Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils.

Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of the dmp operon genes encoding the (methyl)phenol degradative pathway of Pseudomonas sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to para-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways.

Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling.

The Pseudomonas derived sigma(54)-dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators. Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus. Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles. Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenol-binding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR. Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription. Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression.

The sulfhydryl groups of Cys 269 and Cys 272 are critical for the oligomeric state of chloroplast carbonic anhydrase from Pisum sativum.

Chloroplast carbonic anhydrase is dependent on a reducing environment to retain its catalytic activity. To investigate the properties of the three accessible cysteine residues of pea carbonic anhydrase, mutants were made in which Ala or Ser substituted for C165, C269, and C272. The mutants at position 165 were found to be spectroscopically similarly to the wild-type. They have a high catalytic activity, and are also sensitive to oxidation. In contrast, both C269 and C272 were found to be critical both for the structure and for the catalytic activity. All mutants with substitutions at either of these two positions had to be co-overexpressed with GroES/EL chaperones to give soluble enzyme in Escherichia coli. The k(cat) values were decreased by 2 and 3 orders of magnitude for the C272A and C269A mutants, respectively, and the Km values were increased approximately 7 times. However, the binding of the inhibitor ethoxyzolamide was only slightly weakened. The near-UV CD spectra were found to be changed in both sign and intensity compared to that of the wild-type, and the far-UV spectra indicate some loss of alpha-helix structure. Moreover, the quaternary structure was changed from the wild-type octameric to tetrameric in these mutants. The results indicate that mutation of either of these cysteines causes minor structural changes around at least one of the two tryptophans of the subunit. Furthermore, the data demonstrate that C269 and C272 are involved in the interaction between subunits and are necessary for a proper structure at the tetramer-tetramer interface.

Determination of the structural role of the N-terminal domain of human extracellular superoxide dismutase by use of protein fusions.

The N-terminal domain, containing the 49 N-terminal amino-acid residues, of human extracellular superoxide dismutase (hEC-SOD) has been studied after construction of fusion proteins comprised of the defined domain and human carbonic anhydrase II (HCAII). The specific advantage of this technique is that it allows characterization of properties that are intrinsic to the N-terminal domain of hEC-SOD, i.e., the results are not obscured by properties pertaining to the rest of the hEC-SOD molecule. Moreover, the fusion to HCAII allows a rapid and gentle one-step purification by affinity chromatography. When the N-terminal domain was fused to the N-terminal of HCAII ( = FusNN) a well defined structure was formed and the resulting protein was tetrameric. When the same hEC-SOD-derived domain was fused to the C-terminal of HCAII ( = FusNC), no defined structure of the fused domain could be observed, and the resulting protein was monomeric. It was concluded that a 'free' N-terminus is required for formation of the proper structure of the N-terminal domain.

Mercuric ion binding abilities of MerP variants containing only one cysteine.

Using site-directed mutagenesis, Cys14 and Cys17 in MerP were replaced in turn by serine or alanine. All four variants were purified and partially characterized. The The mutant proteins all had one reactive thiol group left. In the absence of external thiols, the protein variants bound between two and four Hg2+, but unlike non-mutant MerP, none of the variants could bind Hg2+ when external thiol was added. This loss of the ability to specifically bind one Hg2+ per protein molecule shows that both cysteine residues 14 and 17 are necessary for binding of Hg2+ when there is competition from other thiol groups.