Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue of the heme binding domain resulted in complete loss of monooxygenase activity of both CYP enzymes, indicating that this cysteine residue is indispensable for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. Considering the important role of CYP enzymes in biotransformation of xenobiotics and the presence of N. virens in estuarine environments that accumulates organic xenobiotics, our results are important in understanding the molecular mechanism of biotransformation in marine polychaetes.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91:1140-1154. 1966.-The control of synthesis of the five enzymes responsible for the conversion of d(-)-mandelate to benzoate by Pseudomonas putida was investigated. The first three compounds occurring in the pathway, d(-)-mandelate, l(+)-mandelate, and benzoylformate, are equipotent inducers of all five enzymes. A nonmetabolizable inducer, phenoxyacetate, also induces synthesis of these enzymes; but, unlike the metabolizable inducer-substrates, it does not elicit synthesis of enzymes that mediate steps in the pathway beyond benzoate. Under conditions of semigratuity, dl-mandelate elicits immediate synthesis at a steady rate of the first two enzymes of the pathway, but two enzymes which act below the level of benzoate are synthesized only after a considerable lag. Succinate and asparagine do not significantly repress the synthesis of the enzymes responsible for mandelate oxidation.     

Peroxisomal β-Oxidation Enzymes

The enzymes involved in -oxidation spiral are schematically classified into two groups. The first group consists of palmitoyl-CoA oxidase, the L-bifunctional protein, which has been called as the bifunctional protein, and 3-ketoacyl-CoA thiolase. The second group consists of the newly confirmed enzymes, branched chain oxidase, the D-bifunctional protein, and sterol carrier protein x. The enzymes of the first group are inducible and act on the straight chain acyl-CoA substrates. But the enzymes of the second group are non-inducible and act on branched chain acyl-CoAs. Accordingly, bile acid formation and oxidation of pristanic acid derived from phytol are catalyzed by the enzymes of the second group but not by those of the first group. The functions of the peroxisomal system and methods of analysis of the enzymes are briefly summarized.     

Induction and multi-sensitive end-product repression in the enzymic pathway degrading mandelate in Pseudomonas fluorescens

1. The first five enzymes involved in the degradation of mandelate in Pseudomonas fluorescens have been examined. 2. Induction is not significantly affected by glucose. 3. The first three enzymes form a group inducible by mandelate and repressible by benzoate, catechol and succinate. 4. The possibility that benzoate and catechol act as repressors only after they have been degraded to succinate is unlikely since mutants blocked at suitable points in the pathway have the same repression pattern as the wild type. 5. It is concluded that synthesis of the enzymes is subject to a multi-sensitive repression mechanism that can be independently activated by benzoate or catechol or succinate. 6. In each case the repression can be largely overcome by increasing the concentration of the inducer. 7. The enzymes of the first group are thus controlled by a dual system in which induction by the first substrate is opposed by repression exerted by the end product of the first group and by the products of succeeding groups.     

Development of Endopeptidase Activities in Maize (Zea mays L.) Endosperms

An activity stain was used after native polyacrylamide gel electrophoresis, and at least 17 different endopeptidase activities were detected in maize (Zea mays L.) endosperm extracts prepared during the first 6 d after imbibition. The enzymes detected were classified into four groups based on their time of appearance and on their mobility in polyacrylamide gels. The first group, which included two enzymes present in dry endosperms, disappeared soon after imbibition. The second group, comprising five activity bands, appeared during the first 2 to 3 d after imbibition and then disappeared. The third set of enzymes increased continuously throughout the experimental period. The fourth group appeared after d 3 and remained at a constant level after that time. The endopeptidase activities were characterized by the effect of specific inhibitors on their activities. The two enzymes of the first group are metalloendopeptidases based on their sensitivity to ethylenediaminetetracetate (EDTA). Enzymes of the second, third, and fourth groups are sulfhydryl-endopeptidases as judged by their sensitivity to antipain, chymostatin, leupeptin, and E-64 and by their requirement for 2-mercaptoethanol. Pepstatin, phenylmethylsulfonyl fluoride, or EDTA had no effect on these enzymes. Many of the second, third, and fourth group enzymes cleaved [alpha]-zein-rich proteins as well as such easily obtained proteins as gelatin (used in our standard assay) and hemoglobin. The second group had a high affinity for [gamma]-zein, whereas none of the bands in the fourth group of enzymes cleaved this type of zein. The two metalloenzymes of the first group cleaved neither [alpha]- nor [gamma]-zeins.     

Homing endonucleases: keeping the house in order.

Homing endonucleases are rare-cutting enzymes encoded by introns and inteins. They have striking structural and functional properties that distinguish them from restriction enzymes. Nomenclature conventions analogous to those for restriction enzymes have been developed for the homing endonucleases. Recent progress in understanding the structure and function of the four families of homing enzymes is reviewed. Of particular interest are the first reported structures of homing endonucleases of the LAGLIDADG family. The exploitation of the homing enzymes in genome analysis and recombination research is also summarized. Finally, the evolution of homing endonucleases is considered, both at the structure-function level and in terms of their persistence in widely divergent biological systems.     

Anaerobic sulfatase-maturating enzymes, first dual substrate radical S-adenosylmethionine enzymes

Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to C(alpha)-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S](2+,+) cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 '-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to C(alpha)-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence.     

Intracellular sites of lipid synthesis and the biogenesis of mitochondria

Experimental data are presented on the intracellular localization in rat liver of three enzymes which are involved in the biosynthesis of phosphatidylethanolamine and diphosphatidylglycerol. These enzymes are phosphatidylserine decarboxylase, CDP-diglyceride-l-alpha-glycerophosphate phosphatidyl transferase, and phosphatidylethanolamine-l-serine phosphatidyl transferase. It was found that the first two enzymes are primarily mitochondrial while the latter enzyme is primarily microsomal. The intracellular sites for the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol are discussed, and the implications of their sites of biosynthesis on the assembly processes involved in the biogenesis of mitochondria are considered.     

Antioxidant metabolism of Xenopus laevis embryos during the first days of development

Reactive oxygen species (ROS) are formed and degraded in all aerobic organisms, but their role during embryonic development has not yet been well established. In this paper, we report the activities of various enzymes involved in antioxidant metabolism during the first 7 days of embryonic development of Xenopus laevis embryos. During the first two days of development, embryo antioxidant metabolism is based on catalase and superoxide dismutase activities. Later, the glutathione system is activated, and the activity of all the enzymes involved increases. The results presented in this study, together with previously reported data, support the hypothesis that antioxidant defences may include enzymes that are genetically regulated, while the other systems that appear to be environmentally modulated become relevant later in development, probably to protect embryos from environmental and toxic factors.     

A comprehensive analysis of the geranylgeranylglyceryl phosphate synthase enzyme family identifies novel members and reveals mechanisms of substrate specificity and quaternary structure organization

Geranylgeranylglyceryl phosphate synthase (GGGPS) family enzymes catalyse the formation of an ether bond between glycerol‐1‐phosphate and polyprenyl diphosphates. They are essential for the biosynthesis of archaeal membrane lipids, but also occur in bacterial species, albeit with unknown physiological function. It has been known that there exist two phylogenetic groups (I and II) of GGGPS family enzymes, but a comprehensive study has been missing. We therefore visualized the variability within the family by applying a sequence similarity network, and biochemically characterized 17 representative GGGPS family enzymes regarding their catalytic activities and substrate specificities. Moreover, we present the first crystal structures of group II archaeal and bacterial enzymes. Our analysis revealed that the previously uncharacterized bacterial enzymes from group II have GGGPS activity like the archaeal enzymes and differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases. The length of the isoprenoid substrate is determined in group II GGGPS enzymes by ‘limiter residues’ that are different from those in group I enzymes, as shown by site‐directed mutagenesis. Most of the group II enzymes form hexamers. We could disrupt these hexamers to stable and catalytically active dimers by mutating a single amino acid that acts as an ‘aromatic anchor’.     

A DNA enzyme that mimics the first step of RNA splicing

We have discovered an artificial DNA enzyme that mimics the first step of RNA splicing. In vitro selection was used to identify DNA enzymes that ligate RNA. One of the new DNA enzymes carries out splicing-related catalysis by specifically recognizing an unpaired internal adenosine and facilitating attack of its 2'-hydroxyl onto a 5'-triphosphate. This reaction forms 2',5'-branched RNA and is analogous to the first step of in vivo RNA splicing, in which a ribozyme cleaves itself with formation of a branched intermediate. Unlike a natural ribozyme, the new DNA enzyme has no 2'-hydroxyl groups to aid in the catalytic mechanism. Our finding has two important implications. First, branch-site adenosine reactivity seems to be mechanistically favored by nucleic acid enzymes. Second, hydroxyl groups are not obligatory components of nucleic acid enzymes that carry out biologically related catalysis.     

Enzymes of the Tryptophan Pathway in Acinetobacter calco-aceticus

All enzymes of the tryptophan synthetic pathway were detectable in extracts from wild-type Acinetobacter calco-aceticus. The levels of these enzymes were determined in extracts from a number of auxotrophs grown under limiting tryptophan. In each case only anthranilate synthetase was found to be present in increased amounts, whereas the specific activities of the remaining enzymes remained unchanged and unaffected by the tryptophan concentration. Derepression of anthranilate synthetase was found to occur as the concentration of tryptophan became limiting. Anthranilate synthetase and phosphoribosyl transferase activities are both feedback-inhibited by tryptophan. Molecular weight determination carried out by gel filtration and zonal centrifugation in sucrose revealed that all the enzymes are less than 100,000, and no molecular aggregates of these enzymes were detected. The data indicate that tryptophan synthesis in Acinetobacter is regulated both by feedback inhibition of the first two enzymes of the pathway and by repression control of anthranilate synthetase.     

Oxidation of Nicotinic Acid by a Bacillus Species: Regulation of Nicotinic Acid and 6-Hydroxynicotinic Acid Hydroxylases

The first two enzymes employed by a Bacillus species for the dissimilation of nicotinic acid are coordinately induced. The inducer of the enzymes appears to be 6-hydroxynicotinic acid, the product of the first enzyme in the pathways. Synthesis of the enzymes is repressed by glucose when ammonium is present in the medium, but not when nicotinic acid is the sole nitrogen source. The possible significance of the coordinate induction and unusual repression is discussed.     

Myxospore coat synthesis in Myxococcus xanthus: enzymes associated with uridine 5''-diphosphate-N-acetylgalactosamine formation during myxospore development.

Activities of the enzymes glutamine synthetase (EC 6.3.1.2.), glucosamine 6-phosphate acetyltransferase (EC 2.3.1.4.), uridine 5'-diphosphate (UDP)-N-acetylglucosamine pyrophosphorylase (EC 2.7.23.), UDP-N-acetylglucosamine 4-epimerase (EC 5.1.3.7.), fructose 1,6-diphosphate phosphatase (EC 3.13.11.), L-glutamine-fructose 6-phosphate transamidase (EC 5.3.1.19.), alkaline phosphatase (EC 3.1.3.1.), and malic dehydrogenase (EC 1.1.1.37) were assayed in partially purified extracts prepared at different stages of myxospore formation and germination in liquid cultures of Myxococcus xanthus. The specific activities of the first six of these enzymes increased 4.5- to 7.5-fold after 2 h of induction with 0.5 M glycerol or 0.2 M dimethyl sulfoxide. The increase in specific activities of these six enzymes was not observed in a mutant unable to be induced with glycerol. During the first 2 to 4 h of induction and during the first hour of germination, the level of these enzymes decreased to the level characteristic of vegetative cells. It is suggested that the six enzymes are responsible for the increased conversion of fructose 1,6-diphosphate to UDP-N-acetylgalactosamine, the major precursor of the myxospore coat.     

Localization of the Enzymes of Quinolizidine Alkaloid Biosynthesis in Leaf Chloroplasts of Lupinus polyphyllus

Studies with purified chloroplasts of Lupinus polyphyllus LINDL. leaflets indicate that the first two enzymes of quinolizidine alkaloid biosynthesis, lysine decarboxylase and 17-oxosparteine synthase, are localized in the chloroplast stroma. Thus, both enzymes share the same subcellular compartment as the biosynthetic pathway of lysine, the precursor of quinolizidine alkaloids. The activity of diaminopimelate decarboxylase, the final enzyme in lysine biosynthesis, is about two to three orders of magnitude higher than that of the enzymes of alkaloid formation.     

Theoretical studies of enzyme mechanisms involving high-valent iron intermediates

Recent theoretical contributions to the elucidation of mechanisms for iron containing enzymes are reviewed. The method used in most of these studies is hybrid density functional theory with the B3LYP functional. Three classes of enzymes are considered, the mononuclear non-heme enzymes, enzymes containing iron dimers, and heme-containing enzymes. Mechanisms for both dioxygen and substrate activations are discussed. The reactions usually go through two half-cycles, where a high-valent intermediate Fe(IV)O species is created in the first half-cycle, and the substrate reactions involving this intermediate occur in the second half-cycle. Similarities between the three classes of enzymes dominate, but significant differences also exist.     

Evolutionary optimisation of enzymes

Aside from the demonstration that individual molecular traits of enzymes can be evolutionarily optimised, the discovery that several traits can be simultaneously optimised is a major advance. The first observations of the effects of evolutionary optimisation at the structural level, through X-ray crystallography, reinforce the view that enzymes are best optimised by evolution and not by design.     

Regulation of early enzymes of ergosterol biosynthesis in Saccharomyces cerevisiae.

In order to determine the regulation mechanisms of ergosterol biosynthesis in yeast, we developed growth conditions leading to high or limiting ergosterol levels in wild type and sterol-auxotrophic mutant strains. An excess of sterol is obtained in anaerobic sterol-supplemented cultures of mutant and wild type strains. A low sterol level is obtained in aerobic growth conditions in mutant strains cultured with optimal sterol supplementation and in wild type strain deprived of pantothenic acid, as well as in anaerobic cultures without sterol supplementation. Measurements of the specific activities of acetoacetyl-CoA thiolase, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase (the first three enzymes of the pathway), show that in cells deprived of ergosterol, acetoacetyl-CoA thiolase and HMG-CoA synthase are generally increased. In an excess of ergosterol, in anaerobiosis, the same enzymes are strongly decreased. A 5-10-fold decrease is observed for acetoacetyl-CoA thiolase and HMG-CoA synthase. In contrast, HMG-CoA reductase is only slightly affected by these conditions. These results show that ergosterol could regulate its own synthesis, at least partially, by repression of the first two enzymes of the pathway. Our results also show that exogenous sterols, even if strongly incorporated by auxotrophic mutant cells, cannot suppress enzyme activities in aerobic growth conditions. Measurement of specific enzyme activities in mutant cells also revealed that farnesyl pyrophosphate thwarts the enhancement of the activities of the two first enzymes.