Dataset Information

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2).

ABSTRACT: We describe the identification of Rex, a novel redox-sensing repressor that appears to be widespread among Gram-positive bacteria. In Streptomyces coelicolor Rex binds to operator (ROP) sites located upstream of several respiratory genes, including the cydABCD and rex-hemACD operons. The DNA-binding activity of Rex appears to be controlled by the redox poise of the NADH/NAD+ pool. Using electromobility shift and surface plasmon resonance assays we show that NADH, but not NAD+, inhibits the DNA-binding activity of Rex. However, NAD+ competes with NADH for Rex binding, allowing Rex to sense redox poise over a range of NAD(H) concentrations. Rex is predicted to include a pyridine nucleotide-binding domain (Rossmann fold), and residues that might play key structural and nucleotide binding roles are highly conserved. In support of this, the central glycine in the signature motif (GlyXGlyXXGly) is shown to be essential for redox sensing. Rex homologues exist in most Gram-positive bacteria, including human pathogens such as Staphylococcus aureus, Listeria monocytogenes and Streptococcus pneumoniae.

SUBMITTER: Brekasis D

PROVIDER: S-EPMC212721 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2).

Brekasis Dimitris D Paget Mark S B MS

The EMBO journal 20030901 18

We describe the identification of Rex, a novel redox-sensing repressor that appears to be widespread among Gram-positive bacteria. In Streptomyces coelicolor Rex binds to operator (ROP) sites located upstream of several respiratory genes, including the cydABCD and rex-hemACD operons. The DNA-binding activity of Rex appears to be controlled by the redox poise of the NADH/NAD+ pool. Using electromobility shift and surface plasmon resonance assays we show that NADH, but not NAD+, inhibits the DNA-b ...[more]

PMID: 12970197

Similar Datasets

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

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2).

Publications

A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets