Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.

Assessment of the fifth ligand-binding repeat (LR5) of the LDL receptor as an analytical reagent for LDL binding.

The fifth ligand binding repeat (LR5) of the low density lipoprotein (LDL) receptor was assessed ex vivo as an 'analytical reagent' to distinguish LDL state, in atherosclerosis risk monitoring. LR5 was immobilized to mercaptoundecanoic acid modified gold surfaces via a glycine linker. Surface plasmon resonance (SPR) was used to monitor LDL binding. Unfolded LR5 was ineffectual as an affinity ligand for LDL but refolded LR5 showed a high affinity for native LDL but little affinity for oxidized LDL. LR5 refolded in the presence of calcium or EDTA gave the equivalent LDL binding capacity. However, EDTA-LR5 was less stable than Ca-LR5 at pH 5 and, from tryptophan fluorescence evidence, they appeared to involve different regions of LR5 and/or LDL in the binding. Involvement of amino acid residues of the calcium cage of LR5 was tested in LDL binding by monitoring calcium ion release with a calcium ionophore. The results were consistent with approximately 7-8 LR5 binding per LDL, of which only some induce calcium release (a maximum of approximately 25 mol% calcium, based on LR5, was released during LDL binding). For LDL binding to the LDL receptor in vivo more than one ligand-binding repeat is needed and this may be consistent with LR5 acting here also at binding sites which other LRs normally occupy in the LDL-LDL receptor complex. This initial study is encouraging for the use of a minimum peptide repeat array based on the conserved region of the LRs as an affinity surface for atherosclerosis risk monitoring.

Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1.

A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

Effects of single-residue substitutions on negative cooperativity in ligand binding to dihydrofolate reductase.

The effects of six amino acid substitutions in Lactobacillus casei dihydrofolate reductase, predominantly in the coenzyme binding site, on catalysis and on the negative cooperativity between NADPH and tetrahydrofolate binding have been determined. Replacement of Leu62, His64 or Leu54 by alanine has no effect on kcat, and produces only modest changes in negative cooperativity. Alanine substitution of His77, which interacts indirectly with the coenzyme adenine ring, leads to a doubling of the negative cooperativity and a consequent doubling of kcat. Replacement of Arg43, which interacts with the coenzyme 2'-phosphate, by alanine, or of Trp21, which interacts with the coenzyme nicotinamide ring, by histidine leads to a 20-100-fold decrease in negative cooperativity. In both mutants there is a decrease in kcat; isotope effects show that product release is largely rate-limiting in R43A, whereas in W21H hydride ion transfer is rate-limiting. 1H NMR has been used to obtain information on the extent of the structural changes produced by the substitutions. This varies from very local effects in H64A to very widespread effects in W21H. These changes are used as the basis for discussion of the mechanisms of the functional effects of the various substitutions. It is suggested that residues in helix C, beta-strand 3 and the beta3-beta4 loop may be involved in the transmission of effects between the coenzyme and substrate binding sites.