In vivo levels of chlorinated hydroquinones in a pentachlorophenol-degrading bacterium.

Sphingomonas chlorophenolica RA-2 is a soil microorganism that can grow on pentachlorophenol (PCP) as a sole carbon source. In this microorganism, PCP is converted to tetrachlorohydroquinone (TCHQ), trichlorohydroquinone, and 2,6-dichlorohydroquinone. The remainder of the pathway has not yet been defined. The ability to grow on PCP as a sole carbon source is remarkable because of the toxicity of PCP and its chlorinated hydroquinone metabolites. Experiments in which the levels of PCP and chlorinated hydroquinones were measured in cells metabolizing [U-14C]PCP revealed that the levels of chlorinated hydroquinones in the cytoplasm are in the low micromolar range. The toxicity of chlorinated hydroquinones was evaluated by exposure of Escherichia coli cells that had been treated with EDTA (to remove the outer membrane) to TCHQ. Significant toxicity due to TCHQ was not apparent until concentrations of 500 microM and higher. Thus, an important part of the explanation for why S. chlorophenolica RA-2 is able to grow on PCP as a sole carbon source is undoubtedly that it can process sufficient carbon for growth without accumulating high levels of toxic intermediates.

Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant.

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.

Exploration of the relationship between tetrachlorohydroquinone dehalogenase and the glutathione S-transferase superfamily.

Tetrachlorohydroquinone dehalogenase is found in Sphingomonas chlorophenolica, a soil bacterium that degrades pentachlorophenol, a widely used wood preservative. This enzyme converts tetrachlorohydroquinone (TCHQ) to trichlorohydroquinone (TriCHQ) and TriCHQ to dichlorohydroquinone (DCHQ) (Xun et al. (1992) J. Bacteriol. 174, 8003-8007). The reducing equivalents for each step are provided by two molecules of glutathione (Xun et al. (1992) Biochem. Biophys. Res. Commun. 182, 361-366). In addition to the expected TriCHQ and DCHQ products, the enzyme also produces substantial amounts of 2,3,5-trichloro-6-S-glutathionylhydroquinone (GS-TriCHQ) and an unidentified isomer of dichloro-S-glutathionylhydroquinone (GS-DCHQ). Treatment of the purified enzyme with dithiothreitol dramatically decreases the formation of GS-TriCHQ and GS-DCHQ. Furthermore, enzyme in freshly-prepared crude extracts forms only very small amounts of GS-TriCHQ and GS-DCHQ. We conclude that GS-TriCHQ and GS-DCHQ are produced by enzyme that has undergone some type of oxidative damage and are therefore not physiologically relevant products. The fact that the oxidative damage can be repaired by DTT suggests that a cysteine or methionine residue may be involved. We have created the C13S and C156S mutants of the enzyme. The C13S mutant converts TCHQ to GS-TriCHQ and GS-DCHQ, rather than to DCHQ. Thus, Cys13 is required for the reductive dehalogenation of TCHQ. A mechanism for the reaction which involves Cys13 is proposed.

Genograms: a psychosocial assessment tool for hospice.

Genograms are a valuable and non-threatening evaluation tool for hospice patients and families. The genogram provides basic information about the family including the role of each member and the family dynamics. As the diagram is drawn, family life cycle issues and relationships between family members become evident. The genogram may go beyond the household to include supportive neighbors, friends, and community resources. Religious and spiritual support is also noted. The information is used to assess family needs and to provide interventions both before the death and during bereavement.