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At least 19 recordsLinked to original sources
PubMed1998

Identification and sequencing of a cytochrome P450 gene cluster from Bradyrhizobium japonicum.

Sequencing of a region from Bradyrhizobium japonicum previously shown to encode for cytochromes P450 revealed a cluster of three complete P450 genes (CYP112, CYP114, and CYP117) plus a partial P450 gene fragment (CYP115P). Present also are five additional open reading frames. The close positioning of the genes suggests that they comprise an operon. Although the biochemical function of the gene products is uncertain, the similarities to other genes suggests an operon involved in terpenoid synthesis. ORF3 has similarity to a [3Fe-4S] ferredoxin from Streptomyces griseolus. ORF4 has strong similarity to members of the short chain alcohol dehydrogenase family, including sterol dehydrogenases from enteric bacteria and to some plant 3-oxoacyl-(acyl carrier protein) reductases. ORF6 has strong similarity to prenyl transferases, including dimethylallyltranstransferase from Escherichia coli. ORF7 bears some similarity to plant genes for ent-kaurene synthase (a precursor of gibberellins), and to bacterial squalene-hopene cyclases. ORF8 has some similarity to a Streptomyces gene for synthesis of the cyclic sesquiterpene pentalenene. The 5' end of the mRNA transcript is 38-39 nucleotides downstream from the center of a motif that bears sequence homology to bacterial fnr promoters. A gus operon fusion to the promoter was expressed anaerobically and symbiotically 6-10-fold greater than aerobically.

Amino Acid Sequence↗
PubMed2001

Inhibition of human tumor cell growth in vivo by an orally bioavailable inhibitor of human farnesyltransferase, BIM-46228.

Oncogenic mutations of the ras gene leading to constitutive activation of downstream effectors have been detected in a wide spectrum of human cancers (pancreas, thyroid, colon, non-small-cell lung cancer). Membrane anchorage of Ras, required for functional activity in signal transduction, is facilitated by post-translational modifications resulting in covalent attachment of a farnesyl group to the cysteine in the C-terminal CAAX motif. This attachment is mediated by farnesyltransferase (FTase). Here, we report a novel FTase inhibitor, BIM-46228, which showed (i) specific inhibition of purified human FTase enzyme, (ii) inhibition of proliferation in vitro in a large spectrum of human tumor cell lines, (iii) inhibition of growth of human tumor xenografts in athymic nude mice treated by per os administration and (iv) the benefits of in vitro combination of its activity with chemotherapy or radiotherapy.

3T3 Cells↗
PubMed1994

Developmental regulation of the expression of genes encoding proteins involved in cholesterol homeostasis.

The developmental patterns of expression of HMG-CoA reductase, farnesyl pyrophosphate synthase, cholesterol 7 alpha-hydroxylase, and LDL receptor were investigated using Northern blotting analysis to quantitate mRNA levels. It was found that HMG-CoA reductase and farnesyl pyrophosphate synthase mRNA levels in brain reached peaks at age 4 days which correlates with the time of peak enzyme activity and the onset of rapid brain growth and myelination. In liver, HMG-CoA reductase and cholesterol 7 alpha-hydroxylase mRNA both rose dramatically at weaning. This is consistent with the concept that de novo synthesized cholesterol is the preferred substrate for cholesterol 7 alpha-hydroxylase and may also be involved in the induction of the enzyme. In testes, HMG-CoA reductase activity was highest at age 21 days and then declined, while LDL receptor mRNA levels rose from age 31 to 120 days. These studies suggest a major role for de novo cholesterol synthesis in developing brain, liver, and testes.

Animals↗
PubMed1992

The prenylation of proteins.

The prenylated proteins represent a newly discovered class of post-translationally modified proteins. The known prenylated proteins include the oncogene product p21ras and other low molecular weight GTP-binding proteins, the nuclear lamins, and the gamma subunit of the heterotrimeric G proteins. The modification involves the covalent attachment of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid moiety in a thioether linkage to carboxyl terminal cysteine. The nature of the attached substituent is dependent on specific sequence information in the carboxyl terminus of the protein. In addition, prenylation entrains other posttranslational modifications forming a reaction pathway. In this article, we review our current understanding of the biochemical reactions involved in prenylation and discuss the possible role of this modification in the control of cellular functions such as protein maturation and cell growth.

Amino Acid Sequence↗
PubMed2002

Predicting amino acid residues responsible for enzyme specificity solely from protein sequences.

We describe a general, modular method for developing protocols to identify the amino acid residues that most likely define the division of a protein superfamily into two subsets. As one possibility, we use PROBE to gather superfamily members and perform an ungapped alignment. We then use a modified BLOSUM62 substitution matrix to determine the discriminating power of each column of aligned residues. The overall method is particularly useful for predicting amino acids responsible for substrate or binding specificity when no structures are available. We apply our method to three pairs of protein classes in three different superfamilies, and present our results, some of which have been experimentally verified. This approach may accelerate the elucidation of enzymic substrate specificity, which is critical for both mechanistic insights into biocatalysis and ultimate application.

Adenylyl Cyclases↗
PubMed2006

Reverse prenyltransferase in the biosynthesis of fumigaclavine C in Aspergillus fumigatus: gene expression, purification, and characterization of fumigaclavine C synthase FGAPT1.

A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.

Aspergillus fumigatus↗
PubMed2006

Synthesis of stable analogues of geranylgeranyl diphosphate possessing a (Z,E,E)-geranylgeranyl side chain, docking analysis, and biological assays for prenyl protein transferase inhibition.

Herein, we report the synthesis of novel stable analogues of geranylgeranyl diphosphate (GGPP), in which the "natural" all-trans geranylgeranyl portion has been replaced by a (Z,E,E)-geranylgeranyl chain. The change in configuration and consequent change in the relative position of the polar portion with the lipophilic side chain did not improve the properties of the E,E,E analogues in their inhibition of geranylgeranyl protein transferase I (GGTase I). However, a significant level of GGTase I inhibition and selectivity for GGTase I over farnesyl transferase (FTase) was maintained the unsubstituted phosphonoacetamidoxy derivative 4 a. This has shed light on the relative importance of the configuration at the C2=C3 double bond among GGPP derivatives. Moreover, the biological activities of all the compounds reported herein, in particular the preferential FTase inhibitory activity shown by compound 6, were in good agreement with the results of docking analysis.

Dimethylallyltranstransferase↗
PubMed1996

Identification of isoprenyl modified proteins metabolically labeled with [3H]farnesyl- and [3H]geranylgeranyl-pyrophosphate.

Here we describe a direct approach for two-dimensional (2-D) gel mapping of proteins that are modified by post-translational isoprenylation in mammalian cells. Briefly, transformed human amnion cells (AMA) and transfected COS-1 cells were metabolically labeled with either [3H]farnesyl-pyrophosphate or [3H]geranylgeranyl-pyrophosphate following treatment with lovastatin, which blocks the synthesis of mevalonic acid. The proteins were then separated by 2-D gel electrophoresis and electrotransferred to nitrocellulose filters. The membranes were immersed in dimethyl ether, containing 10% of 2,5-diphenyloxazole prior to fluorography. Over 40 [3H]farnesyl-labeled proteins and over 25 [3H]geranylgeranylated proteins were identified on the 2-D autoradiograms. Several [3H]farnesyl-labeled proteins exhibited the same coordinates (M(r) and pI) as their [3H]geranylgeranylated counterparts, raising the possibility that they may be substrates for both farnesyl and geranylgeranyl transferase(s). The approach offers high resolution of both farnesylated and geranylgeranylated proteins and it may serve as a powerful tool for the identification of hitherto unknown prenylated proteins as well as for the determination of prenylated protein levels, type of isoprenoid modification, and possible changes in protein prenyltransferase activity.

Amnion↗
PubMed1985

Tumour genes in plants: T-DNA encoded cytokinin biosynthesis.

Gene 4 from the T-region of Ti plasmids is responsible for cytokinin effects in crown gall cells; we investigated whether it codes for an enzyme of hormone biosynthesis. In a first set of experiments, gene 4 from octopine plasmid pTiAch5 and nopaline plasmid pTiC58 was expressed in Escherichia coli, and the gene products were identified by reaction with antiserum raised against a decapeptide derived from the DNA sequence of the gene. Extracts from cells expressing the gene contained high isopentenyl-transferase activity catalyzing the formation of N6-(delta2-isopentenyl)adenosine from 5'-AMP and delta2-isopentenylpyrophosphate. The cytokinin was identified by sequential h.p.l.c. chromatography and mass spectrometry. In a second set of experiments it was shown that crown gall cells contained isopentenyltransferase activity and a protein of mol. wt. 27 000 which was identified as the product of gene 4 by reaction with the antiserum. Isopentenyltransferase activity was specifically inhibited by the antiserum. No comparable enzyme activity or immunoreactive protein was detected in cytokinin-autotrophic, T-DNA free tobacco cells. The results establish that gene 4 from the T-region of octopine and nopaline Ti plasmids codes for an enzyme of cytokinin biosynthesis.

Alkyl and Aryl Transferases↗
PubMed2005

An interaction between apolipoprotein E and TERE1 with a possible association with bladder tumor formation.

TERE1, a recently discovered gene/protein appears to play a role in bladder tumor growth regulation but to date does not have clear functional correlates. The objective of this study was to gain further insight into the function of the TERE1 protein by identifying potential protein to protein interactions with TERE1 and determining whether these interactions are associated with putative growth regulatory pathways and/or bladder tumor formation. Towards this aim, we have performed a bacterial two hybrid assay and isolated interacting clones, which then were sequenced and further examined by affinity chromatography and immunoprecipitation. From among several positive clones, we isolated a putative interacting plasmid containing the C-terminal portion of preapolipoprotein E starting from amino acid number 124 from the pBT-TERE1/pTarget-cDNA bacterial two hybrid system. The C-terminal portion of apoE interaction with the TERE1 was confirmed using ProBond columns by the expression of 6XHis recombinant and (35)S methionine/cysteine labeled proteins. We found that there was ubiquitous expression of the apoE transcript in normal bladder and in various grades and stages of transitional cell carcinoma (TCC) of the bladder. Likewise, we detected the apoE protein in both normal and malignant bladder tissues by Western blot. There was a significant decrease in the apoE protein in 12 of 16 muscle invasive TCCs of the bladder compared to normal bladder mucosa samples. Previous studies in rat fibroblasts have found that expression of apoE can decrease the phosphorylation of the growth factor-related p42/44 MAP kinase. A significant decrease in p44/p42 MAPK phophorylation was also apparent using a phosphorylation specific antibody in human 293 kidney cells upon transfection and expression of apoE. In conclusion, the results from this study suggest that the expression and regulation of the apoE pathway may yield clues toward understanding the function of TERE1.

Apolipoproteins E↗
PubMedDate not supplied

Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum.

Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and 1 was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed.

Antifungal Agents↗
PubMed1994

Isoprenyl diphosphate synthases: protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure.

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain-elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate-rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of alpha-helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.

Alkyl and Aryl Transferases↗
PubMed2003

TERE1, a novel gene affecting growth regulation in prostate carcinoma.

Recently, we isolated a ubiquitously expressed gene designated TERE1, which has a significant effect on the growth regulation in bladder cancer. The TERE1 gene maps to chromosome 1p36.11-1p36.33 between the micro-satellite markers D1S2667 and D1S434, a chromosome locus that has been identified by loss of heterozygosity studies as a site of a putative tumor suppressor gene or genes for multiple tumor types including prostate carcinoma. The expression of the TERE1 transcript and protein was examined in a series of thirty microdissected prostate tumors by semi-quantitative RT/PCR and immunohistochemistry. There was a significant 61% decrease in the TERE1 transcript in prostate carcinoma (CaP) and a distinct loss of the TERE1 protein in metstatic prostate. Though a loss of heterozygosity at chromosome 1p36 was found in 25% of these prostate tumors, there appeared to be no TERE1 mutations present in these tumor samples. Induced TERE1 expression after transduction or transfection of TERE1 constructs into two prostate carcinoma (LNCaP and PC-3) cell lines significantly decreased proliferation up to 80% with a significant increase in the number of cells in G1. Serum factors but not DHT (dihydrotestosterone) appear to regulate the amount of TERE1 protein in the androgen responsive LNCaP cell line. Additionally, we have identified by microarray analysis various growth regulatory genes that are down-regulated or up-regulated in TERE1-transduced PC-3 cells. Altogether, these data suggest that TERE1 maybe significant in prostate cancer growth regulation and the down regulation or absence of TERE1 may be an important component of the phenotype of advanced disease.

Androgens↗
PubMed2006

Structure and function of cis-prenyl chain elongating enzymes.

All carbon skeletons of isoprenoids, whose chain lengths vary widely from geranyl diphosphate (C10) to natural rubber (C>10,000), are synthesized by sequential condensation of isopentenyl diphosphate with an allylic diphosphate through catalytic functions of a group of enzymes commonly called "prenyltransferases." Prenyltransferases are classified into two major groups, trans- or (E)-prenyltransferases and cis- or (Z)-prenyltransferases, according to the geometry of the prenyl chain units in the products. From the year 1987, many genes encoding trans-prenyltransferases were cloned and clearly characterized. In contrast, the structure and detailed mechanism of cis-prenyltransferase was completely unknown until the identification of a gene encoding the undecaprenyl diphosphate (UPP) synthase from Micrococcus luteus B-P 26 in 1998. Not only the primary but also the tertiary structure of the UPP synthase is quite different from that of the trans-prenyltransferases. Multiple alignment of the primary structures of cis-prenyltransferases identified from various organisms reveals five highly conserved regions. Site-directed mutagenesis of the conserved amino acid residues in UPP synthases based on the crystal structure has elucidated the basic catalytic mechanisms. Moreover, comparison of the structures of short-, medium-, and long-chain cis-prenyltransferases reveals important amino acid residues for product chain length determination, which enabled us to understand the regulation mechanism of the ultimate chain length among cis-prenyltransferases.

Amino Acid Sequence↗
PubMed1994

Sequence around the centromere of Saccharomyces cerevisiae chromosome II: similarity of CEN2 to CEN4.

We report the sequence of a 12 kilobase region spanning the centromere of Saccharomyces cerevisiae chromosome II. The sequence from the left arm includes genes for histones H2A and H2B. The sequence from the right arm includes a gene that probably encodes a novel trehalase, as well as the COQ1 gene (for an enzyme involved in coenzyme Q biosynthesis), and an open reading frame with significant similarity to bacterial genes of unknown function. The trehalase gene (YBR0106) on chromosome II is located beside the centromere and transcribed towards it. This is identical to the arrangement of the neutral trehalase gene (NTH1) beside the centromere of chromosome IV. The centromere regions of chromosomes II and IV may therefore have arisen through a duplication of the centromere region of an ancestral chromosome. The YBR0106 and NTH1 proteins are 77% identical in predicted amino acid sequence, but there is no pronounced sequence similarity between the two centromeres (CEN2 and CEN4) outside of the universally conserved CDE I and CDE III elements. The genes flanking the centromere and trehalase genes differ between the two chromosomes, so the similarity between chromosomes II and IV may be less extensive than that recently reported between chromosomes III and XIV.

Alkyl and Aryl Transferases↗
PubMed1994

Effect of squalene synthase inhibition on the expression of hepatic cholesterol biosynthetic enzymes, LDL receptor, and cholesterol 7 alpha hydroxylase.

Squalene synthase catalyzes the committed step in the biosynthesis of sterols. Treating rats with zaragozic acid A, a potent inhibitor of squalene synthase, caused marked increases in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, squalene synthase, and LDL receptor mRNA levels. The increase in HMG-CoA reductase mRNA fully accounted for the increases seen in enzyme protein and activity. Farnesyl pyrophosphate synthase mRNA and activity were only slightly increased by zaragozic acid A, while cholesterol 7 alpha hydroxylase mRNA levels were decreased substantially. When rats were pretreated with zaragozic acid A, there was no change in mRNA levels for the cholesterol biosynthetic enzymes or cholesterol 7 alpha hydroxylase upon subsequent treatment with mevalonolactone. Under these same conditions, the enzymatic activity of HMG-CoA reductase was also unaffected. Mevalonolactone treatment reduced the zaragozic acid A-mediated increase in hepatic LDL receptor mRNA levels. Feeding cholesterol eliminated the zaragozic acid A-induced increase in HMG-CoA reductase mRNA levels. These results suggest that inhibition of squalene synthase decreases the level of a squalene-derived regulatory product, resulting in altered amounts of several mRNAs and coordinate increases in HMG-CoA reductase mRNA, protein, and activity. The increase in HMG-CoA reductase gene expression was closely related to the degree of inhibition of cholesterol synthesis caused by zaragozic acid A.

Animals↗
PubMed1998

Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production.

Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative "mature proteins" from which the transit peptide was deleted, were transformed into a yeast mutant defective for the beta-subunit of type II geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction. In vitro activity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS-PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in induced T. canadensis suspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.

Acetates↗
PubMed1996

Biosynthesis of [14C]geranylgeranyldiphosphate by a prenyl transferase system from a mutant strain of Gibberella fujikuroi.

[14C]Geranylgeranyldiphosphate was produced by a prenyl transferase system extracted from the mutant strain SG4 of Gibberella fujikuroi (wild-type strain IMI 58289), which is blocked in carotenoid biosynthesis. The fungus was grown in liquid medium containing only potato-dextrose broth. Crude extracts of 12- to 14-day-old mycelia in Tris buffer, pH 8.5, were centrifuged at 100,000g. The supernatant was desalted and used to produce [4,8,12,16-14C]geranylgeranyldiphosphate from R,S-[2-14C]mevalonic acid. Maximum yield of [14C]geranylgeranyldiphosphate was obtained using mycelia from the stationary growth phase with incubation of the enzyme preparation at pH 8.5 for 5 h. Routinely, 65% of the biogenetic available R-mevalonic acid was converted to [14C]geranylgeranyldiphosphate, which was identified by cochromatography with authentic [1-3H]geranylgeranyldiphosphate by means of radio-TLC, radio-HPLC, and hydrolysis to geranylgeraniol.

Chromatography, High Pressure Liquid↗