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Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the TNF receptor superfamily. However, the exact roles of TRAF2 and TRAF5 in TNF-induced NF-kappaB activation still remain controversial. To address this issue, we generated TRAF2 and TRAF5 double knockout (DKO) mice. TNF- but not interleukin-1-induced nuclear translocation of NF-kappaB was severely impaired in murine embryonic fibroblasts (MEFs) derived from DKO mice. Moreover, DKO MEFs were more susceptible to TNF-induced cytotoxicity than TRAF2 knockout MEFs. Collectively, these results indicate that both TRAF2 and TRAF5 are involved in TNF-induced NF-kappaB activation and protection from cell death.

Intrinsic resistance toward beta-lactams in methicillin-resistant Staphylococcus aureus strains, a major source of nosocomial infections, is believed to be caused mainly by penicillin-binding protein 2a (PBP2a). This protein resembles other penicillin-binding proteins that are involved in bacterial cell wall biosynthesis and are the targets of active site acylation by beta-lactam antibiotics. PBP2a, however, presumably remains active at therapeutic concentrations of beta-lactams. In this paper, we describe a three-step purification of a soluble form of PBP2a (PBP2a') to apparent homogeneity using anion-and cation-exchange, and dye-ligand affinity chromatographies. Purified PBP2a' was a 74-kDa monomeric protein that appeared to be folded. The protein was evaluated for its enzymatic acylation with beta-lactams initially by fluorescence quenching and then kinetically by radioactive labeling. Using a modified 125I-labeled penicillin V-acylation assay, the apparent Km of PBP2a' for penicillin V was 1.2 mM. Three other beta-lactams, each of which exhibited significant fluorescence quenching, acted as strong competitive inhibitors of penicillin V with apparent Ki values of 123.4, 36.1, and 12.4 microM, respectively. By a new beta-lactam preincubation analysis, these compounds could function as substrates with similar Km values. Also, the acylation rates of different beta-lactams could be readily ascertained. The enzymatic acylation data substantiate the major causative role of PBP2a in the bacterial resistance. The quantitative radioactive acylation assays are potentially useful in screening for a potent inhibitor of the enzyme.

A reductase of Streptomyces fradiae was speculated to catalyze reduction of tylosin to relomycin, an industrially undesirable product. The activity of tylosin reductase was closely related to bacterial growth, suggesting involvement of the enzyme in a primary metabolism. The reductase activity was improved significantly in vivo and in vitro. The enzyme was also partially stabilized in vitro. Using a simple five-step chromatographic procedure, the reductase was purified 480-fold to apparent homogeneity. The purified reductase had a molecular mass of 270 kDa and consisted of two different subunits of 26 and 7 kDa at 1:1 ratio. The enzyme exhibited an absorption maximum at 405 nm and was inhibited by exogenous FAD or FMN, indicating a flavin as its prosthetic group. Tylosin reductase was optimally active at pH 7.0-7.2 and 40 degrees C with NADPH as a preferred electron donor. The Km of the enzyme for tylosin was 1.4 mM and that for NADPH was 0.15 mM. The Vmax for the enzymatic reaction was 917 mumol of tylosin formed/min/mg protein. The enzymatic conversion of tylosin to relomycin was coupled to that of NADPH to NADP+ at a stoichiometric ratio of 1:1. Tylosin reductase showed a broad substrate specificity toward all macrolide aldehydes (as normal and shunt metabolites of tylosin biosynthesis) tested. Thus, the enzyme may have a physiological role of macrolide detoxification for the bacterium.

Muconolactone isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive catabolic steps in bacteria. Separately inducible beta-ketoadipate enol-lactone hydrolases I and II are formed in representatives of Acinetobacter calcoaceticus. When subjected to DEAE-cellulose chromatography, Acinetobacter enol-lactone hydrolase I displays heterogeneous behavior which, in whole or in part, appears to be due to modifications of sulfhydryl groups in the protein; the enzyme is unusual in that its NH2-terminal amino acid is cysteine. Comparison of the NH2-terminal amino acid sequence of Acinetobacter enollactone hydrolase I, reported here, with the corresponding amino acid sequences of Acinetobacter enollactone hydrolase II and Pseudomonas enol-lactone hydrolase indicates that all three proteins have diverged widely from a common evolutionary origin. Sequence comparisons suggest that divergence of the Acinetobacter enol-lactone hydrolase structural genes was achieved by substitution with DNA derived from an ancestral muconolactone isomerase structural gene.

Muconolactone delta-isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive reactions in the beta-ketoadipate pathway of bacteria. An earlier investigation (Yeh, W.K., Davis, G., Fletcher, P., and Ornston, L.N. (1978) J. Biol. Chem. 253, 4920-4923) revealed that the respective NH2-terminal amino acid sequences of Pseudomonas putida muconolactone isomerase and Acinetobacter calcoaceticus beta-ketoadipate enol-lactone hydrolase II are evolutionarily homologous. In this report, we describe the purification of Pseudomonas beta-ketoadipate enol-lactone hydrolase and present evidence indicating that the protein is a trimer composed of identical 11,000-dalton subunits. The NH2-terminal amino acid sequences of Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase have diverged widely from each other, yet the two sequences contain different fragments of an ancestral sequence which is represented in Acinetobacter enol-lactone hydrolase II. The widely divergent Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase sequences each contain unique sets of repeated peptides. In principle, the repetitive sequences might have been introduced by elongation mutations which occurred early in the evolution of the proteins. However, the divergence of Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase is so extreme that the observed sequence repetitions cannot have been conserved from ancestral duplication mutations. Rather, the data favor the interpretation that copies of DNA were substituted into structural genes for the enzymes as they diverged.

gamma-Carboxymuconolactone decarobxylase (EC 4.1.1.44) and muconolactone isomerase (EC 5.3.3.4) mediate chemically analogous reactions in bacteria. The enzymes are inducible, and different metabolites trigger the respective syntheses of the decarboxylases in Acinetobacter calcoaceticus and Pseudomonas putida. The decarobxylases share similar oligomeric structures in which identical subunits of about 13,300 daltons appear to be self-associated into hexamers. Identical residues are found in 18 of the first 36 positions of the enzymes' NH2-terminal amino acid sequences. Thus, genetic rearrangements appear to have placed homologous structural genes for the decarboxylases under different transcriptional control in the two bacterial species. The NH2-terminal amino acid sequences of the decarboxylases and muconolactone isomerases are similar, suggesting that a common ancestral protein gave rise to the enzymes with different (albeit analogous) activities. In addition, the NH2-terminal amino acid sequences of the decarboxylases appear to have been conserved at a second region within the primary structure of the muconolactone isomerases. As has been observed with the two enol-lactone hydrolases (EC 3.1.3.24) of Acinetobacter, the structural genes for the decarboxylases and the isomerases appear to have diverged widely as they were co-selected within a single cell line, In part the divergence appears to have been achieved by mutations in which fragments of DNA within structural genes are replaced with fragments of DNA derived from a co-evolving sequence.

Cells of Pseudomonas putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methyl-cyclohexa-3,5-diene. One of these components has been purified to homogeneity and shown to be a flavoprotein that contains FAD as the only detectable prosthetic group. Fad was removed from the enzyme during purification. However, equilibrium dialysis experiments showed that the enzyme can bind one mol of FAD/mol of enzyme protein. The apparent molecular weight of the enzyme is 46,000, as judged by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. The latter result suggests the presence of a single polypeptide chain. The amino acid composition of the enzyme reveals a relatively high content of the hydrophobic amino acids leucine, isoleucine, and valine and is remarkably similar in composition to the flavoproteins that function in certain monooxygenase enzyme systems. The purified enzyme catalyzes the reduction of dichloroindophenol, nitrobluetetrazolium, ferricyanide, and ferredoxinTOL. Its ability to reduce cytochrome c and to function in the toluene dioxygenase enzyme system is absolutely dependent on the presence of ferredoxinTOL.

Homogeneous beta-ketoadipate succinyl-CoA transferase (EC 2.8.3.6) preparations were obtained from extracts of Acinetobacter calcoaceticus and Pseudomonas putida. Gel filtration indicated that the respective transferases have similar molecular weights of 108,000 and 109,000; each transferase appears to have an alpha 2 beta 2 oligomeric structure formed by association of nonidentical subunits with a molecular weight of about 25,000. The subunits were separated by sodium dodecyl sulfate-gel electrophoresis, and differences in their primary structures were revealed by determination of the NH2-terminal amino acid sequences of the oligomers. The transferases cross-react immunologically and possess similar amino acid compositions. These are remarkably similar to the amino acid compositions of gamma-carboxymuconolactone decarboxylases (EC 4.1.1.44) and beta-ketoadipate enol-lactone hydrolases (EC 3.1.1.24), enzymes that mediate consecutive reactions preceding the transferase step in the beta-ketoadipate pathway.