In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysenedegrading organism Sphingomonas sp. strain CHY-1 were investigated. [14 C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1 a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.

Inwardly rectifying potassium (Kir) channels in Mü ller glia play a critical role in the spatial buffering of potassium ions that accumulate during retinal activity. To this end, Kir channels show a polarized subcellular distribution with the predominant channel subunit in Mü ller glia, Kir4.1, clustered in the endfeet of these cells at the inner limiting membrane. However, the molecular mechanisms underlying their distribution have yet to be identified. Here, we show that laminin, agrin and a-dystroglycan (DG) codistribute with Kir4.1 at the inner limiting membrane in the retina and that laminin-1 induces the clustering of a-DG, syntrophin and Kir4.1 in Mü ller cell cultures. In addition, we found that a-DG clusters were enriched for agrin and sought to investigate the role of agrin in their formation using recombinant C-agrins. Both C-agrin 4,8 and C-agrin 0,0 failed to induce a-DG clustering and neither of them potentiated the a-DG clustering induced by laminin-1. Finally, our data reveal that deletion of the PDZ-ligand domain of Kir4.1 prevents their laminin-induced clustering. These findings indicate that both laminin-1 and a-DG are involved in the distribution of Kir4.1 to specific Mü ller cell membrane domains and that this process occurs via a PDZ-domain-mediated interaction. Thus, in the basal lamina laminin is an essential regulator involved in clearing excess potassium released during neuronal activity, thereby contributing to the maintenance of normal synaptic transmission in the retina.

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an ␣ 3 ␤ 3 heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per ␣ subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent K m for salicylate of 1.1 ؎ 0.2 M. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (K m , 28 ؎ 6 M). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a K i of 0.68 M. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.

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The cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the ATP-binding cassette (ABC) superfamily that functions as a chloride channel. The predicted structure of CFTR protein contains two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD1 and NBD2). The opening of the Cl- channel is directly linked to ATP-driven tight dimerization of CFTR's NBD1 and NBD2 domains. The presence of a heterodimeric interfaces (HI) region in NBD1 and NBD2 generated a head to tail orientation necessary for channel activity. This process was also suggested to promote important conformational changes in the associated transmembrane domains of CFTR, which may impact the CFTR plasma membrane stability. To better understand the role of the individual HI region in this process, we generated recombinant CFTR protein with suppressed HI-NBD1 and HI-NBD2. Our results indicate that HI-NBD2 deletion leads to the loss of the dimerization profile of CFTR that affect its plasma membrane stability. We conclude that, in addition to its role in Cl- transport, HI-NBD2 domain confers membrane stability of CFTR by consolidating its quaternary structure through interactions with HI-NBD1 region.