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PubMed1997

DNA enzymes.

Biological catalysis is dominated by enzymes that are made of protein, but several distinct classes of catalytic RNAs are known to promote chemical transformations that are fundamental to cellular metabolism. Is biological catalysis limited only to these two biopolymers, or is DNA also capable of functioning as an enzyme in nature? To date, no DNA enzymes of natural origin have been found. However, an increasing number of catalytic DNAs, with characteristics that are similar to those of ribozymes, are being produced outside the confines of the cell. An assessment of the potential for structure formation by DNA leads to the conclusion that DNA might have considerable latent potential for enzymatic function.

Base Sequence↗
PubMed2006

Bacterial pathways for degradation of nitroaromatics.

The last one hundred years have seen a massive expansion in the chemicals industry; however, with this progress came the concomitant pollution of the environment with a significant range of xenobiotics.Nitroaromatic compounds form one such category of novel environmental contaminants and are produced through a large number of industrial processes, most notably the pesticides, dyes and explosives industries. Whilst singly nitrated aromatic compounds are usually mineralised in the environment, multiply nitrated aromatics, such as the explosive 2,4,6-trinitrotoluene (TNT), are recalcitrant and highly toxic. The predominant route of biological transformation of aromatic compounds is oxidation; however, the presence of three electron-withdrawing nitro-groups around the ring prevents oxidation, rendering such compounds resistant to biodegradation. The subsequent accumulation of these contaminants has stimulated much research leading to the isolation of bacteria that possess, to varying extents, the ability to remediate explosives and other nitroaromatic pollutants.The extreme environments created by these toxic substances accelerate the evolutionary process and examples of bacteria that have conscripted metabolic enzymes for novel remediatory pathways are included. This Highlight ends with a discussion of the future of nitroaromatic bioremediation including engineering plants to express bacterial enzymes for use in bioremediation programs.

Bacteria↗
PubMed2001

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

Amino Acid Sequence↗
PubMed2003

Engineering the root-soil interface via targeted expression of a synthetic phytase gene in trichoblasts.

For biochemical modification of the root-soil interface, the engineered secretion of stable enzymes from trichoblasts (= root hair bearing rhizodermal cells) is proposed. As a reporter activity, we chose to express a synthetic gene encoding a secretory phytase (PHY) directed by a trichoblast-specific promoter in root hair cells of the crop plant potato. Transgenic plants produced and secreted phytase in sufficient amounts to release phosphate from phytate in liquid medium. When grown in an unsterile substrate containing phytate, transgenic plants accumulated 40% more P in leaves than wild-type plants. The improved P nutrition driven by trichoblast-targeted expression and subsequent secretion of PHY illustrates the potential of using trichoblast-targeted expression of suitable enzymes for future applications in plant nutrition, phytoremediation and molecular farming.

Journal Article↗
PubMed1998

Ionophores and receptors using cation-pi interactions: collarenes.

Cation-pi interactions are important forces in molecular recognition by biological receptors, enzyme catalysis, and crystal engineering. We have harnessed these interactions in designing molecular systems with circular arrangement of benzene units that are capable of acting as ionophores and models for biological receptors. [n]Collarenes are promising candidates with high selectivity for a specific cation, depending on n, because of their structural rigidity and well-defined cavity size. The interaction energies of [n]collarenes with cations have been evaluated by using ab initio calculations. The selectivity of these [n]collarenes in aqueous solution was revealed by using statistical perturbation theory in conjunction with Monte Carlo and molecular dynamics simulations. It has been observed that in [n]collarenes the ratio of the interaction energies of a cation with it and the cation with the basic building unit (benzene) can be correlated to its ion selectivity. We find that collarenes are excellent and efficient ionophores that bind cations through cation-pi interactions. [6]Collarene is found to be a selective host for Li+ and Mg2+, [8]collarene for K+ and Sr2+, and [10]collarene for Cs+ and Ba2+. This finding indicates that [10]collarene and [8]collarene could be used for effective separation of highly radioactive isotopes, 137Cs and 90Sr, which are major constituents of nuclear wastes. More interestingly, collarenes of larger cavity size can be useful in capturing organic cations. [12]Collarene exhibits a pronounced affinity for tetramethylammonium cation and acetylcholine, which implies that it could serve as a model for acetylcholinestrase. Thus, collarenes can prove to be novel and effective ionophores/model-receptors capable of heralding a new direction in molecular recognition and host-guest chemistry.

Benzene Derivatives↗
PubMed1995

Structural features of the precursor to mitochondrial aspartate aminotransferase responsible for binding to hsp70.

The precursor (pmAspAT) and mature (mAspAT) forms of mitochondrial aspartate aminotransferase interact with hsp70 very early during translation when synthesized in either rabbit reticulocyte lysate or wheat germ extract (Lain, B., Iriarte, A., and Martinez-Carrion. (1994) J. Biol. Chem. 269, 15588-15596). The nature of the structural elements responsible for recognition and binding of this protein to hsp70 has been studied by examining the folding and potential association with the chaperone of several engineered forms of this enzyme. Whereas pmAspAT and mAspAT bind hsp70 very early during translation, the cytosolic form of this enzyme (cAspAT) does not interact with hsp70. A fusion protein consisting of the mitochondrial presequence peptide attached to the amino terminus of cAspAT associates with hsp70 only after the protein has acquired its native-like conformation, apparently through binding to the presequence exposed on the surface of the folded protein. Deletion of the amino-terminal segment of mAspAT or its replacement with the corresponding domain from the cytosolic isozyme eliminates the cotranslational binding of hsp70 to the mitochondrial protein. We conclude that both the presequence and NH2-terminal region of pmAspAT represent recognition signals for binding of hsp70 to the newly synthesized mitochondrial precursor. Results from competition studies with synthetic peptides support this conclusion. The ability of hsp70 to discriminate between these two highly homologous proteins probably involves the recognition of specific sequence elements in the NH2-terminal portion of the mitochondrial protein and may relate to their separate localization in the cell. A slower folding rate and higher affinity for cytosolic chaperones may represent evolutionary adaptations of translocated mitochondrial proteins to ensure their efficient importation into the organelle.

Amino Acid Sequence↗
PubMed1996

The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase. Site-directed mutagenesis of effector binding site residues.

D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli catalyzes the first committed step in serine biosynthesis and is allosterically regulated by L-serine, the end product of the pathway. Each subunit of the homotetramer is made up of three distinct domains with one of the intersubunit contacts being between adjacent regulatory domains. Each regulatory domain interface contains two symmetrical serine binding sites such that serine forms hydrogen bonds to both domains across the interface. Previous work (Al-Rabiee, R., Lee, E. J., and Grant, G. A. (1996) J. Biol. Chem. 271, 13013-13017) demonstrated that when adjacent regulatory domains are covalently linked to one another by engineered disulfide bonds, the enzyme was inactivated. Breaking the disulfide bonds by reduction restored enzymatic activity. This study demonstrates that the complementary situation is also true. Site-directed mutagenesis of three residues at the effector binding site, His344, Asn346, and Asn364', render the enzyme increasingly less susceptible to inhibition by the effector. When mutations result in a situation where it is no longer possible to establish a stable hydrogen bonding network across the regulatory domain interface, the inhibitory capacity of the effector is lost. Furthermore, mutations that produce as much as 5 orders of magnitude decrease in the ability of L-serine to inhibit the enzyme have no appreciable effect on the Km or kcat of the enzyme. These observations support the model that predicts that catalytic activity in D-3-phosphoglycerate dehydrogenase is regulated by the movement of adjacent regulatory domains about a flexible hinge and that effector binding tethers the regulatory domains together producing a state that results in a stable, open active site cleft that is no longer able to promote catalysis.

Allosteric Regulation↗
PubMed2006

Roles of Asp75, Asp78, and Glu83 of GTP-dependent phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis.

The roles of Asp(75), Asp(78), and Glu(83) of the (75)DPSDVARVE(83) element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp(78) and Glu(83) are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp(78) influences Tyr(220); Tyr(220) helps to position bound PEP, and Glu(83) interacts with Arg(81). Experimental data on other PEPCKs indicate that Arg(81) binds PEP, and the phosphate of PEP interacts with Mn(2+) of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent K(m) value for Mn(2+) 170-fold. In contrast, Asp(75) is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, V(max) decreased 1.4-4-fold. K(m) values for PEP and Mn(2+) increased 3-9- and 1.2-10-fold, respectively. K(m) values for GDP and bicarbonate changed little. 2) For PEP formation, V(max) increased 1.5-2.7-fold. K(m) values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu(83)-Arg(81) interaction affected Arg(81)-PEP association. D78A change altered the Tyr(220)-PEP interaction. These events perturbed PEP-Mn(2+) interaction and consequently affected catalysis severely. In contrast, substitutions at Asp(75), a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp(75) substitutions affected PEP-Mn(2+) interaction by changing the positions of Asp(78), Arg(81), and Glu(83), which translated to differential effects on two directions.

Amino Acid Sequence↗
PubMed2006

Degradation of organophosphorus neurotoxicity in SY5Y neuroblastoma cells by organophosphorus hydrolase (OPH).

Numerous approaches have been studied to degrade organophosphorus (OP) compounds and ameliorate their toxicity. In the current study, the potential of genetically engineered organophosphorus hydrolase (OPH) enzymes to functionally biotransform OP neurotoxicants was examined by assessing effects of OPH-hydrolyzed OPs on acute and delayed indicators of neurotoxicity. SY5Y human neuroblastoma cells were used as a model test system, as these cells respond distinctly to mipafox, which produces OP-induced delayed neuropathy, and paraoxon, which does not. Short-term effects of four OPH-treated OPs on acetylcholinesterase (AChE) and neuropathy target esterase (NTE) activities were measured in retinoic acid-differentiated or undifferentiated cells, and delayed effects of OPH-treated paraoxon or mipafox on levels of neuronal cytoskeletal proteins in nerve growth factor (NGF)-differentiated cells. The anti-AChE activity of paraoxon (maximum 3 muM) and anti-NTE activity of mipafox (250 muM) in SY5Y cells were prevented by biodegradation with OPH. Anti-AChE activities of mipafox, methyl parathion, and demeton-S were partially ameliorated, depending on OP concentration. Intracellular amounts of the 200-kD neurofilament protein NF200 were unchanged after treatment with OPH-treated or buffer-treated paraoxon, as expected, as this endpoint is insensitive to paraoxon. However, NF200 levels rose in cells treated during late differentiation with OPH-treated mipafox. This finding suggests the existence of a threshold concentration of mipafox below which SY5Y cells can maintain their viability for compensating cellular damage due to mipafox in neurite elongation. These results indicate that OPH may be used to biodegrade OPs and remediate their neurotoxic effects in vitro and that AChE and NTE are suitable detectors for OPH amelioration.

Acetylcholinesterase↗
PubMed1985

Construction of a vector, pRSVcatamb38, for the rapid and sensitive assay of amber suppression in human and other mammalian cells.

We describe the generation of an amber mutation in the chloramphenicol acetyltransferase (cat) gene of the mammalian cell transfection vector pRSVcat (Gorman et.al. (1982), Proc.Natl.Acad.Sci. 79 6777-6791). We have demonstrated the in vivo suppression of this amber mutation in monkey and human cells by co-transfection with a synthetic Xenopus suppressor tRNATyr under the control of the late SV40 promoter. The vector, pRSVcatamb38, may be used to quantitate amber suppression in various mammalian cells.

Acetyltransferases↗
PubMed2002

Molecular basis of cold adaptation.

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold-adapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.

Acclimatization↗
PubMed1987

Some factors affecting the copy number of specific plasmids in Bacillus species.

Some factors affecting the copy number of specific plasmids in different hosts were presented. A low copy number (one copy per chromosome) Kmr Tcr plasmid pTB19 was isolated from thermophilic bacillus. We have constructed 22 derivatives from pTB19 and their copy numbers range from 1 to 214 per chromosome in B. subtilis and B. stearothermophilus. Some recombinant plasmids containing the specific 1.0-MDa EcoRI fragment exhibited high transformation frequency and low copy number in B. stearothermophilus and were stable in the thermophile; on the other hand, those plasmids were unstable in different host B. subtilis, as mentioned earlier. By selecting the best combination of vector plasmid and host strain, both molecular cloning of various enzyme genes (i.e., penicillinase, thermostable alpha-amylase, and thermostable neutral protease) and enhancement of the enzyme production could be easily achieved.

Bacillus↗
PubMed1987

Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors.

Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.

Chromosomes↗