Hideo Koga

Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin

Cytochrome P450cam (CYP101) of Pseudomonas putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate‐dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid‐point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen‐bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Identification of 2Fe-2S cysteine ligands in putidaredoxin

The iron-sulfur center of putidaredoxin is coordinated by four cysteine sulfhydrals. In order to determine which of the six cysteine residues in the protein coordinate the Fe-S center, we have individually mutated cysteine residues 73, 85 and 86 into serines. Of these mutant proteins, only C85S and C73S express holo-protein as evidence by SDS-PAGE and EPR spectroscopy. This leads us to the conclusion that residues 39,45,48, and 86 are the cysteines that coordinate the iron-sulfur center in putidaredoxin.

Lambda phage mutants insensitive to temperature-sensitive repressor: II. Genetic character of ?virC mutant

SummaryThe isolation of λ recombinant carrying virC mutation from newly isolated λ virulent carrying virL virC virR, (Horiuchi et al., 1969) was succeeded and the genetic character of virC mutation producing clear plaque was studied.λ virL virC shows weak-virulent character and produces clear plaque on λCIts lysogen but not on wild type λ lysogen. λ virC shows avirulent character and no plaque is produced on these lysogen. The virC mutation is located very closely to and on the left side of the virR region (Fig. 1) which is presumed to be the operator of the right-side operon including O and P cistrons. The genetic characters of virL, virR and virC, were compared with v1, v2, v3 mutations of classical λ vir (Jacob and Wollman, 1954) and c17 mutation of another type of λ virulent (Da Silva and Jacob, 1968). The results indicate that virL, virC or virR mutation is similar to v2, v1 or v3 mutation, respectively, and an effect of virC mutation on producing virulent character was somewhat similar to that of c17 mutation and was stronger than that of virR mutation. The length of virR regions was suggested to be smaller than one tenth of that of the CI cistron.

Purification and properties of a λ operator-binding protein which is expected to be autorepressor (tof protein) from E. coli carrying λdv plasmid

SummaryIn order to study the mode of action of the tof gene product, which is an “autorepressor” of the bacteriophage λ and plasmid λdv, we have purified a DNA-binding protein which is specifically produced in bacteria carrying λdv. This protein possesses characteristics expected for the product of the tof gene, since it is produced under conditions where cI-repressor is not made, and since it binds to oL and oR operators on the λ phage genome. The molecular weight of the native protein is 16,000–17,000 daltons, and the monomeric molecular weight as measured by gel electrophoresis in the presence of sodium dodecyl sulfate is about 10,000 daltons. Denaturation and renaturation experiments demonstrated that the native protein is a dimer of 10,000-dalton monomers. The λDNA-specific binding protein is not produced in cells carrying i21dv or Φ80dv.

Hydrogen bond network of cytochrome P-450cam: a network connecting the heme group with helix K

During investigations of the structural character of a mutant P-450cam where Glu-286 is replaced with lysine, we obtained evidence of a hydrogen bond network between helix K and the heme group via helix L of P-450cam. This mutant protein loses the ability to maintain the heme group in a proper position, possibly due to a break in the hydrogen bond network.

Complete nucleotide sequence of the 5-exo-hydroxycamphor dehydrogenase gene on the CAM plasmid of Pseudomonas putida (ATCC 17453)

We determined the complete nucleotide sequence of the first gene of Pseudomonas putida cytochrome P-450cam hydroxylase operon, camD, which encodes 5-exo-hydroxycamphor dehydrogenase. This dehydrogenase apparently consists of 361 amino acids and has a molecular mass of 38.4 kDa. Structural relationships to other zinc-containing alcohol dehydrogenases also became evident.

Transcription and replication of lambda bacteriophage virulent derivatives

Abstract λ vir L, λ vir R, and λ vir C synthesized very small amounts of λ-specific mRNA and DNA in λ ind − lysogens. The vir L genomes were subject to replication inhibition, while the vir R genomes partially, and the vir C genomes almost completely, were released from replication inhibition. λ vir LR also showed very little mRNA and DNA synthesis, and produced only small amounts of the viable phages. λ vir CR showed fairly extensive mRNA and DNA synthesis, and significantly produced viable phages. λ vir LC synthesized extensively mRNA and DNA, and produced viable phages as did λ vir LCR. The synthesis of r R mRNA by λ vir CR sus N 7 in the nonpermissive sensitive cells was approximately half that by λc 17 sus N 7 , but exceeded 2-fold that by λc I sus N 7 and the DNA synthesis was approximately 80% of that by λc I.

The mutagenic activity of ethyl N-hydroxycarbamate and its related compounds in Salmonella typhimurium

Alkyl N-hydroxycarbamates exhibited weak but significant mutagenic activity for Salmonella typhimurium TA100. The mutagenic potencies of these N-hydroxycarbamates were ranked thus: ethyl N-hydroxycarbamate greater than propyl N-hydroxycarbamate greater than methyl N-hydroxycarbamate. Acylation of ethyl N-hydroxycarbamate markedly enhanced its mutagenic activity for TA100. The highest mutagenic activity was observed with ethyl N-benzoyloxycarbamate among these acyl derivatives. Almost all the compounds were mutagenic to all the strains TA1535, TA100, TA98, especially to TA100.

Nucleotide sequence of the gene encoding a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid

The camR gene of Pseudomonas putida encodes a repressor which regulates expression of the cytochrome P-450cam hydroxylase operon (camDCAB). We determined the nucleotide sequence of 1134 continuous base pairs, including the camR gene. When comparing the amino acid sequence deduced from the open reading frame of the gene sequence with that of amino-terminal five residues of the cam repressor, purified from Pseudomonas putida, we found that the camR gene encodes a protein of 186 amino acids, with a molecular mass of 20.4 kDa. The start codon for the cam repressor is the rare initiation codon GTG. The cam repressor predicted from the camR sequence contained a region similar to that seen in other DNA-binding proteins.

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH2-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH2-terminal portion of the reductase involving residues 5–35.