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Biomedical subjects

I C Gunsalus

Publications and source records attributed to I C Gunsalus.

At least 19 recordsLinked to original sources
PubMed1992

Reconstitution of cytochrome P4502B4 (LM2) activity with camphor and linalool monooxygenase electron donors.

The P4502B4 (LM2) monooxygenase was shown to bind the P450cam and P450lin redoxins with respective dissociation constants of ca. 500 and 100 microM. When the redoxin reductases are added, the heterogeneous complexes demethylate benzphetamine with HCHO yields and Vmax similar to results with the homologous microsomal reductase. Implications of evolutionary conserved P450 redox electron donor binding site residues are discussed.

Acyclic Monoterpenes↗
PubMed1991

Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region.

Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of salicylate hydroxylase from Pseudomonas putida PpG7.

The salicylate hydroxylase from P. putida PpG7 was purified and characterized. The enzyme appears to be monomeric, and it showed one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 45 kDa. The sequence of the first 25 amino acids of salicylate hydroxylase (PpG7) was determined. Also, the total amino acid composition of salicylate hydroxylase (PpG7) was obtained and compared with that of the known salicylate hydroxylase from P. putida.

Amino Acid Sequence↗
PubMed1990

Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase.

The cytochrome P-450 heme-thiolate monooxygenases that hydroxylate monoterpene hydrocarbon groups are effective models for the cytochrome P-450 family. We have purified and characterized the three proteins from a P-450-dependent linalool 8-methyl hydroxylase in Pseudomonas putida (incognita) strain PpG777. The proteins resemble the camphor 5-exohydroxylase components in chemical and physical properties; however, they show neither immunological cross-reactivity nor catalytic activity in heterogenous recombination. These two systems provide an excellent model to probe more deeply the heme-thiolate reaction center, molecular domains of substrate specificity, redox-pair interactions, and the regulation of the reaction cycle.

Acyclic Monoterpenes↗
PubMed1989

Conformational changes of cytochromes P-450cam and P-450lin induced by high pressure.

Absorbance and fluorescence spectra of bacterial cytochrome P-450cam and cytochrome P-450lin have been studied as a function of pressure. These pressure-induced spectral perturbations fall into two categories, which are interpreted as resulting from denaturation domains and are discussed in terms of protein structural dynamics. The results presented herein support a view that these two bacterial cytochromes have large structural differences and suggest a picture in which the gellike cortex of each protein may play an essential role in stability and function.

Camphor↗
PubMed1989

Plasmid control of the Pseudomonas aeruginosa and Pseudomonas putida phenotypes and of linalool and p-cymene oxidation.

Two Pseudomonas strains (PpG777 and PaG158) were derived from the parent isolate Pseudomonas incognita (putida). Strain PpG777 resembles the parental culture in growth on linalool as a source of carbon and slight growth on p-cymene, whereas PaG158 grows well on p-cymene, but not on linalool or other terpenes tested, and has a P. aeruginosa phenotype. Curing studies indicate that linalool metabolism is controlled by an extrachromosomal element whose loss forms a stable strain PaG158 with the p-cymene growth and P. aeruginosa phenotype characters. The plasmid can be transferred by PpG777 to both P. putida and P. aeruginosa strains. Surprisingly, the latter assume the P. putida phenotype. We conclude that the genetic potential to oxidize p-cymene is inherent in PpG777 but expression is repressed. Similarly, this observation implies that support of linalool oxidation effectively conceals the P. aeruginosa character.

Acyclic Monoterpenes↗
PubMed1988

Nucleotide sequence of plasmid NAH7 gene nahR and DNA binding of the nahR product.

The nah and sal operons of the 80-kilobase-pair (kb) NAH7 plasmid specify catabolism of naphthalene and salicylate under positive regulation by gene nahR. A 1.75-kb fragment (PstI-HindIII) cloned into the pCP13 derivative of vector RK2 complemented in trans five nahR mutations. The fragment sequence contained a 1,122-base-pair open reading frame with a predicted sequence of 374 residues that was rich in basic amino acids with regions similar to known DNA-binding proteins. Clones from the nahR gene region were expressed in mexicells. Plasmid pY1923, carrying the 1.75-kb PstI-HindIII fragment, expressed a protein of Mr ca. 35,000 which bound to the upstream region of gene nahR in a gel electrophoresis DNA-binding assay. Other clones expressed proteins of currently unknown function; pY1311, with the 1.1-kb HindIII fragment, produced a polypeptide with an Mr of 23,000, and pY1812, with the 1.2-kb PstI-SphI fragment, produced a polypeptide (Mr 41,000) which appeared to be a fused nahR-lacZ product.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL pWWO.

The enzyme catechol 2,3-dioxygenase (C23O) encoded by the nahH gene of plasmid NAH7 converts catechol to alpha-hydroxymuconic epsilon-semialdehyde in Pseudomonas putida. We have cloned this structural gene into vectors pUC18 and pKT240, determined the nucleotide sequence and deduced the amino acid sequence. In comparison to the gene xylE of the TOL plasmid pWWO which encodes a similar C230 enzyme [Nakai et al. J. Biol. Chem. (1983b), 2923-2928], the respective G + C contents were 55% and 57%, the nucleotide sequences 81% homologous, and the amino acid homology 85%. The flanking sequences of the two C23O-coding genes also show homology. Clones of the nahH gene in Escherichia coli overproduce the protein product at least ten fold and the gene product was identified by polyacrylamide gel electrophoresis.

Base Sequence↗
PubMed1987

The P450 gene superfamily: recommended nomenclature.

A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge.

Alleles↗
PubMed1986

Regulation of the nah and sal operons of plasmid NAH7: evidence for a new function in nahR.

The catabolism of naphthalene and salicylate is specified by two operons on an 80 Kb metabolic plasmid, NAH7. These operons, nah and sal, are carried on the contiguous 30 Kb EcoRI-A, C fragments, and are under positive control of a regulator region, nahR. Five Nah Sal Tn5 insertion mutants form two complementation groups: A = nahR203, nahR204; and B = nahR201, nahR202, nahR205. The physical and genetic maps assign the nahR location to the 15.7-17.2 Kb region of the EcoRI-A fragment, with suggestion of more than one control gene.

Cloning, Molecular↗
PubMed1986

Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli.

Cytochrome P-450cam catalyzes the stereospecific methylene hydroxylation of camphor to form 5-exohydroxycamphor and is encoded by the camC gene on the CAM plasmid of Pseudomonas putida, ATCC 17453. The cytochrome P-450cam structural gene has been cloned by mutant complementation in P. putida (Koga, H., Rauchfuss, B., and Gunsalus, I. C. (1985) Biochem. Biophys. Res. Commun. 130, 412-417). We report the complete nucleotide sequence of the camC gene along with 155 base pairs of 5' and 175 base pairs of 3' flanking sequence. Upon comparison of the amino acid sequence derived from the gene sequence to the one obtained from the purified protein (Haniu, M., Armes, L. G., Yasunobu, K. T., Shastry, B. A., and Gunsalus, I. C. (1982) J. Biol. Chem. 257, 12664-12671), five differences were found. The most significant was the addition of a Trp and a Thr residue between Val-54 and Arg-55, thereby increasing the amino acid numbering scheme by 2 after Val-54, bringing the total number of amino acids to 414. Other differences were: Gln-274----Glu-276, Ser-359----His-361, and Asn-405----Asp-407. N-terminal amino acid sequence analysis of the cloned cytochrome P-450cam enzyme expressed in Escherichia coli under the lac promoter showed a faithful translation of the hemo-protein, with the N-terminal Met removed by processing as found in P. putida. Purification to homogeneity of the cloned protein was accomplished by the method used for the CAM plasmid-encoded enzyme of P. putida. The G + C content of the camC gene was found to be 59.0%, caused by a preferred usage of G and C terminated codons. The gene encoding putidaredoxin reductase, camA, was located 22 nucleotides downstream from the cytochrome P-450cam gene. The camA gene initiated with a novel GUG codon, the first such initiator documented in Pseudomonas.

Amino Acid Sequence↗
PubMed1986

camR, a negative regulator locus of the cytochrome P-450cam hydroxylase operon.

A 4.27-kilobase insert from a HindIII DNA library of Pseudomonas putida carrying the CAM plasmid allowed coordinate expression of genes camD and camC under control of camR, an upstream regulator. The camC gene specifies cytochrome P-450cam, and camD specifies the 5-exo-alcohol dehydrogenase. A 1.38-kilobase deletion from the insert results in the constitutive expression of genes camC and camD; transformation in trans restores the substrate control, indicating that camR is a negative regulator.

Alcohol Dehydrogenase↗
PubMed1985

The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450.

The crystal structure of Pseudomonas putida cytochrome P-450cam in the ferric, camphor bound form has been determined and partially refined to R = 0.23 at 2.6 A. The single 414 amino acid polypeptide chain (Mr = 45,000) approximates a triangular prism with a maximum dimension of approximately 60 A and a minimum of approximately 30 A. Twelve helical segments (A through L) account for approximately 40% of the structure while antiparallel beta pairs account for only approximately 10%. The unexposed iron protoporphyrin IX is sandwiched between two parallel helices designated the proximal and distal helices. The heme iron atom is pentacoordinate with the axial sulfur ligand provided by Cys 357 which extends from the N-terminal end of the proximal (L) helix. A substrate molecule, 2-bornanone (camphor), is buried in an internal pocket just above the heme distal surface adjacent to the oxygen binding site. The substrate molecule is held in place by a hydrogen bond between the side chain hydroxyl group of Tyr 96 and the camphor carbonyl oxygen atom in addition to complementary hydrophobic contacts between the camphor molecule and neighboring aliphatic and aromatic residues. The camphor is oriented such that the exo-surface of C5 would contact an iron bound, "activated" oxygen atom for stereoselective hydroxylation.

Amino Acid Sequence↗
PubMed1985

P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli.

The gene camC, which encodes the cytochrome P450 monoxygenase protein, was cloned into the shuttle vector pKT240 and recovered as the recombinant pKG201 with a 2.3 kb insert from the CAM plasmid in the PstI site. The gene product is expressed constitutively in P. putida and in E. coli whereas the inverted insert clone lacks expression, indicating absence of an insert promoter.

Animals↗
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↗