Gibberellin Biosynthetic Pathway inGibberella fujikuroi:Evidence for a Gene Cluster

Differential screening of aGibberella fujikuroicDNA library was used to successfully clone and identify genes involved in the pathway of gibberellin biosynthesis. Several cDNA clones that hybridized preferentially to a cDNA probe prepared from mycelium induced for gibberellin production were isolated and characterized. The deduced amino acid sequences of two (identical) clones contained the conserved heme-binding motif of cytochrome P450 monooxygenases (FXXGXXXCXG). One of these cDNA fragments was used as a homologous probe for the screening of a genomic library. A hybridizing 6.7-kb genomicSalI fragment was cloned into pUC19. The sequencing of this clone revealed that a second cytochrome P450 monooxygenase gene was closely linked to the first one. Since at least four cytochrome P450 monooxygenase-catalyzed steps are involved in the synthesis of gibberellins, chromosome walking was performed to find a further gene of this family or other genes involved in gibberellin pathway. Next to the two P450 monooxygenase genes, a putative geranylgeranyl diphosphate synthase gene, the copalyl diphosphate synthase gene, which is the first specific gene of the gibberellin pathway, and a third P450 monooxygenase gene were identified. These results suggest that at least some of the genes involved in the biosynthesis of gibberellins are closely linked in a gene cluster inG. fujikuroi,as has been recently found for other “dispensable” pathways in fungi.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

A novel human cytochrome P450 gene (P450IIB): chromosomal localization and evidence for alternative splicing.

We have isolated from a single human liver cDNA library two clones which are highly homologous (78% over the coding region) to the major phenobarbital-inducible P450 from rat (P450IIB1). This is the first direct demonstration of the presence of the P450IIB gene subfamily in humans. This subfamily is much less extensive than the rodent homologues, but does appear to contain at least two genes. Of the cDNA clones isolated one is apparently normally spliced, whereas the other lacks exon 8 and retains all or part of intron 5. Both clones contain transcribed Alu sequences. The human P450IIB gene has been located to chromosome 19q12----19q13.2 using a probe derived from intron 5, and is close to the CYP 2A locus encoding cytochrome P450IIA2. Restriction fragment length polymorphisms have been found with the enzymes BamHI and MspI which will enable linkage to be determined between these two loci.     

Multiplicity of deoxyribonucleic acid sequences with homology to a cloned complementary deoxyribonucleic acid coding for rat phenobarbital-inducible cytochrome P-450

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.     

Taxol biosynthetic genes

The function and properties of heterologously expressed full-length cDNA clones, isolated from a Taxus cDNA library and specific to Taxol biosynthesis, are summarized. Recombinant enzymes are described that catalyze early steps of the pathway, including taxadiene synthase, taxadien-5alpha-ol-O-acetyltransferase and taxadien-5alpha-yl acetate 10beta-hydroxylase, and that catalyze late steps, including 10-deacetylbaccatin III-10beta-O-acetyltransferase and taxane 2alpha-O-benzoyltransferase. The properties of Taxus geranylgeranyl diphosphate synthase are also described; although this synthase does not mediate a committed step of Taxol biosynthesis, it does provide the universal plastidial diterpenoid precursor, geranylgeranyl diphosphate, for initiating Taxol biosynthesis.     

Isolation of a new human fetal liver cytochrome P450 cDNA clone: evidence for expression of a limited number of forms of cytochrome P450 in human fetal livers

From a human fetal liver cDNA library, a new cDNA clone (lambda HFL10) was isolated using an antiserum to P450 HFLa, which has been isolated from livers of human fetuses. Cytochrome P450 cDNAs, namely lambda hPA6, lamda hP2-1, and lambda hPD4 which were highly homologous to cDNA clones, pHY13, Hp1-1, and phP450j, respectively, were also isolated from the cDNA library of human adult livers. Using these cDNA clones as probes together with Lambda HFL10, Northern blot analysis was conducted to determine whether all of these cytochromes were expressed in human fetal livers. The results clearly showed that only P450 HFL10 mRNA was detected in human fetal livers. This result supports the allegation that there is a much more limited number of forms of cytochrome P450 in human fetal livers than in adult livers.     

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.     

Candidate genes involved in tanshinone biosynthesis in hairy roots of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> revealed by cDNA microarray

Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.     

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。     

Sequence and gene expression of rabbit cytochrome P450 IIC16: comparison to highly related family members.

Cytochrome P450s are heme containing proteins which evolved from an ancestral gene(s) to form a large superfamily of enzymes. We have isolated a unique cDNA from the rabbit P450 IIC subfamily, IIC16, which is 2028 bp in length. Nucleotide sequence determination indicated an ATG start codon 66 bp from the 5' end of the molecule, and an open reading frame coding for a protein of 487 amino acids. P450 IIC16 protein is greater than or equal to 90% identical in sequence to rabbit P450 IIC4, IIC5, and to the partial sequence available for IIC15. Northern and slot blot experiments demonstrated that the P450 IIC16 gene is expressed constitutively in liver, lung, testes, and kidney, and is inducible by phenobarbital in each tissue with the exception of the kidney, where mRNA levels are repressed. Alignment analysis of eight rabbit P450 IIC proteins revealed conserved and variable regions common to all IIC enzymes, and specific areas are suggested which may be important with respect to structure and function.     

Comparison of primary structures deduced from cDNA nucleotide sequences for various forms of liver microsomal cytochrome P-450 from phenobarbital-treated rabbits

cDNA clones, termed pHP2, b32-3, b43, and b43-1, encoding cytochromes P-450 that are expressed in the liver of phenobarbital- (PB-) treated rabbits were isolated, and their nucleotide sequences were determined. pHP2 cDNA contains an open reading frame for a 490-residue protein and is a full-length counterpart of pP-450PBc2 [Leighton, J. K., Debrunner-Vossbrinck, B. A., & Kemper, B. (1984) Biochemistry 23, 204-210]. The b32-3 insert has a sequence for a protein whose primary structure is 91% similar to that of progesterone 21-hydroxylase P-450 1, though this cDNA lacks the sequence encoding the amino-terminal 110 residues. The overlapping clones b43 and b43-1 together encode an ethanol-inducible form of cytochrome P-450, though the amino-terminal five or more residues are missing in the composite b43/b43-1 sequence. Northern blot analysis showed that the b43/b43-1 protein is more strongly inducible by polycyclic aromatic hydrocarbons and isosafrole than by PB, in contrast to the case of the HP2 and b32-3 proteins. A comparison of the primary structures of eight forms of cytochrome P-450, including the HP2, b32-3, and b43/b43-1 proteins, that are expressed in the liver of PB-treated rabbits showed that 149 out of 487-492 amino acid residues are conserved in these cytochromes P-450. The eight forms can be assigned to three rabbit cytochrome P-450 gene subfamilies, P450IIB, P450IIC, and P450IIE. It was also shown that the members of the rabbit P450IIC subfamily can be further classified into three subgroups on the basis of their sequence similarity.