Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in resurrection plants and in plant seeds, and the recent discovery of a dehydration-induced Group 3 LEA-like gene in the nematode Aphelenchus avenae suggests a similar association in anhydrobiotic animals. Despite their importance, little is known about the structure of Group 3 LEA proteins, although computer modeling and secondary structure algorithms predict a largely ␣-helical monomer that forms coiled coil oligomers. We have therefore investigated the structure of the nematode protein, Aav-LEA1, in the first such analysis of a well characterized Group 3 LEA-like protein. Immunoblotting and subunit cross-linking experiments demonstrate limited oligomerization of AavLEA1, but analytical ultracentrifugation and gel filtration show that the vast majority of the protein is monomeric. Moreover, CD, fluorescence emission, and Fourier transform-infrared spectroscopy indicate an unstructured conformation for the nematode protein. Therefore, in solution, no evidence was found to support structure predictions; instead, Aav-LEA1 seems to be natively unfolded with a high degree of hydration and low compactness. Such proteins can, however, be induced to fold into more rigid structures by partner molecules or by altered physiological conditions. Because AavLEA1 is associated with desiccation stress, its Fourier transform-infrared spectrum in the dehydrated state was examined. A dramatic but reversible increase in ␣-helix and, possibly, coiled coil formation was observed on drying, indicating that computer predictions of secondary structure may be correct for the solid state. This unusual finding offers the possibility that structural shifts in Group 3 LEA proteins occur on dehydration, perhaps consistent with their role in anhydrobiosis.
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.
Here we report the first structure of a cocaine-degrading enzyme. The bacterial esterase, cocE, hydrolyzes pharmacologically active (-)-cocaine to a non-psychoactive metabolite with a rate faster than any other reported cocaine esterase (kcat = 7.8 s-1 and KM = 640 nM). Because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose. The crystal structure of cocE, solved by multiple anomalous dispersion (MAD) methods, reveals that cocE is a serine esterase composed of three domains: (i) a canonical alpha/beta hydrolase fold (ii) an alpha-helical domain that caps the active site and (iii) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site was identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which were refined at 1.58 A and 1.63 A resolution, respectively. These structural studies suggest that substrate recognition arises partly from interactions between the benzoyl moiety of cocaine and a highly evolved specificity pocket.
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is one of the most important and widely used military explosives. Due to its manufacture, decommissioning, and disposal it has become a serious pollutant at many sites, particularly across the United States and Germany, and its recalcitrance in the environment has led to its persistence in soil and groundwater. RDX is a potent convulsant due to its effects on the central nervous system and is also a class C, possible, carcinogen (7).For a long time it was thought that RDX biodegradation could occur only under anaerobic conditions (15, 26); however, aerobic degradation of RDX has been more recently demonstrated by bacterial consortia and pure strains. Coryneform bacteria (45), Stenotrophomonas maltophilia strain PB1 (4), and a rhodococcal strain (9) have been shown to utilize RDX as a sole source of nitrogen, generally degrading RDX at higher rates than anaerobic systems do.The first pathway for the aerobic degradation of RDX by a strain of Rhodococcus was proposed very recently (16) and involves denitration as the first step followed by spontaneous ring cleavage (Fig. 1). Positively identified products include nitrite (NO 2 Ϫ ), nitrous oxide (N 2 O), formaldehyde (HCHO), and carbon dioxide. A dead-end intermediate with the molecular formula C 2 H 5 N 3 O 3 was also found to accumulate, indicating that not all the components of RDX were mineralized. This paper reports the isolation and identification of a Rhodococcus rhodochrous strain which can degrade RDX as a nitrogen source and the first cloning and expression of a gene responsible for RDX degradation. This gene is constitutively expressed, and its product has homology to a cytochrome P450. MATERIALS AND METHODSReagents. RDX (Ͼ95% purity as determined by high-performance liquid chromatography HPLC) was kindly provided by the Defence Science and Technology Laboratory (Dstl), Fort Halstead. Other chemicals were of analytical grade and, unless otherwise stated, were obtained from ...
We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa 'AlbudAbs' can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.
The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaine esterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k(cat) = 7.8 s(-1) and K(M) = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocaine metabolite that has been observed in patients who concurrently abuse cocaine and alcohol. The high catalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine and cocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting. Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase, with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanion hole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously known to use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocE has a more deleterious effect on the specificity rate constant (k(cat)/K(M)) than the analogous Y473F mutation of prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism, and reveal the relative contributions of active site residues toward substrate recognition and catalysis. Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55E mutation within the active site of cocE results in a modest (2-fold) improvement in K(M), but a 14-fold loss of k(cat). The pH rate profile of cocE was fit to the ionization of two groups (pK(a1) = 7.7; pK(a2) = 10.4) that likely represent titration of H287 and Y44, respectively. We also describe the crystal structures of both S117A and Y44F mutants of cocE. Finally, urea denaturation studies of cocE by fluorescence and circular dichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transition likely representing pertubation of the active site.
The crystal structures of an acetyl esterase, HerE, and its complex with an inhibitor dimethylarsinic acid have been determined at 1.30-and 1.45-Å resolution, respectively. Although the natural substrate for the enzyme is unknown, HerE hydrolyzes the acetyl groups from heroin to yield morphine and from phenyl acetate to yield phenol. Recently, the activity of the enzyme toward heroin has been exploited to develop a heroin biosensor, which affords higher sensitivity than other currently available detection methods. The crystal structure reveals a single domain with the canonical ␣/ hydrolase fold with an acyl binding pocket that snugly accommodates the acetyl substituent of the substrate and three backbone amides that form a tripartite oxyanion hole. In addition, a covalent adduct was observed between the active site serine and dimethylarsinic acid, which inhibits the enzyme. This crystal structure provides the first example of an As-containing compound in a serine esterase active site and the first example of covalent modification of serine by arsenic. Thus, the HerE complex reveals the structural basis for the broad scope inhibition of serine hydrolases by As(V)-containing organic compounds.Heroin (3,6-diacetylmorphine) is a highly addictive, synthetic derivative of the alkaloid morphine (Fig. 1A). The instability of the 3-acetyl suggests that it can undergo rapid spontaneous hydrolysis to give 6-acetylmorphine. HerE is known to hydrolyze the 6-acetyl of 6-acetylmorphine to yield morphine (k cat ϭ 12.6 Ϯ 0.6 s Ϫ1 , K m ϭ 0.5 Ϯ 0.06 mM) and was originally identified from the Rhodococcus sp. strain H1 by selective growth using heroin as the sole carbon source (1). Identification of this esterase has facilitated development of a coupled enzyme biosensor for heroin detection (Fig. 1A) with higher sensitivity than other currently available technology (2). In brief, hydrolysis of heroin by HerE generates morphine, which is the substrate for NADPH-dependent morphine dehydrogenase. The activity of morphine dehydrogenase is directly monitored by bacterial luciferase, which outputs a bioluminescent signal at 490 nm (Fig. 1A) (2). The higher activity (k cat K m Ϫ1 ) of HerE toward smaller substrates such as phenyl acetate (Fig. 1B) (k cat ϭ 3.0 Ϯ 0.05 s Ϫ1 , K m ϭ 70 Ϯ 8 nM at pH ϭ 6.4) provides a benchmark for engineering the esterase binding pocket to accept bulkier substrates more efficiently. Hence, the crystal structure of this acetyl esterase should provide substantial insights for structure-based design of second generation heroin biosensors with improved sensitivity.In addition, our structure with the inhibitor dimethylarsinic acid (Fig. 1B) demonstrates for the first time the covalent modification of serine by arsenic. Arsenic lies directly below phosphorous in the periodic table and shares some similar properties, but in general, arsenic is more reactive. As(V)-containing organic compounds comprise a known class of broad scope serine esterase/protease inhibitors (3, 4). Usually, arsonic R-As(V)O 3 H 2 and arsin...
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.
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