The nucleotide sequences of the Acinetobacter cakoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the oa-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(ll) ion. The less conserved 13-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.The complete degradation of benzoate by aerobic bacteria can occur by either of two catabolic pathways. In both reaction sequences, benzoate is converted to a nonaromatic cis-diol, 2-hydro-1,2-dihydroxybenzoate, and then to catechol (51) (Fig. 1) (75).In addition to the hydroxylase component, the dioxygenases described above usually contain one or two electron transport proteins. The benzoate 1,2-dioxygenase of P. arvilla has a single iron-sulfur flavoprotein exhibiting an NADH-cytochrome c reductase activity that is responsible for the electron transfer from NADH to the aromatic ring hydroxylase. This enzyme is a 38-kDa polypeptide with one iron-sulfur cluster of the [2Fe-2S] type and one molecule of flavin adenine dinucleotide (FAD) (73,74). The involvement of similar proteins in the electron transfer reactions of benzoate 1,2-dioxygenase from A. calcoaceticus and toluate 1,2-dioxygenase from P. putida have been suggested by our previous genetic studies (19,21,42). In the benzene, toluene, and naphthalene dioxygenase systems, however, two 5385 JOURNAL

Cytokines can initiate and perpetuate human diseases, and are among the best-validated of therapeutic targets. Cytokines can be blocked by the use of soluble receptors; however, the use of this approach for cytokines such as interleukin (IL)-1, IL-4, IL-6 and IL-13 that use multi-component receptor systems is limited because monomeric soluble receptors generally exhibit low affinity or function as agonists. We describe here a generally applicable method to create very high-affinity blockers called 'cytokine traps' consisting of fusions between the constant region of IgG and the extracellular domains of two distinct cytokine receptor components involved in binding the cytokine. Traps potently block cytokines in vitro and in vivo and represent a substantial advance in creating novel therapeutic candidates for cytokine-driven diseases.

The DNA sequence of a 2,391-base-pair HindIII restriction fragment of Acinetobacter calcoaceticus DNA containing the pcaCHG genes is reported. The Protocatechuate 3,4-dioxygenase ( Fig. 1; EC 1.13.11.3) plays an essential role in the utilization of numerous aromatic and hydroaromatic compounds via the P-ketoadipate pathway (45). Ubiquity of the pathway in the natural environment is indicated by the fact that the enzyme is a trait universally shared by fluorescent Pseudomonas species (46) and by members of the family Rhizobiaceae (39). The available evidence points to a single evolutionary origin for protocatechuate 3,4-dioxygenases formed by diverse bacteria (14). Other bacterial intradiol dioxygenases (Fig. 1) act upon catechol. Catechol oxygenase I (40) exhibits narrow substrate specificity and a relatively high Kcat (33) when compared with catechol oxygenase II (10), an enzyme that more readily accommodates chlorocatechol as a substrate and exhibits a relatively low Kcat (Fig. 1).The activity of intradiol dioxygenases depends on ferric ion which is ligated by two histidyl and two tyrosyl side chains within the catalytic subunit of the enzymes (41). Determination of the crystal structure of a Pseudomonas protocatechuate 3,4-dioxygenase has established the position of the iron-ligating histidyl and tyrosyl residues within the primary sequence of the a subunit (34). Like other protocatechuate 3,4-dioxygenases, the Pseudomonas enzyme contains in equimolar amounts the i subunit and an a subunit that contributes to substrate binding. Amino acid sequence comparisons (21,24)

Two structural genes needed for catechol degradation, cat4 and catE, encode the respective enzymes catechol 1,2-dioxygenase (EC 1.13.11.1) and muconate cycloisomerase (EC 5.5.1.1). Catechol is an intermediate in benzoate degradation, and the catA and catB genes are clustered within a 17-kilobase-pair (kbp) region of Acinetobacter calcoacesicus chromosomal DNA containing aHl of the structural genes required for the conversion of benzoate to tricarboxylic acid cycle intermediates. cat4 and catB were transcribed in the same direction and were separated by 3.8 kbp of DNA. The 3.8-kbp sequence revealed that directly downstream from catA and potentially transcribed in the same direction were two open reading frames encoding polypeptides of 48 and 36 kilodaltons (kDa). Genetic disruption of these open reading frames did not discernably alter either catechol metabolism or its regulation. A third open reading frame, beginning 123 bp upstream from catB and transcribed divergently from this gene, was designated catM. This gene was found to encode a 28-kDa trans-acting repressor protein that, in the absence of cis,cis-muconate, prevented expression of the cat structural genes. Constitutive expression of the genes was caused by a mutation substituting Arg-156 with His-156 in the catM-encoded repressor. The repressor protein proved to be a member of a diverse family of procaryotic regulatory proteins which, with rare exception, are transcriptional activators. Repression mediated by catM was not the sole transcriptional control exercised over catA in A. calcoaceticus. Expression of cat4 was elicited by either benzoate or cis,cis-muconate in a genetic background from which catM had been deleted. This induction required DNA in a segment lying 1 kbp upstream from the cat4 gene. It is likely that an additional gene, lying outside the region containing the structural genes necessary for benzoate metabolism, contributes to this control.

The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.