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Properties of peptide chain release factor 2 from Streptomyces coelicolor A3(2): conserved primary structure but no frameshift regulation.

ABSTRACT: A gene was cloned from Streptomyces coelicolor A3(2). It encodes a protein of 368 amino acid residues with a high degree of similarity to prokaryotic release factor 2. However, it has neither an internal stop codon nor the Shine-Dalgarno-like sequence immediately upstream of the assumed frameshift position. The gene is expressed and functional in Escherichia coli as peptide chain release factor 2. The transcription start site is at or adjacent to the translational start site. The size of the mRNA detected by hybridization suggests that the gene (prfB) is monocistronic in S. coelicolor A3(2). However, about 80 bp upstream of the gene there is an operon which is composed of two genes encoding eukaryotic-type serine/threonine kinases.

SUBMITTER: Ogawara H

PROVIDER: S-EPMC177330 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Properties of peptide chain release factor 2 from Streptomyces coelicolor A3(2): conserved primary structure but no frameshift regulation.

Ogawara H H Urabe H H Ohtaki R R Nakamura Y Y

Journal of bacteriology 19950901 18

A gene was cloned from Streptomyces coelicolor A3(2). It encodes a protein of 368 amino acid residues with a high degree of similarity to prokaryotic release factor 2. However, it has neither an internal stop codon nor the Shine-Dalgarno-like sequence immediately upstream of the assumed frameshift position. The gene is expressed and functional in Escherichia coli as peptide chain release factor 2. The transcription start site is at or adjacent to the translational start site. The size of the mRN ...[more]

PMID: 7545157

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Project description:4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2). The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30 degrees C, respectively. The K(m) value for 4-coumarate and k(cat) were determined as 131 +/- 4 micro M and 0.202 +/- 0.007 s(-1), respectively. The K(m) value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (K(m), 190 +/- 2 micro M; k(cat), 0.475 +/- 0.012 s(-1)), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for S. coelicolor A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala(294) and Ala(318) are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.

Dataset Information

Properties of peptide chain release factor 2 from Streptomyces coelicolor A3(2): conserved primary structure but no frameshift regulation.

Publications

Properties of peptide chain release factor 2 from Streptomyces coelicolor A3(2): conserved primary structure but no frameshift regulation.

Similar Datasets

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