A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhLA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhLA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.Xanthobacter spp. are nitrogen-fixing bacteria that are able to grow autotrophically with a mixture of hydrogen and oxygen as an energy source (33). A member of this genus that is able to utilize several halogenated hydrocarbons as carbon sources has been isolated (15). The organism was obtained from an enrichment culture with 1,2-dichloroethane, which is an environmentally important compound with a production volume larger than that of any other industrial halogenated chemical. The 1,2-dichloroethane-degrading bacterium, designated strain GJ10, was found to degrade 1,2-dichloroethane via 2-chloroethanol, 2-chloroacetaldehyde, and chloroacetic acid to glycolate ( Fig. 1) (13, 14). The dehalogenation steps in this sequence were found to be catalyzed by two different hydrolytic dehalogenases (14,17).Conversion of 1,2-dichloroethane was mediated by a haloalkane dehalogenase. This was the first enzyme found to catalyze hydrolytic dehalogenation of chlorinated hydrocarbons. The protein has been purified (17) and crystallized (26), and its three-dimensional structure is now under study. Chloroacetic acid hydrolysis was found to be mediated by a different enzyme. This haloacid dehalogenase has not been purified from strain GJ10, but much information is available about other dehalogenases of this class (22).So far, haloalkane dehalogenases are the only enzymes known to be capable of direct hydrolytic dehalogenation of chlorinated and brominated hydrocarbons, without the requirement for coenzymes or oxygen. The enzyme of X. autotrophicus GJ10 is constitutively expressed to 2 to 3% of the soluble cellular protein (13, 17). It has a remarkably broad substrate range which includes terminally halogenated alkanes with chain lengths up to 4 carbons for chlorinated and up to at least 10 carbons for brominated alkanes. Other haloalkane dehalogenases of broad substrate range have been found in gram-positive haloalkane-utilizing bacteria (11,28,35 So far, no information is available about the genetics of haloalkane-utilizing organisms. Since the system is attractive both for st...
Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains ofAncylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhUA) genes of strains AD20 and AD25 were the same as the sequence of dhL4 from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.
The complete nucleotide sequence of the sit gene encoding the soluble lytic transglycosylase (Slt; EC 3.2.1.-) from Escherichia coli has been determined. The largest open reading frame identified on a 2.5-kb PvuII-Sall fragment indicates that the enzyme is translated as a preprotein of either 654 or 645 amino acids, depending on which of two potential start codons is used. The two possible translation products differ only in the lengths of their predicted signal peptides, 36 or 27 amino acids, respectively. In both cases, processing results in a soluble mature protein of 618 amino acids (Mr = 70,468). The deduced primary structure of the mature protein was confirmed by N-terminal sequencing and determination of the amino acid composition of the isolated transglycosylase. The sit gene contains a high percentage of rare codons, comparable to other low-expressed genes. A hairpin structure that could serve as a transcriptional terminator is located downstream of the sit coding region and precedes the trpR open reading frame at 99.7 min on the E. coli chromosomal map. A computer-assisted search did not reveal any significant sequence similarity to other known carbohydratedegrading enzymes, including lysozymes. Interestingly, a stretch of 151 amino acids at the C terminus of the transglycosylase shows similarity to the N-terminal portion of the internal virion protein D from bacteriophage T7. Overexpression of the slt gene, under the control of the temperature-inducible phage lambda PR promoter, results in a 250-fold overproduction of the mature transglycosylase, whereas after deletion of the signal peptide a 100-fold overproduction of the enzyme is observed in the cytoplasm.The murein polymer of the bacterial cell wall is composed of glycan strands of variable length which are cross-linked by short peptide bridges to form one macromolecule around the cell.A whole set of murein-metabolizing enzymes in Escherichia coli has been identified (for a review, see reference 19). The balanced action of these murein-synthesizing and -degrading enzymes determines the specific shape of the murein sacculus and consequently the shape of the bacterium (12,38,54).In E. coli, four of the nine murein-degrading enzymes that have been characterized so far are potentially capable of degrading the intact polymer and thereby lysing the cell (20). Two of these enzymes are endopeptidases, able to cleave the peptide cross bridges of murein (47, 49), whereas the other two are glycosylases which are able to degrade the glycan strands (18,22,33).The two glycosylases have been reported to show the same enzymatic activity but to differ with respect to their cellular localization, one of them being a soluble enzyme with a molecular mass of 65 kDa and the other being membrane bound with a molecular mass of 35 kDa (18,22,33).Both glycosylases catalyze the cleavage of the ,-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues, as do various lysozymes. However, the bacterial glycosylases also catalyze an intramolecular trans...
Background: Understanding the molecular basis for the mixed profiles of progesterone receptor (PR) ligands will benefit future drug design. Results: Two differing mechanisms for the induction of mixed profiles by 11-steroids are described. Conclusion: Subtle electrostatic and steric factors explain the differing PR activities of 11-steroids. Significance: These observations will impact future drug-design strategies for PR and potentially other nuclear receptors.
The p38α mitogen-activated protein kinase regulates the synthesis of pro-inflammatory cytokines in response to stimulation by a diverse set of stress signals. Various different chemotypes and clinical candidates that inhibit p38α function have been reported over the years. In this publication, the novel structure of p38α cocrystallized with the clinical candidate TAK-715 is reported. Owing to the impact of crystallization conditions on the conformation of protein kinases (and in particular p38α), the structures of complexes of p38α with SB-203580, SCIO-469 and VX-745 have also been determined to enable in-depth comparison of ligand-induced protein conformations. The impact of experimental conditions on p38α-inhibitor complex structures, most importantly soaking versus cocrystallization, is discussed. Analysis of the structures and quantification of the protein-ligand interactions couples ligand-induced protein conformations to the number of interactions and to inhibitor selectivity against the human kinome. This shows that for the design of novel kinase inhibitors, selectivity is best obtained through maximization of the number of interactions throughout the ATP pocket and the exploitation of specific features in the active site.
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