Divergent Cl- and H+ pathways underlie transport coupling and gating in CLC exchangers and channels

The CLC family comprises H+-coupled exchangers and Cl- channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown. Here, we use MD simulations, biochemical and electrophysiological measurements to identify two conserved phenylalanine residues that form an aromatic pathway whose dynamic rearrangements enable H+ movement outside the Cl- pore. These residues are important for H+ transport and voltage-dependent gating in the CLC exchangers. The aromatic pathway residues are evolutionarily conserved in CLC channels where their electrostatic properties and conformational flexibility determine gating. We propose that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters and suggest a unifying mechanism that describes the gating mechanism of both CLC subtypes.

Divergent Cl− and H+ pathways underlie transport coupling and gating in CLC exchangers and channels

The CLC family of anion transporting proteins is comprised of secondary active H+-coupled exchangers and of Cl− channels. Both functional subtypes play key roles in human physiology, and mutations causing their dysfunction lead to numerous genetic disorders. Current models suggest that the CLC exchangers do not utilize a classical ‘ping-pong’ mechanism of antiport, where the transporter sequentially interacts with one substrate at a time. Rather, in the CLC exchangers both substrates bind and translocate simultaneously while moving through partially congruent pathways. How ions of opposite electrical charge bypass each other while moving in opposite directions through a shared permeation pathway remains unknown. Here, we use MD simulations in combination with biochemical and electrophysiological measurements to identify a pair of highly conserved phenylalanine residues that form an aromatic pathway, separate from the Cl− pore, whose dynamic rearrangements enable H+ movement. Mutations of these aromatic residues impair H+ transport and voltage-dependent gating in the CLC exchangers. Remarkably, the role of the aromatic pathway is evolutionarily conserved in CLC channels. Using atomic-scale mutagenesis we show that the electrostatic properties and conformational flexibility of these aromatic residues are essential determinants of channel gating. Our results suggest that Cl− and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters. We propose a unifying mechanism that describes the gating mechanism of CLC exchangers and channels.

A model system of gene-enzyme nomenclature in the genome era applied to aromatic biosynthesis

Background: The accurate annotation of functional roles for newly sequenced genes of genomes is not a simple matter. Function is, of course, related to amino-acid sequence and to domain structure but not always in straightforward ways. Even where given functional roles have been identified experimentally, the application of an uneven and erratic nomenclature has generated confusion on the part of annotators and has produced errors that tend to become progressively compounded in database repositories. Results: The pathway that is deployed in nature for aromatic biosynthesis exemplifies an accumulation of chaotic nomenclature and a variety of annotation dilemmas. We view this pathway as one that is sufficiently complex to pose most of the common problems, and yet is one that at the same time is of a manageable size. A set of guidelines has been developed for naming genes of aromatic-pathway biosynthesis and the corresponding gene products, and we suggest that these can be generalized for application to other metabolic pathways. Conclusion: A system of nomenclature for aromatic biosynthesis is presented that is logical, consistent, and evolutionarily informative.

A model system of gene-enzyme nomenclature in the genome era applied to aromatic biosynthesis

Background: The accurate annotation of functional roles for newly sequenced genes of genomes is not a simple matter. Function is, of course, related to amino-acid sequence and to domain structure but not always in straightforward ways. Even where given functional roles have been identified experimentally, the application of an uneven and erratic nomenclature has generated confusion on the part of annotators and has produced errors that tend to become progressively compounded in database repositories. Results: The pathway that is deployed in nature for aromatic biosynthesis exemplifies an accumulation of chaotic nomenclature and a variety of annotation dilemmas. We view this pathway as one that is sufficiently complex to pose most of the common problems, and yet is one that at the same time is of a manageable size. A set of guidelines has been developed for naming genes of aromatic-pathway biosynthesis and the corresponding gene products, and we suggest that these can be generalized for application to other metabolic pathways. Conclusion: A system of nomenclature for aromatic biosynthesis is presented that is logical, consistent, and evolutionarily informative.

Analysis of the magnetically induced current density of molecules consisting of annelated aromatic and antiaromatic hydrocarbon rings

The aromatic pathway of molecules with annelated aromatic and antiaromatic rings has been studied by calculating magnetically induced current densities.

Limited aromatic pathway genes diversity amongst aromatic compound degrading soil bacterial isolates

Identification and characterization of novel genes belonging to microbial aromatic biodegradation pathway is of great importance as they have been proven versatile biocatalysts. In this study, the selection of 19 environmental bacterial isolates capable to degrade a wide range of aromatic compounds has been screened for the presence of five genes from the lower and the upper aromatic biodegradation pathway using PCR methodology. In the case of 4-oxalocrotonate tautomerase and toluene dioxygenases, although present in the most of environmental isolates, very limited diversity of the genes has been encountered. Highly conserved sequences of these genes in environmental samples revealed high homology with gene sequences of the characterized corresponding genes from Pseudomonas putida species. The screen using degenerate primers based on known catechol-and naphthalene dioxygenases sequences resulted in a limited number of amplified fragments. Only two catechol 2,3-dioxygenase from two Bacillus isolates were amplified and showed no significant similarities with dioxygenases from characterized organisms, but 80-90% identities with partial catechol 2,3-dioxygenase sequences from uncultured organisms. Potentially three novel catechol 1,2-dioxygenases were identified from Bacillus sp. TN102, Gordonia sp. TN103 and Rhodococcus sp. TN112. Highly homologous tautomerase and toluene dioxygenases amongst environmental samples isolated from the contaminated environment suggested horizontal gene transfer while limited success in PCR detection of the other three genes indicates that these isolates may still be a source of novel genes.

Evaluating anthranilate synthase as a herbicide target

Attempts to discover new active ingredients and target sites within the aromatic pathway have resulted in the synthesis of potent enzyme inhibitors, but no herbicides. As an aid in identifying a new target for inhibitor design and screening, we have determined the mode of action of a compound (6-methyl anthranilate) that exhibits noncommercial levels of herbicidal activity. Our evidence suggests that 6-methyl anthranilate is converted in vivo, by traversing the tryptophan biosynthetic sequence, to 4-methyl tryptophan, which inhibits anthranilate synthase. Inhibitors synthesized by design and those found by target-based screening converged on analogs of tryptophan and anthranilate. None, however, was more herbicidal than 6-methyl anthranilate.