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Functional analysis of Rex, a sensor of the NADH/NAD+ redox poise in Streptomyces coelicolor

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posted on 2023-06-07, 15:44 authored by Claire Michelle Strain-Damerell
Maintenance of the intracellular NADH/NAD+ redox poise is vital for energy generation in cells. Gram-positive bacteria, including the antibiotic-producing organism, Streptomyces coelicolor, have evolved a regulatory protein Rex that both senses this ratio and mediates an adaptive response to changes in it. Rex is a dimeric redox-sensitive transcriptional repressor. It is capable of binding to both NAD+ and NADH, although only NADH is an effector, causing dissociation of the protein from operator (ROP) sites. As NADH levels rise during oxygen limitation Rex dissociates from its target genes allowing expression, which helps to restore the NADH/NAD+ ratio. Microarray-based expression studies had suggested that Rex regulated only a small number of genes. In this work, however, ChIP-on-chip analyses revealed 38 genes that are potential regulon members. Analysis of the Rex binding sites in S. coelicolor revealed new insights into the mode of binding and show that Rex can bind with low affinity to incomplete half sites. This work also focused on characterising two key Rex targets, ndh and nuoA-N, that encode non-proton-translocating and proton translocating NADH dehydrogenases, respectively. Whereas nuoAN is not essential and was not expressed in liquid media, ndh was essential for growth. Depletion of NDH from growing cells led to the induction of Rex target genes confirming that ndh and Rex play key roles in maintaining redox homeostasis. Structure-based dissection of Rex, via a close homologue in Thermus aquaticus, identified a key interaction between the NADH- and DNAbinding domains of Rex. An R29-D203’ salt-bridge, that traverses the NADH binding and DNA binding domains of Rex, appeared to stabilise the DNA-bound form of Rex, but is ‘broken’ in the presence of NADH. In the NADH-bound form of Rex, D203 alternatively interacts with Y111, which in turn interacts with the nicotinamide ring of NADH. In order to assess the importance of individual subunits in the dimeric Rex, a single-chain derivative was constructed and the NADH binding and DNA binding domains individually disrupted.

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  • Published version

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205.0

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  • Biochemistry Theses

Qualification level

  • doctoral

Qualification name

  • dphil

Language

  • eng

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University of Sussex

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  • Yes

Legacy Posted Date

2011-02-08

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