Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.     

Avian kidney mitochondrial hemeprotein P-4501α: isolation,characterization and NADPH-ferredoxin reductase-dependent activity

We describe the isolation of cytochrome P-450_1α from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-450_1α could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-450_1α traveled as a single band in SDS gel electrophoresis with an apparent M_r = 57 000. The absolute spectrum of the P-450_1α(Fe³⁺) form gave a λ_max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-450_1α which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275–8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933–3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-450_1α catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3β-ol at the C-1 position exclusively with a turnover number of 0.03 min^?1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.     

Cloning and expression of genes for lysozyme and a 20-kDal protein of colitis bacteriophage

BamHI fragments of colitis phage DNA were cloned in pBR322 DNA, and the recombinant clones carrying the lysozyme gene were identified by lysozyme activity. The inserted DNA was 1.2 kb long and when expressed in minicells it produced lysozyme and a 20-kDal protein. Colitis-phage-specific mRNAs which hybridized to the insert were 0.5 kb and 0.7 kb long and were translated into lysozyme and a 20-kDal protein, respectively, in a cell-free system derived from rice embryos. They were transcribed as monocistronic mRNAs using the internal promoters present on the inserted DNA.     

A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: Purification,characterization, and expression

Background

Dienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli.

Methods

The enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene.

Results

The enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 °C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C₄) to laurate (C₁₂). The k_cat/K_m ratios for trans-dienelactone and p-nitrophenyl caprylate (C₈), the best substrate, were 92.5 and 54.7 s⁻¹ μM⁻¹, respectively.

Conclusions

The enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site.

General significance

The enzyme is the first characterized archaeal dienelactone hydrolase.