业应用瓶颈及其解决途径;并对P450酶未来的应用前景进行了展望。     

Genome mining approach for the discovery of novel cytochrome P450 biocatalysts

Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon–hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.     

Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding

Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-methoxyresorufin O-demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli) on the basis of 7-methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K/V193M/K170Q yielded a kcat > five times faster than wild type P450 1A2 in steady-state kinetic analysis using either isolated membrane fractions or purified, reconstituted enzymes. The enhanced catalytic activity was not attributed to changes in substrate affinity. The kinetic hydrogen isotope effect of the triple mutant did not change from wild type enzyme and suggests that C-H bond cleavage is rate-limiting in both enzymes. Homology modeling, based on an X-ray structure of rabbit P450 2C5, suggests that the locations of mutated residues are not close to the substrate binding site and therefore that structural elements outside of this site play roles in changing the catalytic activity. This approach has potential value in understanding P450 1A2 and generating engineered enzymes with enhanced catalytic activity.     

细胞色素P450 2B4的结构及其催化反应

细胞色素P450是广泛存在于动物、植物和微生物中的含亚铁血红素单加氧酶,参与致癌作用和药物代谢、类固醇激素合成、脂溶性维生素代谢、多不饱和脂肪酸转换为生物活性分子等生理过程。P450能够催化完成伯、仲碳氢键羟基化、烯烃和芳烃环氧化、碳碳键耦合和断裂、α羟基化(去烷基化和杂原子氧化)、还原、1,2-迁移(卤素、氢和苯)等有机反应。本文综述了P450 2B4的结构与功能,讨论了细胞色素P450 2B4的活性中心和底物识别位点、与底物反应和产物释放的机理,以及P450在有机合成中的应用。     

Hormonal regulation of cytochrome P450 enzymes, cholesterol side-chain cleavage and 17 alpha-hydroxylase/C17-20 lyase in Leydig cells

Testosterone biosynthesis in Leydig cells is dependent on two cytochrome P450 enzymes, cholesterol side-chain cleavage (P450scc) and 17 alpha-hydroxylase/C17-20 lyase (P450(17 alpha]. The expression of these two enzymes is differentially regulated by LH acting via its second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), and by specific steroid hormones. P450scc is constitutively expressed in normal mouse Leydig cells and in MA-10 tumor Leydig cells. Chronic cAMP stimulation increases the steady state levels of P450scc mRNA and de novo P450scc protein synthesis. In contrast, cAMP is obligatory for de novo synthesis of P450(17 alpha) in normal mouse Leydig cells; P450(17 alpha) synthesis ceases in the absence of luteinizing hormone or cAMP. MA-10 tumor Leydig cells do not express P450(17 alpha) even after treatment with cAMP. The amount of P450(17 alpha) in Leydig cells is negatively regulated by testosterone acting by two distinct mechanisms. At low concentrations, testosterone acts via the androgen receptor to repress cAMP-induced synthesis of P450(17 alpha), whereas at high concentrations this steroid increases the rate of degradation of the enzyme by an oxygen-mediated mechanism. Both constitutive and cAMP-induced synthesis of P450scc protein and steady state levels of mRNA are modulated by glucocorticoids. In normal mouse Leydig cells, glucocorticoids repress P450scc synthesis and steady state levels of P450scc mRNA, whereas glucocorticoids stimulate P450scc synthesis and levels of P450scc mRNA in the tumor Leydig cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex reactions catalyzed by cytochrome P450 enzymes

Cytochrome P450 (P450) enzymes are some of the most versatile redox proteins known. The basic P450 reactions include C-hydroxylation, heteroatom oxygenation, heteroatom release (dealkylation), and epoxide formation. Mechanistic explanations for these reactions have been advanced. A number of more complex P450 reactions also occur, and these can be understood largely in the context of the basic chemical mechanisms and subsequent rearrangements. The list discussed here updates a 2001 review and includes chlorine oxygenation, aromatic dehalogenation, formation of diindole products, dimer formation via Diels-Alder reactions of products, ring coupling and also ring formation, reductive activation (e.g., aristolochic acid), ring contraction (piperidine nitroxide radical), oxidation of troglitazone, cleavage of amino oxazoles and a 1,2,4-oxadiazole ring, bioactivation of a dihydrobenzoxathiin, and oxidative aryl migration.     

Activation of procarcinogens by human cytochrome P450 enzymes

Enzymatic transformation of most chemical carcinogens is requisite to the formation of electrophiles that cause genotoxicity, and the cytochrome P450 (P450) enzymes are the most prominent enzymes involved in such activation reactions. During the past 15 years the human P450 enzymes have been extensively characterized. Considerable evidence exists that the variation in activity of these enzymes can have important consequences in the actions of drugs. Other studies have been concerned with the activation of procarcinogens by human P450s. Assignments of roles of particular P450s in the metabolism of chemical carcinogens are discussed, along with the current state of evidence for relationships of particular P450s with human cancer.     

Expression of Cytochrome P450 Side-Chain Cleavage Enzyme and 3β-Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450_SCC) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) has been studied in the rat brain. P450_SCC transforms cholesterol into pregnenolone and 3β-HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non-steroidogenic tissue lung have been used as controls. The expression of P450_SCC and 3β-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450_SCC and 3β-HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450_SCC and 3β-HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450_SCC and 3β-HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.     

Characterization of adrenal ACTH signaling pathway and steroidogenic enzymes in Erhualian and Pietrain pigs with different plasma cortisol levels

Our previous study demonstrated significant difference in the basal plasma cortisol levels between Erhualian (EHL) and Pietrain (PIE) pigs, implicating fundamental breed difference in adrenocortical function. The objectives of the present study were therefore to characterize the expression pattern of proteins involved in adrenal ACTH signaling and, including melanocortin type 2 receptor (MC2R), cAMP response element binding protein (CREB) and phosphorylated CREB (pCREB), steroidogenic acute regulatory protein (StAR), as well as that of the key enzymes involved in steroidogenesis in EHL and PIE pigs, in association with the plasma corticotrophin (ACTH) and cortisol levels. The plasma concentrations of the substrates for adrenal steroidogenesis, cholesterol and low-density lipoprotein (LDL) cholesterol, did not differ between breeds. Plasma concentration of ACTH and the adrenal contents of MC2R mRNA and protein were similar in two breeds of pigs, whereas the basal plasma concentrations of cortisol in EHL pigs were 1.5 folds higher than that in PIE pigs. The higher basal plasma cortisol levels in EHL pigs were found to be accompanied with the higher expression of ACTH post-receptor signaling components, cAMP, pCREB and StAR, as well as the higher expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17alpha-hydroxylase cytochrome P450 (P450(17alpha)), 21-hydroxylase cytochrome P450 (P450c21) and 11beta-hydroxylase cytochrome P450 (P450(11beta)). These results indicated that the enhanced cAMP/PKA/pCREB-signaling system and augmented expression of StAR and steroidogenic enzymes are major attributes to the higher basal plasma cortisol concentrations in pigs.     

Immunolocalization of steroidogenic enzymes in gonads of European eel, Anguilla anguilla (L.)

The localization of cytochrome P450 cholesterol side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase (P450arom) was investigated using polyclonal antibodies during gonad development in wild European eels, Anguilla anguilla (L.), from the River Po Delta (Ferrara, Italy). The first steroidogenic cells, observed in undifferentiated gonads of 14–16 cm yellow eels, showed no P450scc, 3β-HSD or P450arom activity, but positive regions appeared in head kidney insulae from this stage until the silver eel stage. In undifferentiated gonads of 16–20 cm yellow eels the steroidogenic cells were positive to all enzymes. Pre-Leydig steroidogenic cells, identified in Syrski organs of yellow eels of 22–26 cm evolving into testes, were positive to 3β-HSD and P450scc, but negative to P450arom. However, steroidogenic cells in Syrski organs evolving towards ovaries and in small but fully differentiated ovaries were positive to all enzymes. Immature testes of yellow and silver eels had Leydig cells positive to P450scc and 3β-HSD; the same reactions were also observed in some Sertoli cells of silver eel testes containing meiotic cells. Sex differentiation in A. anguilla apparently occurs through an initial female stage controlled by P450arom activity. Leydig and Sertoli cells appear involved in different steps of hormonal control of spermatogenesis: Leydig cells begin their steroidogenic activity before meiosis, while Sertoli cells begin their activity during meiosis.     

Purification of cytochrome P-450 from bovine adrenocortical mitochondria

One soluble cytochrome P.450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The purified enzyme catalyses side-chain cleavage of cholesterol and to a much lesser extent 11β-hydroxylation (<13% side-chain cleavage) but shows no 18-hydroxylase activity. The molecular weight of this P.450 is approximately 800,000.     

A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin.

Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.