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I C Gunsalus

Publications and source records attributed to I C Gunsalus.

At least 37 records · Page 2Linked to original sources
PubMed1985

Regulation of naphthalene catabolic genes of plasmid NAH7.

Tn5 insertion mutations defining a regulatory gene, nahR, of the naphthalene catabolic pathway encoded by the NAH7 plasmid were mapped within a small NAH7 region only a few hundred bases upstream of the nahG gene, the most promoter-proximal gene of the nahGHIJK operon. The nahR mutations blocked the induction of both the nahABCDEF and nahGHIJK operons, and the defect was completely corrected in the presence of the wild-type allele in a trans position. The pleiotropic, recessive, and negative nature of these mutations indicates that the nahR gene specifies a regulatory element which is required to activate both nah operons.

Chromosome Mapping↗
PubMed1984

Learning.

Explore the source record for details and available documents.

Bacterial Physiological Phenomena↗
PubMed1983

Heme binding and substrate-protected cysteine residues in P-450cam.

Reactions of the Pseudomonas putida cytochrome P-450-substrate complex or enzyme alone with 14C-labeled iodoacetic acid have been investigated at pH 7.0. After subsequent conversion of all of the cysteine residues to S-beta-carboxymethylcysteinyl residues, tryptic peptides of the derivative were separated by either high performance liquid chromatography or two dimensional electrophoresis, and their amino acid compositions and partial sequences were determined. All but cysteine residue-134 reacted to some extent. This result implicated residue-134 as the thiol group which is the axial heme ligand since the heme was intact in all of the derivatives made. Reaction of the enzyme-substrate complex with "cold" iodoacetic acid followed by substrate removal and reaction with 14C-labeled iodoacetic acid resulted in radiolabeling of mainly cysteine-240. This suggested cysteine-240 to be an active site cysteine residue.

Binding Sites↗
PubMed1983

Electron microscope heteroduplex mapping of naphthalene oxidation genes on the NAH7 and SAL1 plasmids.

Introduction of the transposon Tn5 to serve as a marker allows electron microscope heteroduplex mapping of the naphthalene oxidation genes on the approximately 83-kb NAH7 and the related approximately 85-kb SAL1 plasmids. The electron microscope-mapped gene positions on the NAH7 plasmid are in close agreement with those mapped previously by restriction digestion. The SAL1 plasmid can be considered as a mutant NAH7 plasmid which fails to direct the conversion of naphthalene to salicylate because of a mutational block but retains intact coding sequences for salicylate oxidation. Analysis of heteroduplex molecules formed between the SAL1 and NAH7::Tn5 EcoRI fragments and the known NAH7/SAL1 homology strongly suggest that the SAL1 DNA is completely homologous to NAH7 DNA except that a approximately 2.5-kb DNA segment constituting most of the nahA gene is replaced by approximately 4.6-kb nonhomologous DNA.

Chromosome Mapping↗
PubMed1983

Cloning of genes for naphthalene metabolism in Pseudomonas putida.

Plasmid pIG7 DNA cloned in Pseudomonas putida with the broad-host-range vectors pRK290 and pKT240 expresses the genes encoding nephthalene oxidation in the presence of the intermediate substrate, salicylate, or the gratuitous inducer, anthranilate. Two operons, nahAF and nahGK, cloned from the EcoRI fragment A (25 kilobases) are under wild-type regulation by the nahR locus. Deletion plasmids provide a restriction map of both operons. Double transformants containing structural and regulatory cistron nahR in trans are used to demonstrate positive control of expression.

Cloning, Molecular↗
PubMed1982

Amino acid sequence of the Pseudomonas putida cytochrome P-450. I. Sequences of tryptic and clostripain peptides.

In order to elucidate the complete amino acid sequence of Pseudomonas putida cytochrome P-450, tryptic digestion was performed on the S-carboxymethylated enzyme. Although cleavage did not occur at every lysyl and arginyl bond, 31 tryptic peptides ranging in size from 1 to 55 residues were isolated. These were sequenced by manual Edman degradation and carboxypeptidase digestion. Overlaps of some od these tryptic peptides were obtained by data obtained from partial Edman degradation and amino acid composition of the clostripain cleavage products. These results, together with data from the cyanogen bromide and acid cleavage peptides reported in the accompanying paper, established the complete amino acid sequence of P. putida cytochrome P-450.

Amino Acid Sequence↗
PubMed1982

Active site studies of cytochrome P=450CAM. I. Specific cysteine labeling with the affinity reagent isobornyl bromoacetate as a model for substrate binding.

A model is presented suggesting a function of specific cysteine residue of cytochrome P-450CAM in binding the substrate camphor, via a thiohemiketal bond, for its correct orientation to the heme iron and for the subsequent transfer of nascent product to facilitate its release. This model was developed to explain the results of affinity labeling with isobornyl bromoacetate. This reagent couples to the proteins via a thioether bond to cysteine, eliciting a type I transition in the difference spectrum. Formation of this covalent complex, which is strongly inhibited by the substrate, can be monitored by quantitation of S-carboxymethylcysteine in acid hydrolyzates. While addition of one equivalent of label yields 0.3 equivalents of the cysteine derivative after 5 min, increasing to 0.8 equivalents after 24 h, the spectral shift decays with time. Kinetic analysis of the spectral decay and of covalent coupling strongly suggests that thioether bond formation occurs at the substrate binding-site, in a reaction step prior to, and distinct from, the step associated with the spectral decay. The P-450CAM derivative, when titrated with camphor, produced again a type I spectrum virtually identical with the spectrum of the native P-450CAM-substrate complex. While the model presented here is not the only possible interpretation of the results, it is fully consistent with them and provides an excellent framework for further study of the catalytic mechanism of P-450CAM.

Affinity Labels↗
PubMed1982

Plasmid gene organization: naphthalene/salicylate oxidation.

Genes for naphthalene metabolism are localized on nah7, an 83-kilobase (kb) plasmid, in two gene clusters under salicylate control. Polar mutations formed by insertion of the transposon Tn5 permit detection of the transcription direction and the gene organization within two approximately 10-kb DNA segments separated by a approximately 7-kb regulatory gene region. The gene cluster specifying conversion of naphthalene to salicylate lies near the left initiation of a 25-kb DNA fragment A released by EcoRI; that for the salicylate pathway via catechol meta-fission lies near the right terminus with extension into the adjoining 5.9-kb fragment C. The genetic organization and regulation resemble the tol plasmid-encoded "upper" and "lower" pathways of toluene/xylene oxidation in Pseudomonas putida mt2.

Chromosome Mapping↗
PubMed1981

Apoprotein formation and heme reconstitution of cytochrome P-450cam.

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.

Apoproteins↗
PubMed1981

Cobalt-substituted cytochrome P-450cam.

Reconstitution of the apo-cytochrome with cobalt protoporphyrin provides a faithful P-450cam analogue as characterized by optical, ligand-binding, and enzymatic parameters. The thiol and cyanide complexes exhibit Soret "hyper" spectra, not previously observed in cobalt porphyrins. Substrate-induced spectral changes and limited stereospecific hydroxylation activity are retained in the cobalt P-450cam. The EPR (electron paramagnetic resonance) spectra of the reduced cobaltous protein indicate clearly an endogenous axial ligand other than a nitrogenous base and support an assignment of thiolate coordination. A thiolate ligand is also indicated by EPR measurements in the oxygenated cobaltous analogue. By analogy, these studies suggest that the native ferrous and oxygenated P-450cam states retain a thiolate axial ligand.

Apoproteins↗
PubMed1979

The effect of cytochrome P-450cam on the NMR relaxation rate of water protons.

Cytochrome P-450cam in the native, substrate-free state (Fe3+, S = 1/2) substantially reduces the NMR relaxation times, T1 and T2, of water protons. Temperature and frequency dependences of T1 and T2 were measured; they are consistent with a model of one or two protons exchanging between a binding site on a heme ligand and bulk water. The relevant parameters of this model have been deduced from the data. The spin relaxation time of the heme iron, tau S similar to 0.5 ns at 25 degrees C, is unusually long for a low spin ferric heme protein but is compatible with the line widths measured for paramagnetically shifted heme resonances. The proton residence time on the ligand, tau M similar to 1 microsecond at 25 degrees C, follows an Arrhenius law with activation energy EM similar to 15 kcal/mol. A scalar hyperfine interaction A/h = 2.2 MHz (3.1 MHz for one-proton exchange) of the found proton(s) with the heme iron is deduced from the difference between T1 and T2 observed in the fast exchange limit. The iron-proton distance is found to be 2.9 A (2.6 A for one-proton exchange). Variation of pH between pH 6.4 and 8.6 does not affect T1. The bearing of these results on the question of the axial heme ligand is discussed.

Binding Sites↗
PubMed1979

Chromosomal mobilization from a recA mutant of Pseudomonas putida.

A methyl methane sulfonate sensitive mutant of P. putida strain PpG1 is also extremely sensitive to UV-rays, compared to parent wild type cells. This mutant behaves typically as recombination less (recA) mutants of Escherichia coli and Pseudomonas aeruginosa, since as a recipient, it exhibits extremely low frequency of recombination following conjugational, transductional, and transformational gene transfer. Sex factor plasmids such as K-XYL or TOL can mobilize chromosomal genes equally well both from recA+ and recA801 donor cells, suggesting that host recombination functions are not necessary for mobilization of chromosomal genes by such plasmids.

Bacterial Proteins↗
PubMed1979

Testing cosolvent cryoenzymology on multi-enzyme systems.

Fluid mixed solvents appear to supply an effective tool for investigation of enzyme catalysis at subzero temperatures. Labile reaction intermediates may be stabilized, characterized and separated. Cycling of reaction processes and side reactions can be slowed or stopped to permit single turnover of individual molecular events, and kinetic response to temperature and other physical parameters can provide dynamic and thermodynamic analysis of single enzyme systems. Multienzyme systems can furnish reliable probes of potential, limitations and possible procedural artefacts of the method. Amply explored examples are supplied by the studies of two components of the soluble camphor hydroxylase and the hepatic R450LM2 in solution and in the subcellular microsomal organelles, to reveal labile single enzyme compounds and multienzyme processes.

Cold Temperature↗