The GCN2 eIF2␣ kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2 ؊/؊ knockout strain of mice. Gcn2 ؊/؊ mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2 ؊/؊ mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2 ؊/؊ mice failed to show the normal induction of eIF2␣ phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2␣ and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2 ؊/؊ mice.

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 Å) and flaviolin-bound (1.62 Å) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.

The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wildtype strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.Biosynthetic pathways for many antibiotics in bacteria and plants are often associated with the presence of cytochromes P450 (P450, CYP).3 The CYPs perform late-stage stereo-and regiospecific metabolic reactions, such as hydroxylation, epoxidation, dehydrogenation, and C-C bond coupling, to modify the parent structural skeletons to give biosynthetic intermediates and/or bioactive products (1-4). Members of the genus Streptomyces produce a wide variety of bioactive secondary metabolites used in human and veterinary therapeutics, including a majority of the medically useful antibiotics (5, 6). Although terpenoids are one of the largest classes of naturally occurring, biologically active substances, with more than 50,000 known compounds, including anti-cancer drugs, antiparasitic, and antibacterial agents, only a relative handful of such terpene metabolites have been isolated from Streptomyces spp (7,8).P450 monooxygenases are a superfamily of heme proteins utilizing atmospheric dioxygen and electrons supplied by NAD(P)H almost always through redox partner proteins to generate, most commonly, oxidized organic products and a molecule of water (9). P450s play an important role in drug metabolism and in the biosynthesis of steroids, lipids, vitamins, antibiotics, and other natural secondary metabolites.Streptomyces coelicolor A3 (2) is genetically one of the most studied actinomycetes and has been a model for genetic analysis of antibiotic production. The complete genome sequence of S. coelicolor has revealed the presence of 18 CYP, 6 ferredoxin, and 4 ferredoxin reductase genes (10, 11). The endogenous biological function of CYP170A1, belonging to the 18-member P450 CYPome of S. coelicolor, has not previously been identified (12). 4 The sco5223 gene encoding CYP170A1 in S. coelicolor is part of a two-gene cluster with a sesquiterpene cyclase gene (sco5222) with which it shares a four-nucleotide ATGA transcriptional overlap at its 5Ј-end. The sco5222 gene product has been shown to catalyze the synthesis of a novel sesquiterpene, epi-isozizaene (1, Fig. 1), by cyclization of the universal sesquiterpene synthase substrate farne...

Background: Steroid 21-hydroxylase deficiency accounts for ϳ95% of individuals with congenital adrenal hyperplasia (CAH). Results: The bovine cytochrome P450 21A2 (CYP21A2) crystal structure complexed with the substrate 17-hydroxyprogesterone was determined to 3.0 Å resolution. Conclusion:The structure reveals the binding mode of two molecules of the steroid substrate and accurate residue locations in the protein.Significance: The structure of CYP21A2 enhances our understanding of CAH.

Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligandfree CYP170A1 (2.6 Å ) and complex of endogenous substrate (epiisozizaene) with CYP170A1 (3.3 Å ). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 ␣-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an ␣-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.Cytochrome P450 monooxygenases (CYP or P450) 2 are members of the most structurally diverse and functionally versatile superfamily of heme-containing enzymes with more than 10,000 known genes distributed among all biological kingdoms. CYP proteins have extremely diverse primary sequences and are grouped into different families when they have less than 40% amino acid sequence identity. Only the trans-cysteine ligand coordinating with the heme iron atom is conserved among all P450s (1). CYP enzymes are involved both in endogenous biosynthetic functions and in the metabolism of xenobiotic chemicals (2) and catalyze the oxidation of a wide variety of substrates with both regiospecificity and stereospecificity. There a few examples, however, of CYP enzymes that exhibit some degree of non-stereo-specific hydroxylation, for example, the oxidation of norbornane by CYP2B1 (3).Alabaflavenone monooxygenase was first identified from the soil bacterium Streptomyces coelicolor A3 (2), which is the most studied member of the Streptomyces genus of bacteria and produces a chemically diverse array of different secondary metabolites, including antibiotics, pigments, siderophores, hopanoids, and other lipids (4). CYP170A1 is a member of a two-gene operon also containing a sesquiterpene cyclase that converts farnesyl diphosphate (FPP) to the tricyclic hydrocarbon epi-isozizaene (5). This monooxygenase has been clearly shown to catalyze the conversion of...

The genome sequence of the giant virus Acanthamoeba polyphaga mimivirus revealed the presence of two putative cytochrome P450 (CYP) genes. The product of one of the two predicted CYP genes (YP_143162) showed low-level homology to sterol 14-demethylase (CYP51) and contained a C-terminal polypeptide domain of unknown function. YP_143162 expression (without an N-terminal membrane binding domain) in Escherichia coli yields a CYP protein which gives a reduced CO difference maximum at 448 nm and was formally demonstrated as the first viral cytochrome P450. Analysis of binding of lipid and sterol substrates indicated no perturbation in CYP heme environment, and an absence of activity was seen when 14-methyl sterols were used as a substrate. The function of the CYP protein and its C-terminal domain remain unknown. taxidϭ10239&optϭVirus).Acanthamoeba polyphaga mimivirus is the largest known virus, which grows in amoeba (5). In 2004, the 1.2-Mbp genome of mimivirus (GenBank accession no. AY653733) was sequenced (9). Its genome is larger than that of several bacteria and archaea and is predicted to encode 911 proteins, among which only 298 have predicted functions. Many atypical proteins are predicted to be encoded by the mimivirus genome, including key protein translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (9). Interestingly, among genes never yet reported to occur in a virus, mimivirus contained two putative gene sequences predicted to encode cytochrome P450 enzymes (GenBank accession no. YP_142886 and YP_143162, also known as MIMI_L532 and MIMI_L808, respectively). First, YP_142886 is a putative protein of 468 amino acids in length. In a BLASTP search, this putative CYP protein showed homology to a range of bacterial P450 proteins, including a P450 protein from Chloroflexus aurantiacus (23% identity) and CYP171 from Streptomyces peucetius (23% identity). Additionally, YP_142886 also showed homology at the same level to nematode P450 proteins, including Caenorhabditis briggsae CYP37B1 (25% identity) and a P450 protein from the sea squirt Ciona intestinalis similar to the CYP4 family (24% identity). Efforts in our laboratory to express the YP_142886 gene and verify that it indeed encodes a cytochrome P450 have been unsuccessful, but additional attempts are in progress. The mimivirus protein YP_143886 was designated CYP5254A1 by David Nelson (http://drnelson.utmem .edu/CytochromeP450.html).The second putative mimivirus CYP protein (YP_143162) showed in a BLASTP search the strongest homology to CYP51 proteins (7) from a variety of organisms, including protozoal CYP51 proteins from, e.g., Leishmania major (23% identity); plant CYP51 proteins from, e.g., Arabidopsis thaliana (22% identity); and fungal CYP51 proteins from, e.g., Aspergillus fumigatus (21% identity). This homology is low, strongly suggesting the absence of a functional link. Further analysis of the YP_143162 709-amino-acid sequence revealed this putative CYP protein to be appro...

Background: Fish (and human) P450 17A1 catalyze both steroid 17␣-hydroxylation and 17␣,20-lyase reactions. A second fish P450, 17A2 (51% identical), catalyzes only 17␣-hydroxylation. Results: Crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1 are very similar. Conclusion: In kinetic analysis, the two-step oxidation of progesterone is more distributive than for pregnenolone. Significance: Small structural differences are associated with activities of the two fish P450s.

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