Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD).
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A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity. (+info)
Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.
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BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC). RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate. CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems. (+info)
Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14. A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily.
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A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 degrees C and displays a broad substrate specificity with a preference for substituted cyclohexanols. When incubated with a diastereomeric mixture of (4R)- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the (6S)-carveol stereoisomers only. Kinetic studies with pure stereoisomers showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea suggests that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexible C-terminal part involved in monoterpene binding. (+info)
Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases.
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Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins. (+info)
The gene encoding hydroxypyruvate reductase (GRHPR) is mutated in patients with primary hyperoxaluria type II.
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Primary hyperoxaluria type II (PH2) is a rare monogenic disorder that is characterized by a lack of the enzyme that catalyzes the reduction of hydroxypyruvate to D-glycerate, the reduction of glyoxylate to glycolate and the oxidation of D-glycerate to hydroxypyruvate. The disease is characterized by an elevated urinary excretion of oxalate and L-glycerate. The increased oxalate excretion can cause nephrolithiasis and nephrocalci-nosis and can, in some cases, result in renal failure and systemic oxalate deposition. We identified a glyoxylate reductase/hydroxypyruvate reductase (GRHPR) cDNA clone from a human liver expressed sequence tag (EST) library. Nucleotide sequence analysis identified a 1198 nucleotide clone that encoded a 984 nucleotide open reading frame. The open reading frame encodes a predicted 328 amino acid protein with a mass of 35 563 Da. Transient transfection of the cDNA clone into COS cells verified that it encoded an enzyme with hydroxy-pyruvate reductase, glyoxylate reductase and D-glycerate dehydrogenase enzymatic activities. Database analysis of human ESTs reveals widespread tissue expression, indicating that the enzyme may have a previously unrecognized role in metabolism. The genomic structure of the human GRHPR gene was determined and contains nine exons and eight introns and spans approximately 9 kb pericentromeric on chromosome 9. Four PH2 patients representing two pairs of siblings from two unrelated families were analyzed for mutations in GRHPR by single strand conformation polymorphism analysis. All four patients were homozygous for a single nucleotide deletion at codon 35 in exon 2, resulting in a premature stop codon at codon 45. The cDNA that we have identified represents the first characterization of an animal GRHPR sequence. The data we present will facilitate future genetic testing to confirm the clinical diagnosis of PH2. These data will also facilitate heterozygote testing and prenatal testing in families affected with PH2 to aid in genetic counseling. (+info)
Isoform-selective metabolism of mianserin by cytochrome P-450 2D.
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The involvement of cytochrome P-450 (CYP) 2D isoforms in the metabolism of mianserin and the stereoselectivity of their catalytic activities were investigated by using five CYP2D isoforms (CYP2D1, 2D2, 2D3, 2D4, and 2D6). Using RS-mianserin as a substrate, we found that five CYP2D isoforms had similar levels of 8-hydroxylation activity. However, N-demethylation activity differed among the isoforms; CYP2D3 and 2D4 efficiently demethylated RS-mianserin compared with the other three isoforms. N-Oxidation activity was specific to CYP2D1 although its level was relatively low. Another metabolite, assigned as 8-hydroxy-N-desmethylmianserin by liquid chromatography/mass spectrometry analysis, was formed by CYP2D4 and 2D6. The metabolism exhibited stereoselectivity. CYP2D1 and 2D4 selectively 8-hydroxylated the R(-)-enantiomer, and CYP2D6 predominately N-demethylated R(-)-enantiomer. N-Oxidation by CYP2D1 was specific to R(-)-enantiomer. In conclusion, CYP2D isoforms are involved in several metabolic pathways of mianserin acting in an isoform-specific manner. Stereoselectivity of the catalytic activities was clearly observed in the reactions of CYP2D1, 2D4, and 2D6. (+info)
Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members.
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Complementary DNA clones encoding human aflatoxin B(1) aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine. The AKR1C family members proved not to be as widely expressed as the other reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone. As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further. Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting. Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules. Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions. (+info)
The conversion gradient at HIS4 of Saccharomyces cerevisiae. I. Heteroduplex rejection and restoration of Mendelian segregation.
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In Saccharomyces cerevisiae, some gene loci manifest gradients in the frequency of aberrant segregation in meiosis, with the high end of each gradient corresponding to a hotspot for DNA double-strand breaks (DSBs). The slope of a gradient is reduced when mismatch repair functions fail to act upon heteroduplex DNA-aberrant segregation frequencies at the low end of the gradient are higher in the absence of mismatch repair. Two models for the role of mismatch repair functions in the generation of meiotic "conversion gradients" have been proposed. The heteroduplex rejection model suggests that recognition of mismatches by mismatch repair enzymes limits hybrid DNA flanking the site of a DSB. The restoration-conversion model proposes that mismatch repair does not affect the length of hybrid DNA, but instead increasingly favors restoration of Mendelian segregation over full conversion with increasing distance from the DSB site. In our experiment designed to distinguish between these two models, data for one subset of well repairable mismatches in the HIS4 gene failed to show restoration-type repair but did indicate reduction in the length of hybrid DNA, supporting the heteroduplex rejection model. However, another subset of data manifested restoration-type repair, indicating a relationship between Holliday junction resolution and mismatch repair. We also present evidence for the infrequent formation of symmetric hybrid DNA during meiotic DSB repair. (+info)